Tabla espec. clave
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M, R||WB, Mplex, PIA, DB, ChIP-seq, ChIP||Rb||Affinity Purified||Polyclonal Antibody|
|Presentation||Purified rabbit polyclonal in buffer containing 0.1 M Tris-Glycine (pH 7.4) 150 mM NaCl with 0.05% sodium azide.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at 2-8° C from date of receipt.
For maximum recovery of product, centrifuge the vial prior to removing the cap.
|Material Size||100 µL|
Ficha datos de seguridad (MSDS)
Referencias bibliográficas | 85 Disponible | Ver todas las referencias
|Visión general referencias||Aplicación||Especie||Pub Med ID|
|TP53 engagement with the genome occurs in distinct local chromatin environments via pioneer factor activity. |
Sammons, MA; Zhu, J; Drake, AM; Berger, SL
Genome research 25 179-88 2015
Despite overwhelming evidence that transcriptional activation by TP53 is critical for its tumor suppressive activity, the mechanisms by which TP53 engages the genome in the context of chromatin to activate transcription are not well understood. Using a compendium of novel and existing genome-wide data sets, we examined the relationship between TP53 binding and the dynamics of the local chromatin environment. Our analysis revealed three distinct categories of TP53 binding events that differ based on the dynamics of the local chromatin environment. The first class of TP53 binding events occurs near transcriptional start sites (TSS) and is defined by previously characterized promoter-associated chromatin modifications. The second class comprises a large cohort of preestablished, promoter-distal enhancer elements that demonstrates dynamic histone acetylation and transcription upon TP53 binding. The third class of TP53 binding sites is devoid of classic chromatin modifications and, remarkably, fall within regions of inaccessible chromatin, suggesting that TP53 has intrinsic pioneer factor activity and binds within structurally inaccessible regions of chromatin. Intriguingly, these inaccessible TP53 binding sites feature several enhancer-like properties in cell types within the epithelial lineage, indicating that TP53 binding events include a group of "proto-enhancers" that become active enhancers given the appropriate cellular context. These data indicate that TP53, along with TP63, may act as pioneer factors to specify epithelial enhancers. Further, these findings suggest that rather than following a global cell-type invariant stress response program, TP53 may tune its response based on the lineage-specific epigenomic landscape.
|Mutation of histone H3 serine 86 disrupts GATA factor Ams2 expression and precise chromosome segregation in fission yeast. |
Lim, KK; Ong, TY; Tan, YR; Yang, EG; Ren, B; Seah, KS; Yang, Z; Tan, TS; Dymock, BW; Chen, ES
Scientific reports 5 14064 2015
Eukaryotic genomes are packed into discrete units, referred to as nucleosomes, by organizing around scaffolding histone proteins. The interplay between these histones and the DNA can dynamically regulate the function of the chromosomal domain. Here, we interrogated the function of a pair of juxtaposing serine residues (S86 and S87) that reside within the histone fold of histone H3. We show that fission yeast cells expressing a mutant histone H3 disrupted at S86 and S87 (hht2-S86AS87A) exhibited unequal chromosome segregation, disrupted transcriptional silencing of centromeric chromatin, and reduced expression of Ams2, a GATA-factor that regulates localization of the centromere-specific histone H3 variant CENP-A. We found that overexpression of ams2(+) could suppress the chromosome missegregation phenotype that arose in the hht2-S86AS87A mutant. We further demonstrate that centromeric localization of SpCENP-A(cnp1-1) was significantly compromised in hht2-S86AS87A, suggesting synergism between histone H3 and the centromere-targeting domain of SpCENP-A. Taken together, our work presents evidence for an uncharacterized serine residue in fission yeast histone H3 that affects centromeric integrity via regulating the expression of the SpCENP-A-localizing Ams2 protein. [173/200 words].
|Ancestral Chromatin Configuration Constrains Chromatin Evolution on Differentiating Sex Chromosomes in Drosophila. |
Zhou, Q; Bachtrog, D
PLoS genetics 11 e1005331 2015
Sex chromosomes evolve distinctive types of chromatin from a pair of ancestral autosomes that are usually euchromatic. In Drosophila, the dosage-compensated X becomes enriched for hyperactive chromatin in males (mediated by H4K16ac), while the Y chromosome acquires silencing heterochromatin (enriched for H3K9me2/3). Drosophila autosomes are typically mostly euchromatic but the small dot chromosome has evolved a heterochromatin-like milieu (enriched for H3K9me2/3) that permits the normal expression of dot-linked genes, but which is different from typical pericentric heterochromatin. In Drosophila busckii, the dot chromosomes have fused to the ancestral sex chromosomes, creating a pair of 'neo-sex' chromosomes. Here we collect genomic, transcriptomic and epigenomic data from D. busckii, to investigate the evolutionary trajectory of sex chromosomes from a largely heterochromatic ancestor. We show that the neo-sex chromosomes formed less than 1 million years ago, but nearly 60% of neo-Y linked genes have already become non-functional. Expression levels are generally lower for the neo-Y alleles relative to their neo-X homologs, and the silencing heterochromatin mark H3K9me2, but not H3K9me3, is significantly enriched on silenced neo-Y genes. Despite rampant neo-Y degeneration, we find that the neo-X is deficient for the canonical histone modification mark of dosage compensation (H4K16ac), relative to autosomes or the compensated ancestral X chromosome, possibly reflecting constraints imposed on evolving hyperactive chromatin in an originally heterochromatic environment. Yet, neo-X genes are transcriptionally more active in males, relative to females, suggesting the evolution of incipient dosage compensation on the neo-X. Our data show that Y degeneration proceeds quickly after sex chromosomes become established through genomic and epigenetic changes, and are consistent with the idea that the evolution of sex-linked chromatin is influenced by its ancestral configuration.
|Rational design and validation of a Tip60 histone acetyltransferase inhibitor. |
Gao, C; Bourke, E; Scobie, M; Famme, MA; Koolmeister, T; Helleday, T; Eriksson, LA; Lowndes, NF; Brown, JA
Scientific reports 4 5372 2014
Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer.
|DNA replication and transcription programs respond to the same chromatin cues. |
Lubelsky, Y; Prinz, JA; DeNapoli, L; Li, Y; Belsky, JA; MacAlpine, DM
Genome research 24 1102-14 2014
DNA replication is a dynamic process that occurs in a temporal order along each of the chromosomes. A consequence of the temporally coordinated activation of replication origins is the establishment of broad domains (greater than 100 kb) that replicate either early or late in S phase. This partitioning of the genome into early and late replication domains is important for maintaining genome stability, gene dosage, and epigenetic inheritance; however, the molecular mechanisms that define and establish these domains are poorly understood. The modENCODE Project provided an opportunity to investigate the chromatin features that define the Drosophila replication timing program in multiple cell lines. The majority of early and late replicating domains in the Drosophila genome were static across all cell lines; however, a small subset of domains was dynamic and exhibited differences in replication timing between the cell lines. Both origin selection and activation contribute to defining the DNA replication program. Our results suggest that static early and late replicating domains were defined at the level of origin selection (ORC binding) and likely mediated by chromatin accessibility. In contrast, dynamic domains exhibited low ORC densities in both cell types, suggesting that origin activation and not origin selection governs the plasticity of the DNA replication program. Finally, we show that the male-specific early replication of the X chromosome is dependent on the dosage compensation complex (DCC), suggesting that the transcription and replication programs respond to the same chromatin cues. Specifically, MOF-mediated hyperacetylation of H4K16 on the X chromosome promotes both the up-regulation of male-specific transcription and origin activation.
|The chromatin landscape of Drosophila: comparisons between species, sexes, and chromosomes. |
Brown, EJ; Bachtrog, D
Genome research 24 1125-37 2014
The chromatin landscape is key for gene regulation, but little is known about how it differs between sexes or between species. Here, we study the sex-specific chromatin landscape of Drosophila miranda, a species with young sex chromosomes, and compare it with Drosophila melanogaster. We analyze six histone modifications in male and female larvae of D. miranda (H3K4me1, H3K4me3, H3K36me3, H4K16ac, H3K27me3, and H3K9me2), and define seven biologically meaningful chromatin states that show different enrichments for transcribed and silent genes, repetitive elements, housekeeping, and tissue-specific genes. The genome-wide distribution of both active and repressive chromatin states differs between males and females. In males, active chromatin is enriched on the X, relative to females, due to dosage compensation of the hemizygous X. Furthermore, a smaller fraction of the euchromatic portion of the genome is in a repressive chromatin state in males relative to females. However, sex-specific chromatin states appear not to explain sex-biased expression of genes. Overall, conservation of chromatin states between male and female D. miranda is comparable to conservation between D. miranda and D. melanogaster, which diverged greater than 30 MY ago. Active chromatin states are more highly conserved across species, while heterochromatin shows very low levels of conservation. Divergence in chromatin profiles contributes to expression divergence between species, with ∼26% of genes in different chromatin states in the two species showing species-specific or species-biased expression, an enrichment of approximately threefold over null expectation. Our data suggest that heteromorphic sex chromosomes in males (that is, a hypertranscribed X and an inactivated Y) may contribute to global redistribution of active and repressive chromatin marks between chromosomes and sexes.
|MOF-associated complexes ensure stem cell identity and Xist repression. |
Chelmicki, T; Dündar, F; Turley, MJ; Khanam, T; Aktas, T; Ramírez, F; Gendrel, AV; Wright, PR; Videm, P; Backofen, R; Heard, E; Manke, T; Akhtar, A
eLife 3 e02024 2014
Histone acetyl transferases (HATs) play distinct roles in many cellular processes and are frequently misregulated in cancers. Here, we study the regulatory potential of MYST1-(MOF)-containing MSL and NSL complexes in mouse embryonic stem cells (ESCs) and neuronal progenitors. We find that both complexes influence transcription by targeting promoters and TSS-distal enhancers. In contrast to flies, the MSL complex is not exclusively enriched on the X chromosome, yet it is crucial for mammalian X chromosome regulation as it specifically regulates Tsix, the major repressor of Xist lncRNA. MSL depletion leads to decreased Tsix expression, reduced REX1 recruitment, and consequently, enhanced accumulation of Xist and variable numbers of inactivated X chromosomes during early differentiation. The NSL complex provides additional, Tsix-independent repression of Xist by maintaining pluripotency. MSL and NSL complexes therefore act synergistically by using distinct pathways to ensure a fail-safe mechanism for the repression of X inactivation in ESCs.DOI: http://dx.doi.org/10.7554/eLife.02024.001.
|Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory. |
Hait, NC; Wise, LE; Allegood, JC; O'Brien, M; Avni, D; Reeves, TM; Knapp, PE; Lu, J; Luo, C; Miles, MF; Milstien, S; Lichtman, AH; Spiegel, S
Nature neuroscience 17 971-80 2014
FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2(-/-) mice have lower levels of hippocampal sphingosine-1-phosphate, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, sphingosine-1-phosphate and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories.
|Neural stem cell differentiation is dictated by distinct actions of nuclear receptor corepressors and histone deacetylases. |
Castelo-Branco, G; Lilja, T; Wallenborg, K; Falcão, AM; Marques, SC; Gracias, A; Solum, D; Paap, R; Walfridsson, J; Teixeira, AI; Rosenfeld, MG; Jepsen, K; Hermanson, O
Stem cell reports 3 502-15 2014
Signaling factors including retinoic acid (RA) and thyroid hormone (T3) promote neuronal, oligodendrocyte, and astrocyte differentiation of cortical neural stem cells (NSCs). However, the functional specificity of transcriptional repressor checkpoints controlling these differentiation programs remains unclear. Here, we show by genome-wide analysis that histone deacetylase (HDAC)2 and HDAC3 show overlapping and distinct promoter occupancy at neuronal and oligodendrocyte-related genes in NSCs. The absence of HDAC3, but not HDAC2, initiated a neuronal differentiation pathway in NSCs. The ablation of the corepressor NCOR or HDAC2, in conjunction with T3 treatment, resulted in increased expression of oligodendrocyte genes, revealing a direct HDAC2-mediated repression of Sox8 and Sox10 expression. Interestingly, Sox10 was required also for maintaining the more differentiated state by repression of stem cell programming factors such as Sox2 and Sox9. Distinct and nonredundant actions of NCORs and HDACs are thus critical for control of lineage progression and differentiation programs in neural progenitors.
|Mof-associated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation. |
Ravens, S; Fournier, M; Ye, T; Stierle, M; Dembele, D; Chavant, V; Tora, L
eLife 3 2014
The histone acetyltransferase (HAT) Mof is essential for mouse embryonic stem cell (mESC) pluripotency and early development. Mof is the enzymatic subunit of two different HAT complexes, MSL and NSL. The individual contribution of MSL and NSL to transcription regulation in mESCs is not well understood. Our genome-wide analysis show that i) MSL and NSL bind to specific and common sets of expressed genes, ii) NSL binds exclusively at promoters, iii) while MSL binds in gene bodies. Nsl1 regulates proliferation and cellular homeostasis of mESCs. MSL is the main HAT acetylating H4K16 in mESCs, is enriched at many mESC-specific and bivalent genes. MSL is important to keep a subset of bivalent genes silent in mESCs, while developmental genes require MSL for expression during differentiation. Thus, NSL and MSL HAT complexes differentially regulate specific sets of expressed genes in mESCs and during differentiation.
|The onset of C. elegans dosage compensation is linked to the loss of developmental plasticity. |
Custer, LM; Snyder, MJ; Flegel, K; Csankovszki, G
Developmental biology 385 279-90 2014
Dosage compensation (DC) equalizes X-linked gene expression between sexes. In Caenorhabditis elegans, the dosage compensation complex (DCC) localizes to both X chromosomes in hermaphrodites and downregulates gene expression 2-fold. The DCC first localizes to hermaphrodite X chromosomes at the 30-cell stage, coincident with a developmental transition from plasticity to differentiation. To test whether DC onset is linked to loss of developmental plasticity, we established a timeline for the accumulation of DC-mediated chromatin features on X (depletion of histone H4 lysine 16 acetylation (H4K16ac) and enrichment of H4K20 monomethylation (H4K20me1)) in both wild type and developmentally delayed embryos. Surprisingly, we found that H4K16ac is depleted from the X even before the 30-cell stage in a DCC-independent manner. This depletion requires the activities of MES-2, MES-3, and MES-6 (a complex similar to the Polycomb Repressive Complex 2), and MES-4. By contrast, H4K20me1 becomes enriched on X chromosomes several cell cycles after DCC localization to the X, suggesting that it is a late mark in DC. MES-2 also promotes differentiation, and mes-2 mutant embryos exhibit prolonged developmental plasticity. Consistent with the hypothesis that the onset of DC is linked to differentiation, DCC localization and H4K20me1 accumulation on the X chromosomes are delayed in mes mutant hermaphrodite embryos. Furthermore, the onset of hermaphrodite-specific transcription of sdc-2 (which triggers DC) is delayed in mes-2 mutants. We propose that as embryonic blastomeres lose their developmental plasticity, hermaphrodite X chromosomes transition from a MES protein-regulated state to DCC-mediated repression.
|Uncoupling transcription from covalent histone modification. |
Zhang, H; Gao, L; Anandhakumar, J; Gross, DS
PLoS genetics 10 e1004202 2014
It is widely accepted that transcriptional regulation of eukaryotic genes is intimately coupled to covalent modifications of the underlying chromatin template, and in certain cases the functional consequences of these modifications have been characterized. Here we present evidence that gene activation in the silent heterochromatin of the yeast Saccharomyces cerevisiae can occur in the context of little, if any, covalent histone modification. Using a SIR-regulated heat shock-inducible transgene, hsp82-2001, and a natural drug-inducible subtelomeric gene, YFR057w, as models we demonstrate that substantial transcriptional induction (greater than 200-fold) can occur in the context of restricted histone loss and negligible levels of H3K4 trimethylation, H3K36 trimethylation and H3K79 dimethylation, modifications commonly linked to transcription initiation and elongation. Heterochromatic gene activation can also occur with minimal H3 and H4 lysine acetylation and without replacement of H2A with the transcription-linked variant H2A.Z. Importantly, absence of histone modification does not stem from reduced transcriptional output, since hsp82-ΔTATA, a euchromatic promoter mutant lacking a TATA box and with threefold lower induced transcription than heterochromatic hsp82-2001, is strongly hyperacetylated in response to heat shock. Consistent with negligible H3K79 dimethylation, dot1Δ cells lacking H3K79 methylase activity show unimpeded occupancy of RNA polymerase II within activated heterochromatic promoter and coding regions. Our results indicate that large increases in transcription can be observed in the virtual absence of histone modifications often thought necessary for gene activation.
|Radiation-induced alterations of histone post-translational modification levels in lymphoblastoid cell lines. |
Maroschik, B; Gürtler, A; Krämer, A; Rößler, U; Gomolka, M; Hornhardt, S; Mörtl, S; Friedl, AA
Radiation oncology (London, England) 9 15 2014
Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them.A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients.The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual γ-H2AX foci after 24 h.So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels.
|LSD1 regulates pluripotency of embryonic stem/carcinoma cells through histone deacetylase 1-mediated deacetylation of histone H4 at lysine 16. |
Yin, F; Lan, R; Zhang, X; Zhu, L; Chen, F; Xu, Z; Liu, Y; Ye, T; Sun, H; Lu, F; Zhang, H
Molecular and cellular biology 34 158-79 2014
LSD1 is essential for the maintenance of pluripotency of embryonic stem (ES) or embryonic carcinoma/teratocarcinoma (EC) cells. We have previously developed novel LSD1 inhibitors that selectively inhibit ES/EC cells. However, the critical targets of LSD1 remain unclear. Here, we found that LSD1 interacts with histone deacetylase 1 (HDAC1) to regulate the proliferation of ES/EC cells through acetylation of histone H4 at lysine 16 (H4K16), which we show is a critical substrate of HDAC1. The LSD1 demethylase and HDAC1 deacetylase activities were both inactivated if one of them in the complex was chemically inhibited in ES/EC cells or in reconstituted protein complexes. Loss of HDAC1 phenocopied the selective growth-inhibitory effects and increased the levels of H3K4 methylation and H4K16 acetylation of LSD1 inactivation on ES/EC cells. Reduction of acetylated H4K16 by ablation of the acetyltransferase males absent on the first (MOF) is sufficient to rescue the growth inhibition induced by LSD1 inactivation. While LSD1 or HDAC1 inactivation caused the downregulation of Sox2 and Oct4 and induction of differentiation genes, such as FOXA2 or BMP2, depletion of MOF restored the levels of Sox2, Oct4, and FoxA2 in LSD1-deficient cells. Our studies reveal a novel mechanism by which LSD1 acts through the HDAC1- and MOF-mediated regulation of H4K16 acetylation to maintain the pluripotency of ES/EC cells.
|Epstein-Barr virus-mediated transformation of B cells induces global chromatin changes independent to the acquisition of proliferation. |
Hernando, H; Islam, AB; Rodríguez-Ubreva, J; Forné, I; Ciudad, L; Imhof, A; Shannon-Lowe, C; Ballestar, E
Nucleic acids research 42 249-63 2014
Epstein-Barr virus (EBV) infects and transforms human primary B cells inducing indefinite proliferation. To investigate the potential participation of chromatin mechanisms during the EBV-mediated transformation of resting B cells we performed an analysis of global changes in histone modifications. We observed a remarkable decrease and redistribution of heterochromatin marks including H4K20me3, H3K27me3 and H3K9me3. Loss of H4K20me3 and H3K9me3 occurred at constitutive heterochromatin repeats. For H3K27me3 and H3K9me3, comparison of ChIP-seq data revealed a decrease in these marks in thousands of genes, including clusters of HOX and ZNF genes, respectively. Moreover, DNase-seq data comparison between resting and EBV-transformed B cells revealed increased endonuclease accessibility in thousands of genomic sites. We observed that both loss of H3K27me3 and increased accessibility are associated with transcriptional activation. These changes only occurred in B cells transformed with EBV and not in those stimulated to proliferate with CD40L/IL-4, despite their similarities in the cell pathways involved and proliferation rates. In fact, B cells infected with EBNA-2 deficient EBV, which have much lower proliferation rates, displayed similar decreases for heterochromatic histone marks. Our study describes a novel phenomenon related to transformation of B cells, and highlights its independence of the pure acquisition of proliferation.
|Lunasin sensitivity in non-small cell lung cancer cells is linked to suppression of integrin signaling and changes in histone acetylation. |
Inaba, J; McConnell, EJ; Davis, KR
International journal of molecular sciences 15 23705-24 2014
Lunasin is a plant derived bioactive peptide with both cancer chemopreventive and therapeutic activity. We recently showed lunasin inhibits non-small cell lung cancer (NSCLC) cell proliferation in a cell-line-specific manner. We now compared the effects of lunasin treatment of lunasin-sensitive (H661) and lunasin-insensitive (H1299) NSCLC cells with respect to lunasin uptake, histone acetylation and integrin signaling. Both cell lines exhibited changes in histone acetylation, with H661 cells showing a unique increase in H4K16 acetylation. Proximity ligation assays demonstrated lunasin interacted with integrins containing αv, α5, β1 and β3 subunits to a larger extent in the H661 compared to H1299 cells. Moreover, lunasin specifically disrupted the interaction of β1 and β3 subunits with the downstream signaling components phosphorylated Focal Adhesion Kinase (pFAK), Kindlin and Intergrin Linked Kinase in H661 cells. Immunoblot analyses demonstrated lunasin treatment of H661 resulted in reduced levels of pFAK, phosphorylated Akt and phosphorylated ERK1/2 whereas no changes were observed in H1299 cells. Silencing of αv expression in H661 cells confirmed signaling through integrins containing αv is essential for proliferation. Moreover, lunasin was unable to further inhibit proliferation in αv-silenced H661 cells. This indicates antagonism of integrin signaling via αv-containing integrins is an important component of lunasin's mechanism of action.
|Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination. |
Tang, Jiangbo, et al.
Nat. Struct. Mol. Biol., 20: 317-25 (2013) 2013
The pathogenic sequelae of BRCA1 mutation in human and mouse cells are mitigated by concomitant deletion of 53BP1, which binds histone H4 dimethylated at Lys20 (H4K20me2) to promote nonhomologous end joining, suggesting that a balance between BRCA1 and 53BP1 regulates DNA double strand-break (DSB) repair mechanism choice. Here we document that acetylation is a key determinant of this balance. TIP60 acetyltransferase deficiency reduced BRCA1 at DSB chromatin with commensurate increases in 53BP1, whereas HDAC inhibition yielded the opposite effect. TIP60-dependent H4 acetylation diminished 53BP1 binding to H4K20me2 in part through disruption of a salt bridge between H4K16 and Glu1551 in the 53BP1 Tudor domain. Moreover, TIP60 deficiency impaired homologous recombination and conferred sensitivity to PARP inhibition in a 53BP1-dependent manner. These findings demonstrate that acetylation in cis to H4K20me2 regulates relative BRCA1 and 53BP1 DSB chromatin occupancy to direct DNA repair mechanism.
|The epigenome of evolving Drosophila neo-sex chromosomes: dosage compensation and heterochromatin formation. |
Zhou, Q; Ellison, CE; Kaiser, VB; Alekseyenko, AA; Gorchakov, AA; Bachtrog, D
PLoS biology 11 e1001711 2013
Sex chromosomes originated from autosomes but have evolved a highly specialized chromatin structure. Drosophila Y chromosomes are composed entirely of silent heterochromatin, while male X chromosomes have highly accessible chromatin and are hypertranscribed as a result of dosage compensation. Here, we dissect the molecular mechanisms and functional pressures driving heterochromatin formation and dosage compensation of the recently formed neo-sex chromosomes of Drosophila miranda. We show that the onset of heterochromatin formation on the neo-Y is triggered by an accumulation of repetitive DNA. The neo-X has evolved partial dosage compensation and we find that diverse mutational paths have been utilized to establish several dozen novel binding consensus motifs for the dosage compensation complex on the neo-X, including simple point mutations at pre-binding sites, insertion and deletion mutations, microsatellite expansions, or tandem amplification of weak binding sites. Spreading of these silencing or activating chromatin modifications to adjacent regions results in massive mis-expression of neo-sex linked genes, and little correspondence between functionality of genes and their silencing on the neo-Y or dosage compensation on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those becoming heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities along the ancestral chromosome that formed the sex chromosome to adopt active or repressive chromatin configurations. Our findings have broad implications for current models of sex chromosome evolution, and demonstrate how mechanistic constraints can limit evolutionary adaptations. Our study also highlights how evolution can follow predictable genetic trajectories, by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome.
|Elucidating combinatorial histone modifications and crosstalks by coupling histone-modifying enzyme with biotin ligase activity. |
Lau, PN; Cheung, P
Nucleic acids research 41 e49 2013
Histone post-translational modifications (PTMs) often form complex patterns of combinations and cooperate to specify downstream biological processes. In order to systemically analyse combinatorial PTMs and crosstalks among histone PTMs, we have developed a novel nucleosome purification method called Biotinylation-assisted Isolation of CO-modified Nucleosomes (BICON). This technique is based on physical coupling of the enzymatic activity of a histone-modifying enzyme with in vivo biotinylation by the biotin ligase BirA, and using streptavidin to purify the co-modified nucleosomes. Analysing the nucleosomes isolated by BICON allows the identification of PTM combinations that are enriched on the modified nucleosomes and function together within the nucleosome context. We used this new approach to study MSK1-mediated H3 phosphorylation and found that MSK1 not only directly phosphorylated H3, but also induced hyperacetylation of both histone H3 and H4 within the nucleosome. Moreover, we identified a novel crosstalk pathway between H3 phosphorylation and H4 acetylation on K12. Involvement of these acetyl marks in MSK1-mediated transcription was further confirmed by chromatin immunoprecipitation assays, thus validating the biological relevance of the BICON results. These studies serve as proof-of-principle for this new technical approach, and demonstrate that BICON can be further adapted to study PTMs and crosstalks associated with other histone-modifying enzymes.
|Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis. |
Qian, MX; Pang, Y; Liu, CH; Haratake, K; Du, BY; Ji, DY; Wang, GF; Zhu, QQ; Song, W; Yu, Y; Zhang, XX; Huang, HT; Miao, S; Chen, LB; Zhang, ZH; Liang, YN; Liu, S; Cha, H; Yang, D; Zhai, Y; Komatsu, T; Tsuruta, F; Li, H; Cao, C; Li, W; Li, GH; Cheng, Y; Chiba, T; Wang, L; Goldberg, AL; Shen, Y; Qiu, XB
Cell 153 1012-24 2013
Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here, we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes ("spermatoproteasomes") contain a spermatid/sperm-specific α subunit α4 s/PSMA8 and/or the catalytic β subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis.
|Msl2 is a novel component of the vertebrate DNA damage response. |
Lai, Z; Moravcová, S; Canitrot, Y; Andrzejewski, LP; Walshe, DM; Rea, S
PloS one 8 e68549 2013
hMSL2 (male-specific lethal 2, human) is a RING finger protein with ubiquitin ligase activity. Although it has been shown to target histone H2B at lysine 34 and p53 at lysine 351, suggesting roles in transcription regulation and apoptosis, its function in these and other processes remains poorly defined. To further characterize this protein, we have disrupted the Msl2 gene in chicken DT40 cells. Msl2(-/-) cells are viable, with minor growth defects. Biochemical analysis of the chromatin in these cells revealed aberrations in the levels of several histone modifications involved in DNA damage response pathways. DNA repair assays show that both Msl2(-/-) chicken cells and hMSL2-depleted human cells have defects in non-homologous end joining (NHEJ) repair. DNA damage assays also demonstrate that both Msl2 and hMSL2 proteins are modified and stabilized shortly after induction of DNA damage. Moreover, hMSL2 mediates modification, presumably ubiquitylation, of a key DNA repair mediator 53BP1 at lysine 1690. Similarly, hMSL1 and hMOF (males absent on the first) are modified in the presence of hMSL2 shortly after DNA damage. These data identify a novel role for Msl2/hMSL2 in the cellular response to DNA damage. The kinetics of its stabilization suggests a function early in the NHEJ repair pathway. Moreover, Msl2 plays a role in maintaining normal histone modification profiles, which may also contribute to the DNA damage response.
|Time- and residue-specific differences in histone acetylation induced by VPA and SAHA in AML1/ETO-positive leukemia cells. |
Barbetti, V; Gozzini, A; Cheloni, G; Marzi, I; Fabiani, E; Santini, V; Dello Sbarba, P; Rovida, E
Epigenetics 8 210-9 2013
We analyzed the activity of the histone deacetylase inhibitor (HDACi) suberoyl-anilide hydroxamic acid (SAHA) on Kasumi-1 acute myeloid leukemia (AML) cells expressing AML1/ETO. We also compared the effects of SAHA to those of valproic acid (VPA), a short-chain fatty acid HDACi. SAHA and VPA induced histone H3 and H4 acetylation, myeloid differentiation and massive early apoptosis. The latter effects were not determined by either drug in AML cell lines, such as NB4 or THP-1, not expressing AML1/ETO. SAHA was more rapid and effective than VPA in increasing H3 and H4 acetylation in total Kasumi-1 cell lysates and more effective than VPA in inducing acetylation of H4K8, H4K12, H4K16 residues. At the promoter of IL3, a transcriptionally-silenced target of AML1/ETO, SAHA was also more rapid than VPA in inducing total H4, H4K5, H4K8 and H3K27 acetylation, while VPA was more effective than SAHA at later times in inducing acetylation of total H4, H4K12, H4K16, as well as total H3. Consistent with these differences, SAHA induced the expression of IL3 mRNA more rapidly than VPA, while the effect of VPA was delayed. These differences might be exploited to design clinical trials specifically directed to AML subtypes characterized by constitutive HDAC activation. Our results led to include SAHA, an FDA-approved drug, among the HDACi active in the AML1/ETO-expressing AML cells.
|Conservation and de novo acquisition of dosage compensation on newly evolved sex chromosomes in Drosophila. |
Alekseyenko, AA; Ellison, CE; Gorchakov, AA; Zhou, Q; Kaiser, VB; Toda, N; Walton, Z; Peng, S; Park, PJ; Bachtrog, D; Kuroda, MI
Genes & development 27 853-8 2013
Dosage compensation has arisen in response to the evolution of distinct male (XY) and female (XX) karyotypes. In Drosophila melanogaster, the MSL complex increases male X transcription approximately twofold. X-specific targeting is thought to occur through sequence-dependent binding to chromatin entry sites (CESs), followed by spreading in cis to active genes. We tested this model by asking how newly evolving sex chromosome arms in Drosophila miranda acquired dosage compensation. We found evidence for the creation of new CESs, with the analogous sequence and spacing as in D. melanogaster, providing strong support for the spreading model in the establishment of dosage compensation.
|Expansion of oligodendrocyte progenitor cells following SIRT1 inactivation in the adult brain. |
Rafalski, VA; Ho, PP; Brett, JO; Ucar, D; Dugas, JC; Pollina, EA; Chow, LM; Ibrahim, A; Baker, SJ; Barres, BA; Steinman, L; Brunet, A
Nature cell biology 15 614-24 2013
Oligodendrocytes-the myelin-forming cells of the central nervous system-can be regenerated during adulthood. In adults, new oligodendrocytes originate from oligodendrocyte progenitor cells (OPCs), but also from neural stem cells (NSCs). Although several factors supporting oligodendrocyte production have been characterized, the mechanisms underlying the generation of adult oligodendrocytes are largely unknown. Here we show that genetic inactivation of SIRT1, a protein deacetylase implicated in energy metabolism, increases the production of new OPCs in the adult mouse brain, in part by acting in NSCs. New OPCs produced following SIRT1 inactivation differentiate normally, generating fully myelinating oligodendrocytes. Remarkably, SIRT1 inactivation ameliorates remyelination and delays paralysis in mouse models of demyelinating injuries. SIRT1 inactivation leads to the upregulation of genes involved in cell metabolism and growth factor signalling, in particular PDGF receptor α (PDGFRα). Oligodendrocyte expansion following SIRT1 inactivation is mediated at least in part by AKT and p38 MAPK-signalling molecules downstream of PDGFRα. The identification of drug-targetable enzymes that regulate oligodendrocyte regeneration in adults could facilitate the development of therapies for demyelinating injuries and diseases, such as multiple sclerosis.
|H4K16 acetylation marks active genes and enhancers of embryonic stem cells, but does not alter chromatin compaction. |
Taylor, GC; Eskeland, R; Hekimoglu-Balkan, B; Pradeepa, MM; Bickmore, WA
Genome research 23 2053-65 2013
Compared with histone H3, acetylation of H4 tails has not been well studied, especially in mammalian cells. Yet, H4K16 acetylation is of particular interest because of its ability to decompact nucleosomes in vitro and its involvement in dosage compensation in flies. Here we show that, surprisingly, loss of H4K16 acetylation does not alter higher-order chromatin compaction in vivo in mouse embryonic stem cells (ESCs). As well as peaks of acetylated H4K16 and KAT8 histone acetyltransferase at the transcription start sites of expressed genes, we report that acetylation of H4K16 is a new marker of active enhancers in ESCs and that some enhancers are marked by H3K4me1, KAT8, and H4K16ac, but not by acetylated H3K27 or EP300, suggesting that they are novel EP300 independent regulatory elements. Our data suggest a broad role for different histone acetylation marks and for different histone acetyltransferases in long-range gene regulation.
|Sir3 and epigenetic inheritance of silent chromatin in Saccharomyces cerevisiae. |
Motwani, T; Poddar, M; Holmes, SG
Molecular and cellular biology 32 2784-93 2012
Epigenetic mechanisms maintain the specific characteristics of differentiated cells by ensuring the inheritance of gene expression patterns through DNA replication and mitosis. We examined the mechanism of epigenetic inheritance of Sir protein-dependent transcriptional silencing in Saccharomyces cerevisiae by examining gene expression and molecular markers of silencing at the silent mating type loci under conditions of limiting Sir3 protein. We observed that silencing at HMR, as previously reported for HML, is epigenetically inherited. This inheritance is accompanied by an increased ability of previously silenced cells to retain or recruit limiting Sir3 protein to cis-acting silencer sequences. We also observed that the low H4-K16 histone acetylation and H3-K79 methylation associated with a silenced HMR locus persist in recently derepressed cells for several generations at levels of Sir3 insufficient to maintain these marks in long-term-derepressed cells. The unique ability of previously silenced cells to retain Sir3 protein, maintain silencing-specific histone modifications, and repress HMR transcription at levels of Sir3 insufficient to mediate these effects in long-term-derepressed cells suggests that a cis-acting, chromatin-based mechanism drives epigenetic inheritance at this locus.
|Epigenetic control of cell cycle-dependent histone gene expression is a principal component of the abbreviated pluripotent cell cycle. |
Medina, R; Ghule, PN; Cruzat, F; Barutcu, AR; Montecino, M; Stein, JL; van Wijnen, AJ; Stein, GS
Molecular and cellular biology 32 3860-71 2012
Self-renewal of human pluripotent embryonic stem cells proceeds via an abbreviated cell cycle with a shortened G(1) phase. We examined which genes are modulated in this abbreviated period and the epigenetic mechanisms that control their expression. Accelerated upregulation of genes encoding histone proteins that support DNA replication is the most prominent gene regulatory program at the G(1)/S-phase transition in pluripotent cells. Expedited expression of histone genes is mediated by a unique chromatin architecture reflected by major nuclease hypersensitive sites, atypical distribution of epigenetic histone marks, and a region devoid of histone octamers. We observed remarkable differences in chromatin structure--hypersensitivity and histone protein modifications--between human embryonic stem (hES) and normal diploid cells. Cell cycle-dependent transcription factor binding permits dynamic three-dimensional interactions between transcript initiating and processing factors at 5' and 3' regions of the gene. Thus, progression through the abbreviated G(1) phase involves cell cycle stage-specific chromatin-remodeling events and rapid assembly of subnuclear microenvironments that activate histone gene transcription to promote nucleosomal packaging of newly replicated DNA during stem cell renewal.
|Caenorhabditis elegans dosage compensation regulates histone H4 chromatin state on X chromosomes. |
Wells, MB; Snyder, MJ; Custer, LM; Csankovszki, G
Molecular and cellular biology 32 1710-9 2012
Dosage compensation equalizes X-linked gene expression between the sexes. This process is achieved in Caenorhabditis elegans by hermaphrodite-specific, dosage compensation complex (DCC)-mediated, 2-fold X chromosome downregulation. How the DCC downregulates gene expression is not known. By analyzing the distribution of histone modifications in nuclei using quantitative fluorescence microscopy, we found that H4K16 acetylation (H4K16ac) is underrepresented and H4K20 monomethylation (H4K20me1) is enriched on hermaphrodite X chromosomes in a DCC-dependent manner. Depletion of H4K16ac also requires the conserved histone deacetylase SIR-2.1, while enrichment of H4K20me1 requires the activities of the histone methyltransferases SET-1 and SET-4. Our data suggest that the mechanism of dosage compensation in C. elegans involves redistribution of chromatin-modifying activities, leading to a depletion of H4K16ac and an enrichment of H4K20me1 on the X chromosomes. These results support conserved roles for histone H4 chromatin modification in worm dosage compensation analogous to those seen in flies, using similar elements and opposing strategies to achieve differential 2-fold changes in X-linked gene expression.
|BCL6 controls neurogenesis through Sirt1-dependent epigenetic repression of selective Notch targets. |
Tiberi, Luca, et al.
Nat. Neurosci., (2012) 2012
During neurogenesis, neural stem/progenitor cells (NPCs) undergo an irreversible fate transition to become neurons. The Notch pathway is important for this process, and repression of Notch-dependent Hes genes is essential for triggering differentiation. However, Notch signaling often remains active throughout neuronal differentiation, implying a change in the transcriptional responsiveness to Notch during the neurogenic transition. We identified Bcl6, an oncogene, as encoding a proneurogenic factor that is required for proper neurogenesis of the mouse cerebral cortex. BCL6 promoted the neurogenic conversion by switching the composition of Notch-dependent transcriptional complexes at the Hes5 promoter. BCL6 triggered exclusion of the co-activator Mastermind-like 1 and recruitment of the NAD(+)-dependent deacetylase Sirt1, which was required for BCL6-dependent neurogenesis. The resulting epigenetic silencing of Hes5 led to neuronal differentiation despite active Notch signaling. Our findings suggest a role for BCL6 in neurogenesis and uncover Notch-BCL6-Sirt1 interactions that may affect other aspects of physiology and disease.
|Chromatin Immunoprecipitation (ChIP)||Mouse||23160044|
|FoxA1 and glucocorticoid receptor crosstalk via histone H4K16 acetylation at a hormone regulated enhancer. |
Belikov S, Holmqvist PH, Astrand C, Wrange O
Experimental cell research 318 61-74. Epub 2011 Oct 6. 2012
The forkhead transcription factor FoxA1 participates in many gene regulatory events with steroid hormone receptors, one example being the integrated mouse mammary tumor virus (MMTV) promoter. Its enhancer harbors several FoxA1 binding sites. FoxA1 promotes glucocorticoid receptor (GR)-DNA binding and transcription. Here we analyze the regulatory capacity of GR, FoxA1 and hormone in quantitative terms when reconstituted with the MMTV enhancer in Xenopus oocytes. By titrating each component we demonstrate that FoxA1 is required for hormone induction at low GR concentration and that FoxA1 is a potent enhancer of GR-induced transcription. Conversely, specific DNA binding of FoxA1 at low intranuclear concentration is highly responsive to minute levels of hormone-activated GR while increased FoxA1 concentration results in constitutive binding. When bound to DNA, FoxA1 induces DNase I hypersensitivity, this is accompanied by increased acetylation, specifically at histone H4K16. Expression of FoxA1 deletion mutants demonstrated its DNA binding domain to be sufficient for DNA binding in vivo. The C-terminal and N-terminal domains both contribute to chromatin remodeling while the latter is more important for GR mediated transcription. Thus FoxA1 is primarily responsible for the chromatin presetting while GR supports chromatin presetting at low hormone concentration and transcriptional induction at high hormone concentration.Copyright © 2011 Elsevier Inc. All rights reserved.
|Differential response of cancer cells to HDAC inhibitors trichostatin A and depsipeptide. |
Chang, J; Varghese, DS; Gillam, MC; Peyton, M; Modi, B; Schiltz, RL; Girard, L; Martinez, ED
British journal of cancer 106 116-25 2012
Over the last decade, several drugs that inhibit class I and/or class II histone deacetylases (HDACs) have been identified, including trichostatin A, the cyclic depsipeptide FR901228 and the antibiotic apicidin. These compounds have had immediate application in cancer research because of their ability to reactivate aberrantly silenced tumour suppressor genes and/or block tumour cell growth. Although a number of HDAC inhibitors are being evaluated in preclinical cancer models and in clinical trials, little is known about the differences in their specific mechanism of action and about the unique determinants of cancer cell sensitivity to each of these inhibitors.Using a combination of cell viability assays, HDAC enzyme activity measurements, western blots for histone modifications, microarray gene expression analysis and qRT-PCR, we have characterised differences in trichostatin A vs depsipeptide-induced phenotypes in lung cancer, breast cancer and skin cancer cells and in normal cells and have then expanded these studies to other HDAC inhibitors.Cell viability profiles across panels of lung cancer, breast cancer and melanoma cell lines showed distinct sensitivities to the pan-inhibitor TSA compared with the class 1 selective inhibitor depsipeptide. In several instances, the cell lines most sensitive to one inhibitor were most resistant to the other inhibitor, demonstrating these drugs act on at least some non-overlapping cellular targets. These differences were not explained by the HDAC selectivity of these inhibitors alone since apicidin, which is a class 1 selective compound similar to depsipeptide, also showed a unique drug sensitivity profile of its own. TSA had greater specificity for cancer vs normal cells compared with other HDAC inhibitors. In addition, at concentrations that blocked cancer cell viability, TSA effectively inhibited purified recombinant HDACs 1, 2 and 5 and moderately inhibited HDAC8, while depsipeptide did not inhibit the activity of purified HDACs in vitro but did in cellular extracts, suggesting a potentially indirect action of this drug. Although both depsipeptide and TSA increased levels of histone acetylation in cancer cells, only depsipeptide decreased global levels of transcriptionally repressive histone methylation marks. Analysis of gene expression profiles of an isogenic cell line pair that showed discrepant sensitivity to depsipeptide, suggested that resistance to this inhibitor may be mediated by increased expression of multidrug resistance genes triggered by exposure to chemotherapy as was confirmed by verapamil studies.Although generally thought to have similar activities, the HDAC modulators trichostatin A and depsipeptide demonstrated distinct phenotypes in the inhibition of cancer cell viability and of HDAC activity, in their selectivity for cancer vs normal cells, and in their effects on histone modifications. These differences in mode of action may bear on the future therapeutic and research application of these inhibitors.
|Lactate, a product of glycolytic metabolism, inhibits histone deacetylase activity and promotes changes in gene expression. |
Latham, T; Mackay, L; Sproul, D; Karim, M; Culley, J; Harrison, DJ; Hayward, L; Langridge-Smith, P; Gilbert, N; Ramsahoye, BH
Nucleic acids research 40 4794-803 2012
Chemical inhibitors of histone deacetylase (HDAC) activity are used as experimental tools to induce histone hyperacetylation and deregulate gene transcription, but it is not known whether the inhibition of HDACs plays any part in the normal physiological regulation of transcription. Using both in vitro and in vivo assays, we show that lactate, which accumulates when glycolysis exceeds the cell's aerobic metabolic capacity, is an endogenous HDAC inhibitor, deregulating transcription in an HDAC-dependent manner. Lactate is a relatively weak inhibitor (IC(50) 40 mM) compared to the established inhibitors trichostatin A and butyrate, but the genes deregulated overlap significantly with those affected by low concentrations of the more potent inhibitors. HDAC inhibition causes significant up and downregulation of genes, but genes that are associated with HDAC proteins are more likely to be upregulated and less likely to be downregulated than would be expected. Our results suggest that the primary effect of HDAC inhibition by endogenous short-chain fatty acids like lactate is to promote gene expression at genes associated with HDAC proteins. Therefore, we propose that lactate may be an important transcriptional regulator, linking the metabolic state of the cell to gene transcription.
|Genome-wide H4 K16 acetylation by SAS-I is deposited independently of transcription and histone exchange. |
Heise, F; Chung, HR; Weber, JM; Xu, Z; Klein-Hitpass, L; Steinmetz, LM; Vingron, M; Ehrenhofer-Murray, AE
Nucleic acids research 40 65-74 2012
The MYST HAT Sas2 is part of the SAS-I complex that acetylates histone H4 lysine 16 (H4 K16Ac) and blocks the propagation of heterochromatin at the telomeres of Saccharomyces cerevisiae. In this study, we investigated Sas2-mediated H4 K16Ac on a genome-wide scale. Interestingly, H4 K16Ac loss in sas2Δ cells outside of the telomeric regions showed a distinctive pattern in that there was a pronounced decrease of H4 K16Ac within the majority of open reading frames (ORFs), but little change in intergenic regions. Furthermore, regions of low histone H3 exchange and low H3 K56 acetylation showed the most pronounced loss of H4 K16Ac in sas2Δ, indicating that Sas2 deposited this modification on chromatin independently of histone exchange. In agreement with the effect of Sas2 within ORFs, sas2Δ caused resistance to 6-azauracil, indicating a positive effect on transcription elongation in the absence of H4 K16Ac. In summary, our data suggest that Sas2-dependent H4 K16Ac is deposited into chromatin independently of transcription and histone exchange, and that it has an inhibitory effect on the ability of PolII to travel through the body of the gene.
|Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context. |
Alekseyenko, AA; Ho, JW; Peng, S; Gelbart, M; Tolstorukov, MY; Plachetka, A; Kharchenko, PV; Jung, YL; Gorchakov, AA; Larschan, E; Gu, T; Minoda, A; Riddle, NC; Schwartz, YB; Elgin, SC; Karpen, GH; Pirrotta, V; Kuroda, MI; Park, PJ
PLoS genetics 8 e1002646 2012
The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.
|Deacetylation of H4-K16Ac and heterochromatin assembly in senescence. |
Contrepois, K; Thuret, JY; Courbeyrette, R; Fenaille, F; Mann, C
Epigenetics & chromatin 5 15 2012
Cellular senescence is a stress response of mammalian cells leading to a durable arrest of cell proliferation that has been implicated in tumor suppression, wound healing, and aging. The proliferative arrest is mediated by transcriptional repression of genes essential for cell division by the retinoblastoma protein family. This repression is accompanied by varying degrees of heterochromatin assembly, but little is known regarding the molecular mechanisms involved.We found that both deacetylation of H4-K16Ac and expression of HMGA1/2 can contribute to DNA compaction during senescence. SIRT2, an NAD-dependent class III histone deacetylase, contributes to H4-K16Ac deacetylation and DNA compaction in human fibroblast cell lines that assemble striking senescence-associated heterochromatin foci (SAHFs). Decreased H4-K16Ac was observed in both replicative and oncogene-induced senescence of these cells. In contrast, this mechanism was inoperative in a fibroblast cell line that did not assemble extensive heterochromatin during senescence. Treatment of senescent cells with trichostatin A, a class I/II histone deacetylase inhibitor, also induced rapid and reversible decondensation of SAHFs. Inhibition of DNA compaction did not significantly affect the stability of the senescent state.Variable DNA compaction observed during senescence is explained in part by cell-type specific regulation of H4 deacetylation and HMGA1/2 expression. Deacetylation of H4-K16Ac during senescence may explain reported decreases in this mark during mammalian aging and in cancer cells.
|Recruitment of Rpd3 to the telomere depends on the protein arginine methyltransferase Hmt1. |
Milliman, EJ; Yadav, N; Chen, YC; Muddukrishna, B; Karunanithi, S; Yu, MC
PloS one 7 e44656 2012
In the yeast Saccharomyces cerevisiae, the establishment and maintenance of silent chromatin at the telomere requires a delicate balance between opposing activities of histone modifying enzymes. Previously, we demonstrated that the protein arginine methyltransferase Hmt1 plays a role in the formation of yeast silent chromatin. To better understand the nature of the Hmt1 interactions that contribute to this phenomenon, we carried out a systematic reverse genetic screen using a null allele of HMT1 and the synthetic genetic array (SGA) methodology. This screen revealed interactions between HMT1 and genes encoding components of the histone deacetylase complex Rpd3L (large). A double mutant carrying both RPD3 and HMT1 deletions display increased telomeric silencing and Sir2 occupancy at the telomeric boundary regions, when comparing to a single mutant carrying Hmt1-deletion only. However, the dual rpd3/hmt1-null mutant behaves like the rpd3-null single mutant with respect to silencing behavior, indicating that RPD3 is epistatic to HMT1. Mutants lacking either Hmt1 or its catalytic activity display an increase in the recruitment of histone deacetylase Rpd3 to the telomeric boundary regions. Moreover, in such loss-of-function mutants the levels of acetylated H4K5, which is a substrate of Rpd3, are altered at the telomeric boundary regions. In contrast, the level of acetylated H4K16, a target of the histone deacetylase Sir2, was increased in these regions. Interestingly, mutants lacking either Rpd3 or Sir2 display various levels of reduction in dimethylated H4R3 at these telomeric boundary regions. Together, these data provide insight into the mechanism whereby Hmt1 promotes the proper establishment and maintenance of silent chromatin at the telomeres.
|Regulating repression: roles for the sir4 N-terminus in linker DNA protection and stabilization of epigenetic states. |
Kueng, S; Tsai-Pflugfelder, M; Oppikofer, M; Ferreira, HC; Roberts, E; Tsai, C; Roloff, TC; Sack, R; Gasser, SM
PLoS genetics 8 e1002727 2012
Silent information regulator proteins Sir2, Sir3, and Sir4 form a heterotrimeric complex that represses transcription at subtelomeric regions and homothallic mating type (HM) loci in budding yeast. We have performed a detailed biochemical and genetic analysis of the largest Sir protein, Sir4. The N-terminal half of Sir4 is dispensable for SIR-mediated repression of HM loci in vivo, except in strains that lack Yku70 or have weak silencer elements. For HM silencing in these cells, the C-terminal domain (Sir4C, residues 747-1,358) must be complemented with an N-terminal domain (Sir4N; residues 1-270), expressed either independently or as a fusion with Sir4C. Nonetheless, recombinant Sir4C can form a complex with Sir2 and Sir3 in vitro, is catalytically active, and has sedimentation properties similar to a full-length Sir4-containing SIR complex. Sir4C-containing SIR complexes bind nucleosomal arrays and protect linker DNA from nucleolytic digestion, but less effectively than wild-type SIR complexes. Consistently, full-length Sir4 is required for the complete repression of subtelomeric genes. Supporting the notion that the Sir4 N-terminus is a regulatory domain, we find it extensively phosphorylated on cyclin-dependent kinase consensus sites, some being hyperphosphorylated during mitosis. Mutation of two major phosphoacceptor sites (S63 and S84) derepresses natural subtelomeric genes when combined with a serendipitous mutation (P2A), which alone can enhance the stability of either the repressed or active state. The triple mutation confers resistance to rapamycin-induced stress and a loss of subtelomeric repression. We conclude that the Sir4 N-terminus plays two roles in SIR-mediated silencing: it contributes to epigenetic repression by stabilizing the SIR-mediated protection of linker DNA; and, as a target of phosphorylation, it can destabilize silencing in a regulated manner.
|Oncogene Ras/phosphatidylinositol 3-kinase signaling targets histone H3 acetylation at lysine 56. |
Liu, Y; Wang, DL; Chen, S; Zhao, L; Sun, FL
The Journal of biological chemistry 287 41469-80 2012
It is well established that the small GTPase Ras promotes tumor initiation by activating at least three different mediators: Raf, PI3K, and Ras-like (Ral) guanine nucleotide exchange factors. However, the exact mechanisms that underlie these different Ras signaling pathways, which are involved in tumor progression, remain to be elucidated. In this study, we report that the Ras-PI3K pathway, but not Raf or the Ral guanine nucleotide exchange factors, specifically targets the acetylation of H3 at lysine 56 (H3K56ac), thereby regulating tumor cell activity. We demonstrate that the Ras-PI3K-induced reduction in H3K56ac is associated with the proliferation and migration of tumor cells by targeting the transcription of tumor-associated genes. The depletion of the histone deacetyltransferases Sirt1 and Sirt2 rescues the Ras-PI3K-induced decrease in H3K56ac, gene transcription, tumor cell proliferation, and tumor cell migration. Furthermore, we demonstrate that the Ras-PI3K-AKT pathway regulates H3K56ac via the MDM2-dependent degradation of CREB-binding protein/p300. Taken together, the results of this study demonstrate that the Ras-PI3K signaling pathway targets specific epigenetic modifications in tumor cells.
|Epigenetic reprogramming as a key contributor to melanocyte malignant transformation. |
Molognoni, F; Cruz, AT; Meliso, FM; Morais, AS; Souza, CF; Xander, P; Bischof, JM; Costa, FF; Soares, MB; Liang, G; Jones, PA; Jasiulionis, MG
Epigenetics 6 450-64 2011
Melanoma progression requires deregulation of gene expression by currently uncharacterized epigenetic mechanisms. A mouse model based on changes in cell microenvironment was developed by our group to study melanocyte malignant transformation. Melanoma cell lines (4C11- and 4C11+) were obtained as result of 5 sequential anchorage blockades of non-tumorigenic melan-a melanocytes. Melan-a cells submitted to 4 de-adhesion cycles were also established (4C), are non-tumorigenic and represent an intermediary phase of tumor progression. The aim of this work was to identify factors contributing to epigenetic modifications in early and later phases of malignant transformation induced by anchorage impediment. Epigenetic alterations occur early in tumorigenesis; 4C cell line shows changes in global and gene-specific DNA methylation and histone marks. Many histone modifications differ between melan-a, 4C, 4C11- (non-metastatic melanoma cell line) and 4C11+ (metastatic melanoma cell line) which could be associated with changes in gene and microRNA expression. These epigenetic alterations seem to play a key role in malignant transformation since melanocytes treated with 5-Aza-2'-deoxycytidine before each anchorage blockade do not transform. Some epigenetic changes seem to be also responsible for the maintenance of malignant phenotype, since melanoma cell lines (4C11- and 4C11+) treated in vitro with 5-Aza-2'-deoxycytidine or Trichostatin A showed reduction of tumor growth in vivo. Changes in gene expression reflecting cell adaptation to new environment were also observed. We propose a model in which sustained microenvironmental stress in melanocytes results in epigenetic reprogramming. Thus, after adaptation, cells may acquire epigenetic marks that could contribute to the establishment of a malignant phenotype.
|Adjacent gene pairing plays a role in the coordinated expression of ribosome biogenesis genes MPP10 and YJR003C in Saccharomyces cerevisiae. |
Arnone, JT; McAlear, MA
Eukaryotic cell 10 43-53 2011
The rRNA and ribosome biogenesis (RRB) regulon from Saccharomyces cerevisiae contains some 200 genes, the expression of which is tightly regulated under changing cellular conditions. RRB gene promoters are enriched for the RRPE and PAC consensus motifs, and a significant fraction of RRB genes are found as adjacent gene pairs. A genetic analysis of the MPP10 promoter revealed that both the RRPE and PAC motifs are important for coordinated expression of MPP10 following heat shock, osmotic stress, and glucose replenishment. The association of the RRPE binding factor Stb3 with the MPP10 promoter was found to increase after glucose replenishment and to decrease following heat shock. Similarly, bulk histone H3 clearing and histone H4K12 acetylation levels at the MPP10 promoter were found to increase or decrease following glucose replenishment or heat shock, respectively. Interestingly, substitutions in the PAC and RRPE sequences at the MPP10 promoter were also found to impact the regulated expression of the adjacent RRB gene YJR003, whose promoter lies in the opposite orientation and some 3.8 kb away. Furthermore, the regulated expression of YJR003C could be disrupted by inserting a reporter cassette that increased its distance from MPP10. Given that a high incidence of gene pairing was also found within the ribosomal protein (RP) and RRB regulons across different yeast species, our results indicate that immediately adjacent positioning of genes can be functionally significant for their coregulated expression.Artículo Texto completo
|HBO1 is required for H3K14 acetylation and normal transcriptional activity during embryonic development. |
Kueh, AJ; Dixon, MP; Voss, AK; Thomas, T
Molecular and cellular biology 31 845-60 2011
We report here that the MYST histone acetyltransferase HBO1 (histone acetyltransferase bound to ORC; MYST2/KAT7) is essential for postgastrulation mammalian development. Lack of HBO1 led to a more than 90% reduction of histone 3 lysine 14 (H3K14) acetylation, whereas no reduction of acetylation was detected at other histone residues. The decrease in H3K14 acetylation was accompanied by a decrease in expression of the majority of genes studied. However, some genes, in particular genes regulating embryonic patterning, were more severely affected than "housekeeping" genes. Development of HBO1-deficient embryos was arrested at the 10-somite stage. Blood vessels, mesenchyme, and somites were disorganized. In contrast to previous studies that reported cell cycle arrest in HBO1-depleted cultured cells, no defects in DNA replication or cell proliferation were seen in Hbo1 mutant embryo primary fibroblasts or immortalized fibroblasts. Rather, a high rate of cell death and DNA fragmentation was observed in Hbo1 mutant embryos, resulting initially in the degeneration of mesenchymal tissues and ultimately in embryonic lethality. In conclusion, the primary role of HBO1 in development is that of a transcriptional activator, which is indispensable for H3K14 acetylation and for the normal expression of essential genes regulating embryonic development.Artículo Texto completo
|Aryl hydrocarbon receptor ligands of widely different toxic equivalency factors induce similar histone marks in target gene chromatin. |
Ovesen, JL; Schnekenburger, M; Puga, A
Toxicological sciences : an official journal of the Society of Toxicology 121 123-31 2011
Posttranslational histone modifications are a critical regulatory mechanism of gene transcription. Previous studies from our laboratory have shown that contingent on binding to its cognate promoter motifs in the Cyp1a1 gene, activation of the aryl hydrocarbon receptor (AHR) by benzo[a]pyrene (BaP) treatment induces histone modifications in the Cyp1a1 promoter that are required for activation of gene transcription. Here, we have studied different AHR ligands, including polychlorinated biphenyls (PCBs) of different toxic equivalency factors (TEF), to determine whether changes in histone modifications are linked to different levels of Cyp1a1 expression or dependent on AHR-ligand affinity. We find that all ligands lead to the same pattern of histone modifications in a relationship that parallels the strength of their AHR-ligand affinity. Thus, whereas PCB126 (TEF 0.1), 3-methylcholanthrene, β-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) initiate a pattern of histone marks similar to those induced by BaP, PCB77 (TEF 0.0001) causes a lower level of change in the same marks and requires a longer activation time than PCB126, BaP, or TCDD. In contrast, the non-dioxin-like PCB153 recruits AHR to the Cyp1a1 enhancer causing a displacement of enhancer-associated histone H3 but does not cause the other observed histone mark changes nor does it induce transcription. These results indicate that AHR recruitment to the promoter is not sufficient to induce the histone modifications needed to activate gene expression and show that there is a good correlation between the regulatory chromatin changes associated with ligand-induced AHR target gene transcription and the resultant toxicity of the ligand.
|Broad chromosomal domains of histone modification patterns in C. elegans. |
Liu, T; Rechtsteiner, A; Egelhofer, TA; Vielle, A; Latorre, I; Cheung, MS; Ercan, S; Ikegami, K; Jensen, M; Kolasinska-Zwierz, P; Rosenbaum, H; Shin, H; Taing, S; Takasaki, T; Iniguez, AL; Desai, A; Dernburg, AF; Kimura, H; Lieb, JD; Ahringer, J; Strome, S; Liu, XS
Genome research 21 227-36 2011
Chromatin immunoprecipitation identifies specific interactions between genomic DNA and proteins, advancing our understanding of gene-level and chromosome-level regulation. Based on chromatin immunoprecipitation experiments using validated antibodies, we define the genome-wide distributions of 19 histone modifications, one histone variant, and eight chromatin-associated proteins in Caenorhabditis elegans embryos and L3 larvae. Cluster analysis identified five groups of chromatin marks with shared features: Two groups correlate with gene repression, two with gene activation, and one with the X chromosome. The X chromosome displays numerous unique properties, including enrichment of monomethylated H4K20 and H3K27, which correlate with the different repressive mechanisms that operate in somatic tissues and germ cells, respectively. The data also revealed striking differences in chromatin composition between the autosomes and between chromosome arms and centers. Chromosomes I and III are globally enriched for marks of active genes, consistent with containing more highly expressed genes, compared to chromosomes II, IV, and especially V. Consistent with the absence of cytological heterochromatin and the holocentric nature of C. elegans chromosomes, markers of heterochromatin such as H3K9 methylation are not concentrated at a single region on each chromosome. Instead, H3K9 methylation is enriched on chromosome arms, coincident with zones of elevated meiotic recombination. Active genes in chromosome arms and centers have very similar histone mark distributions, suggesting that active domains in the arms are interspersed with heterochromatin-like structure. These data, which confirm and extend previous studies, allow for in-depth analysis of the organization and deployment of the C. elegans genome during development.Artículo Texto completo
|Integrative analysis of gene amplification in Drosophila follicle cells: parameters of origin activation and repression. |
Kim, JC; Nordman, J; Xie, F; Kashevsky, H; Eng, T; Li, S; MacAlpine, DM; Orr-Weaver, TL
Genes & development 25 1384-98 2011
In metazoans, how replication origins are specified and subsequently activated is not well understood. Drosophila amplicons in follicle cells (DAFCs) are genomic regions that undergo rereplication to increase DNA copy number. We identified all DAFCs by comparative genomic hybridization, uncovering two new amplicons in addition to four known previously. The complete identification of all DAFCs enabled us to investigate these in vivo replicons with respect to parameters of transcription, localization of the origin recognition complex (ORC), and histone acetylation, yielding important insights into gene amplification as a metazoan replication model. Significantly, ORC is bound across domains spanning 10 or more kilobases at the DAFC rather than at a specific site. Additionally, ORC is bound at many regions that do not undergo amplification, and, in contrast to cell culture, these regions do not correlate with high gene expression. As a developmental strategy, gene amplification is not the predominant means of achieving high expression levels, even in cells capable of amplification. Intriguingly, we found that, in some strains, a new amplicon, DAFC-22B, does not amplify, a consequence of distant repression of ORC binding and origin activation. This repression is alleviated when a fragment containing the origin is placed in different genomic contexts.
|FOXP3 orchestrates H4K16 acetylation and H3K4 trimethylation for activation of multiple genes by recruiting MOF and causing displacement of PLU-1. |
Katoh, H; Qin, ZS; Liu, R; Wang, L; Li, W; Li, X; Wu, L; Du, Z; Lyons, R; Liu, CG; Liu, X; Dou, Y; Zheng, P; Liu, Y
Molecular cell 44 770-84 2011
Both H4K16 acetylation and H3K4 trimethylation are required for gene activation. However, it is still largely unclear how these modifications are orchestrated by transcriptional factors. Here, we analyzed the mechanism of the transcriptional activation by FOXP3, an X-linked suppressor of autoimmune diseases and cancers. FOXP3 binds near transcriptional start sites of its target genes. By recruiting MOF and displacing histone H3K4 demethylase PLU-1, FOXP3 increases both H4K16 acetylation and H3K4 trimethylation at the FOXP3-associated chromatins of multiple FOXP3-activated genes. RNAi-mediated silencing of MOF reduced both gene activation and tumor suppression by FOXP3, while both somatic mutations in clinical cancer samples and targeted mutation of FOXP3 in mouse prostate epithelial cells disrupted nuclear localization of MOF. Our data demonstrate a pull-push model in which a single transcription factor orchestrates two epigenetic alterations necessary for gene activation and provide a mechanism for somatic inactivation of the FOXP3 protein function in cancer cells.
|Rapid and reversible light-mediated chromatin modifications of Arabidopsis phytochrome A locus. |
Jang, IC; Chung, PJ; Hemmes, H; Jung, C; Chua, NH
The Plant cell 23 459-70 2011
Recent genome-wide surveys showed that acetylation of H3K9 and H3K27 is correlated with gene activation during deetiolation of Arabidopsis thaliana seedlings, but less is known regarding changes in the histone status of repressed genes. Phytochrome A (phyA) is the major photoreceptor of deetiolation, and phyA expression is reversibly repressed by light. We found that in adult Arabidopsis plants, phyA activation in darkness was accompanied by a significant enrichment in the phyA transcription and translation start sites of not only H3K9/14ac and H3K27ac but also H3K4me3, and there was also moderate enrichment of H4K5ac, H4K8ac, H4K12ac, and H4K16ac. Conversely, when phyA expression was repressed by light, H3K27me3 was enriched with a corresponding decline in H3K27ac; moreover, demethylation of H3K4me3 and deacetylation of H3K9/14 were also seen. These histone modifications, which were focused around the phyA transcription/translation start sites, were detected within 1 h of deetiolation. Mutant analysis showed that HDA19/HD1 mediated deacetylation of H3K9/14 and uncovered possible histone crosstalk between H3K9/14ac and H3K4me3. Neither small RNA pathways nor the circadian clock affected H3 modification status of the phyA locus, and DNA methylation was unchanged by light. The presence of activating and repressive histone marks suggests a mechanism for the rapid and reversible regulation of phyA by dark and light.
|DNA methyltransferase 3b preferentially associates with condensed chromatin. |
Kashiwagi, K; Nimura, K; Ura, K; Kaneda, Y
Nucleic acids research 39 874-88 2011
In mammals, DNA methylation is catalyzed by DNA methyltransferases (DNMTs) encoded by Dnmt1, Dnmt3a and Dnmt3b. Since, the mechanisms of regulation of Dnmts are still largely unknown, the physical interaction between Dnmt3b and chromatin was investigated in vivo and in vitro. In embryonic stem cell nuclei, Dnmt3b preferentially associated with histone H1-containing heterochromatin without any significant enrichment of silent-specific histone methylation. Recombinant Dnmt3b preferentially associated with nucleosomal DNA rather than naked DNA. Incorporation of histone H1 into nucleosomal arrays promoted the association of Dnmt3b with chromatin, whereas histone acetylation reduced Dnmt3b binding in vitro. In addition, Dnmt3b associated with histone deacetylase SirT1 in the nuclease resistant chromatin. These findings suggest that Dnmt3b is preferentially recruited into hypoacetylated and condensed chromatin. We propose that Dnmt3b is a 'reader' of higher-order chromatin structure leading to gene silencing through DNA methylation.
|Altered levels of histone deacetylase OsHDT1 affect differential gene expression patterns in hybrid rice. |
Li, C; Huang, L; Xu, C; Zhao, Y; Zhou, DX
PloS one 6 e21789 2011
Hybrids between different inbred varieties display novel patterns of gene expression resulted from parental variation in allelic nucleotide sequences. To study the function of chromatin regulators in hybrid gene expression, the histone deacetylase gene OsHDT1 whose expression displayed a circadian rhythm was over-expressed or inactivated by RNAi in an elite rice parent. Increased OsHDT1 expression did not affect plant growth in the parent but led to early flowering in the hybrid. Nonadditive up-regulation of key flowering time genes was found to be related to flowering time of the hybrid. Over-expression of OsHDT1 repressed the nonadditive expression of the key flowering repressors in the hybrid (i.e. OsGI and Hd1) inducing early flowering. Analysis of histone acetylation suggested that OsHDT1 over-expression might promote deacetylation on OsGI and Hd1 chromatin during the peak expression phase. High throughput differential gene expression analysis revealed that altered OsHDT1 levels affected nonadditive expression of many genes in the hybrid. These data demonstrate that nonadditive gene expression was involved in flowering time control in the hybrid rice and that OsHDT1 level was important for nonadditive or differential expression of many genes including the flowering time genes, suggesting that OsHDT1 may be involved in epigenetic control of parental genome interaction for differential gene expression.
|Modulations of hMOF autoacetylation by SIRT1 regulate hMOF recruitment and activities on the chromatin. |
Lu, L; Li, L; Lv, X; Wu, XS; Liu, DP; Liang, CC
Cell research 21 1182-95 2011
A wide variety of nuclear regulators and enzymes are subjected to acetylation of the lysine residue, which regulates different aspects of protein functions. The MYST family histone acetyltransferase, human ortholog of MOF (hMOF), plays critical roles in transcription activation by acetylating nucleosomal H4K16. In this study, we found that hMOF acetylates itself in vitro and in vivo, and the acetylation is restricted to the conserved MYST domain (C2HC zinc finger and HAT), of which the K274 residue is the major autoacetylation site. Furthermore, the class III histone deacetylase SIRT1 was found to interact with the MYST domain of hMOF through the deacetylase catalytic region and deacetylate autoacetylated hMOF. In vitro binding assays showed that non-acetylated hMOF robustly binds to nucleosomes while acetylation decreases the binding ability. In HeLa cells, the recruitment of hMOF to the chromatin increases in response to SIRT1 overexpression and decreases after knockdown of SIRT1. The acetylation mimic mutation K274Q apparently decreases the chromatin recruitment of hMOF as well as the global H4K16Ac level in HeLa cells. Finally, upon SIRT1 knockdown, hMOF recruitment to the gene body region of its target gene HoxA9 decreases, accompanied with decrease of H4K16Ac at the same region and repression of HoxA9 transcription. These results suggest a dynamic interplay between SIRT1 and hMOF in regulating H4K16 acetylation.
|Ordered transcriptional factor recruitment and epigenetic regulation of tnf-alpha in necrotizing acute pancreatitis. |
Sandoval J, Pereda J, Rodriguez JL, Escobar J, Hidalgo J, Joosten LA, Franco L, Sastre J, López-Rodas G
Cell Mol Life Sci 67 1687-97. Epub 2010 Feb 4. 2010
Tauhe expression of the critical initiator cytokine TNF-alpha was strongly upregulated in vivo in acute necrotic pancreatitis (AP) in rodents and in vitro in TNF-alpha activated acinar AR42J cells. Upregulation of tnf-alpha, inos, icam-1 and il-6 occurred both in TNF-alpha receptor 1 and 2 knock-out mice, but not in TNF-alpha knock-out mice, in cerulein-induced acute pancreatitis. Chromatin immunoprecipitation analysis showed that transcriptional factors (ELK-1, SP1, NF-kappaB and EGR-1) and chromatin modification complexes (HDAC1, HDAC2, GCN5, PCAF and CBP) were recruited and/or released from the promoter in a strictly ordered mechanism. Activation of tnf-alpha gene was also accompanied by an ordered increased level of histone H3K9, H3K14 and H3K18-acetylation and H3K4 methylation, as well as H4K5 acetylation. A better knowledge of the molecular mechanisms that control tnf-alpha gene regulation will provide deeper understanding of the initiation and development of the inflammatory processes occurring in acute pancreatitis triggered by TNF-alpha cytokine.
|MOF and H4 K16 acetylation play important roles in DNA damage repair by modulating recruitment of DNA damage repair protein Mdc1. |
Li, X; Corsa, CA; Pan, PW; Wu, L; Ferguson, D; Yu, X; Min, J; Dou, Y
Molecular and cellular biology 30 5335-47 2010
MOF (MYST1) is the major enzyme to catalyze acetylation of histone H4 lysine 16 (K16) and is highly conserved through evolution. Using a conditional knockout mouse model and the derived mouse embryonic fibroblast cell lines, we showed that loss of Mof led to a global reduction of H4 K16 acetylation, severe G(2)/M cell cycle arrest, massive chromosome aberration, and defects in ionizing radiation-induced DNA damage repair. We further showed that although early DNA damage sensing and signaling by ATM were normal in Mof-null cells, the recruitment of repair mediator protein Mdc1 and its downstream signaling proteins 53bp1 and Brca1 to DNA damage foci was completely abolished. Mechanistic studies suggested that Mof-mediated H4 K16 acetylation and an intact acidic pocket on H2A.X were essential for the recruitment of Mdc1. Removal of Mof and its associated proteins phenocopied a charge-neutralizing mutant of H2A.X. Given the well-characterized H4-H2A trans interactions in regulating higher-order chromatin structure, our study revealed a novel chromatin-based mechanism that regulates the DNA damage repair process.
|Asf1 can promote trimethylation of H3 K36 by Set2. |
Lin, LJ; Minard, LV; Johnston, GC; Singer, RA; Schultz, MC
Molecular and cellular biology 30 1116-29 2010
Asf1 is a conserved histone H3/H4 chaperone that can assemble and disassemble nucleosomes and promote histone acetylation. Set2 is an H3 K36 methyltransferase. The functions of these proteins intersect in the context of transcription elongation by RNA polymerase II: both contribute to the establishment of repressive chromatin structures that inhibit spurious intragenic transcription. Here we characterize further interactions between budding yeast (Saccharomyces cerevisiae) Asf1 and Set2 using assays of intragenic transcription, H3/H4 posttranslational modification, coding region cross-linking of Asf1 and Set2, and cooccurrence of Asf1 and Set2 in protein complexes. We find that at some genes Asf1 and Set2 control chromatin metabolism as components of separate pathways. However, the existence of a low-abundance complex containing both proteins suggests that Asf1 and Set2 can more directly collaborate in chromatin regulation. Consistent with this possibility, we show that Asf1 stimulates Set2 occupancy of the coding region of a highly transcribed gene by a mechanism that depends on Asf1 binding to H3/H4. This function of Asf1 promotes the switch from di- to trimethylation of H3 K36 at that gene. These results support the view that Set2 function in chromatin metabolism can intimately involve histone chaperone Asf1.Artículo Texto completo
|An evolutionary consequence of dosage compensation on Drosophila melanogaster female X-chromatin structure? |
Zhang, Y; Oliver, B
BMC genomics 11 6 2010
X chromosomes are subject to dosage compensation in Drosophila males. Dosage compensation requires cis sequence features of the X chromosome that are present in both sexes by definition and trans acting factors that target chromatin modifying machinery to the X specifically in males. The evolution of this system could result in neutral X chromatin changes that will be apparent in females.We find that the general chromatin structure of female X chromosomes is distinct from autosomes. Additionally, specific histone marks associated with dosage compensation and active chromatin marks on the male X chromosome are also enriched on the X chromosomes of females, albeit to a lesser degree.Our data indicate that X chromatin structure is fundamentally different from autosome structure in both sexes. We suggest that the differences between the X chromosomes and autosomes in females are a consequence of mechanisms that have evolved to ensure sufficient X chromosome expression in the soma of males.Artículo Texto completo
|Differential regulation of HIC1 target genes by CtBP and NuRD, via an acetylation/SUMOylation switch, in quiescent versus proliferating cells. |
Van Rechem, C; Boulay, G; Pinte, S; Stankovic-Valentin, N; Guérardel, C; Leprince, D
Molecular and cellular biology 30 4045-59 2010
The tumor suppressor gene HIC1 encodes a transcriptional repressor involved in regulatory loops modulating P53-dependent and E2F1-dependent cell survival, growth control, and stress responses. Despite its importance, few HIC1 corepressors and target genes have been characterized thus far. Using a yeast two-hybrid approach, we identify MTA1, a subunit of the NuRD complex, as a new HIC1 corepressor. This interaction is regulated by two competitive posttranslational modifications of HIC1 at lysine 314, promotion by SUMOylation, and inhibition by acetylation. Consistent with the role of HIC1 in growth control, we demonstrate that HIC1/MTA1 complexes bind on two new target genes, Cyclin D1 and p57KIP2 in quiescent but not in growing WI38 cells. In addition, HIC1/MTA1 and HIC1/CtBP complexes differentially bind on two mutually exclusive HIC1 binding sites (HiRE) on the SIRT1 promoter. SIRT1 transcriptional activation induced by short-term serum starvation coincides with loss of occupancy of the distal sites by HIC1/MTA1 and HIC1/CtBP. Upon longer starvation, both complexes are found but on a newly identified proximal HiRE that is evolutionarily conserved and specifically enriched with repressive histone marks. Our results decipher a mechanistic link between two competitive posttranslational modifications of HIC1 and corepressor recruitment to specific genes, leading to growth control.Artículo Texto completo
|The MYST family histone acetyltransferase regulates gene expression and cell cycle in malaria parasite Plasmodium falciparum. |
Miao, J; Fan, Q; Cui, L; Li, X; Wang, H; Ning, G; Reese, JC; Cui, L
Molecular microbiology 78 883-902 2010
Histone lysine acetylation, normally associated with euchromatin and active genes, is regulated by different families of histone acetyltransferases (HATs). A single Plasmodium falciparum MYST (PfMYST) HAT was expressed as a long and a short version in intraerythrocytic stages. Whereas the recombinant PfMYST expressed in prokaryotes and insect cells did not show HAT activity, recombinant PfMYST purified from the parasites exhibited a predilection to acetylate histone H4 in vitro at K5, K8, K12 and K16. Tagging PfMYST with the green fluorescent protein at the C-terminus showed that PfMYST protein was localized in both the nucleus and cytoplasm. Consistent with the importance of H4 acetylation in var gene expression, PfMYST was recruited to the active var promoter. Attempts to disrupt PfMYST were not successful, suggesting that PfMYST is essential for asexual intraerythrocytic growth. However, overexpression of the long, active or a truncated, non-active version of PfMYST by stable integration of the expression cassette in the parasite genome resulted in changes of H4 acetylation and cell cycle progression. Furthermore, parasites with PfMYST overexpression showed changes in sensitivity to DNA-damaging agents. Collectively, this study showed that PfMYST plays important roles in cellular processes such as gene activation, cell cycle control and DNA repair.
|Proliferating cell nuclear antigen (PCNA) is required for cell cycle-regulated silent chromatin on replicated and nonreplicated genes. |
Miller, A; Chen, J; Takasuka, TE; Jacobi, JL; Kaufman, PD; Irudayaraj, JM; Kirchmaier, AL
The Journal of biological chemistry 285 35142-54 2010
In Saccharomyces cerevisiae, silent chromatin is formed at HMR upon the passage through S phase, yet neither the initiation of DNA replication at silencers nor the passage of a replication fork through HMR is required for silencing. Paradoxically, mutations in the DNA replication processivity factor, POL30, disrupt silencing despite this lack of requirement for DNA replication in the establishment of silencing. We tested whether pol30 mutants could establish silencing at either replicated or non-replicated HMR loci during S phase and found that pol30 mutants were defective in establishing silencing at HMR regardless of its replication status. Although previous studies tie the silencing defect of pol30 mutants to the chromatin assembly factors Asf1p and CAF-1, we found pol30 mutants did not exhibit a gross defect in packaging HMR into chromatin. Rather, the pol30 mutants exhibited defects in histone modifications linked to ASF1 and CAF-1-dependent pathways, including SAS-I- and Rtt109p-dependent acetylation events at H4-K16 and H3-K9 (plus H3-K56; Miller, A., Yang, B., Foster, T., and Kirchmaier, A. L. (2008) Genetics 179, 793-809). Additional experiments using FLIM-FRET revealed that Pol30p interacted with SAS-I and Rtt109p in the nuclei of living cells. However, these interactions were disrupted in pol30 mutants with defects linked to ASF1- and CAF-1-dependent pathways. Together, these results imply that Pol30p affects epigenetic processes by influencing the composition of chromosomal histone modifications.
|Genome-wide mapping of histone H4 serine-1 phosphorylation during sporulation in Saccharomyces cerevisiae. |
Govin, J; Schug, J; Krishnamoorthy, T; Dorsey, J; Khochbin, S; Berger, SL
Nucleic acids research 38 4599-606 2010
We previously showed that histone H4 serine-1 phosphorylation (H4S1ph) is evolutionarily conserved during gametogenesis, and contributes to post-meiotic nuclear compaction and to full completion of sporulation in the yeast Saccharomyces cerevisiae. Previous studies showed that H4S1ph and another modification of the same histone, H4 acetylation (H4ac), do not occur together and have opposing roles during DNA double-strand break (DSB) repair. In this study, we investigated the relationship between these marks during yeast sporulation. H4S1ph and H4ac co-exist globally during later stages of sporulation, in contrast to DSB repair. Genome-wide mapping during sporulation reveals accumulation of both marks over promoters of genes. Prevention of H4S1ph deposition delays the decline in transcription that normally occurs during spore maturation. Taken together, our results indicate that H4S1ph deposition reinforces reduced transcription that coincides with full spore compaction, without disrupting the local acetylation signature. These studies indicate distinctive features of a histone H4 modification marking system during sporulation compared with DSB repair.Artículo Texto completo
|Recognition imaging of acetylated chromatin using a DNA aptamer. |
Lin L, Fu Q, Williams BA, Azzaz AM, Shogren-Knaak MA, Chaput JC, Lindsay S
Biophys J 97 1804-7. 2009
Histone acetylation plays an important role in the regulation of gene expression. A DNA aptamer generated by in vitro selection to be highly specific for histone H4 protein acetylated at lysine 16 was used as a recognition element for atomic force microscopy-based recognition imaging of synthetic nucleosomal arrays with precisely controlled acetylation. The aptamer proved to be reasonably specific at recognizing acetylated histones, with recognition efficiencies of 60% on-target and 12% off-target. Though this selectivity is much poorer than the >2000:1 equilibrium specificity of the aptamer, it is a large improvement on the performance of a ChIP-quality antibody, which is not selective at all in this application, and it should permit high-fidelity recognition with repeated imaging. The ability to image the precise location of posttranslational modifications may permit nanometer-scale investigation of their effect on chromatin structure.Artículo Texto completo
|p8/nupr1 regulates DNA-repair activity after double-strand gamma irradiation-induced DNA damage. |
Gironella M, Malicet C, Cano C, Sandi MJ, Hamidi T, Tauil RM, Baston M, Valaco P, Moreno S, Lopez F, Neira JL, Dagorn JC, Iovanna JL
J Cell Physiol 221 594-602. 2009
The stress protein p8 is a small, highly basic, unfolded, and multifunctional protein. We have previously shown that most of its functions are exerted through interactions with other proteins, whose activities are thereby enhanced or repressed. In this work we describe another example of such mechanism, by which p8 binds and negatively regulates MSL1, a histone acetyl transferase (HAT)-associated protein, which in turn binds the DNA-damage-associated 53BP1 protein to facilitate DNA repair following DNA gamma-irradiation. Contrary to the HAT-associated activity, MSL1-dependent DNA-repair activity is almost completely dependent on 53BP1 expression. The picture that has emerged from our findings is that 53BP1 could be a scaffold that gets the HAT MSL1-dependent DNA-repair activity to the sites of DNA damage. Finally, we also found that, although p8 expression is transiently activated after gamma-irradiation, it is eventually submitted to sustained down-regulation, presumably to allow development of MSL1-associated DNA-repair activity. We conclude that interaction of MSL1 with 53BP1 brings MSL1-dependent HAT activity to the vicinity of damaged DNA. MSL1-dependent HAT activity, which is negatively regulated by the stress protein p8, induces chromatin remodeling and relaxation allowing access to DNA of the repair machinery.
|Core promoter acetylation is not required for high transcription from the phosphoenolpyruvate carboxylase promoter in maize. |
Horst, I; Offermann, S; Dreesen, B; Niessen, M; Peterhansel, C
Epigenetics & chromatin 2 17 2009
Acetylation of promoter nucleosomes is tightly correlated and mechanistically linked to gene activity. However, transcription is not necessary for promoter acetylation. It seems, therefore, that external and endogenous stimuli control histone acetylation and by this contribute to gene regulation. Photosynthetic genes in plants are excellent models with which to study the connection between stimuli and chromatin modifications because these genes are strongly expressed and regulated by multiple stimuli that are easily manipulated. We have previously shown that acetylation of specific histone lysine residues on the photosynthetic phosphoenolpyruvate carboxylase (Pepc) promoter in maize is controlled by light and is independent of other stimuli or gene activity. Acetylation of upstream promoter regions responds to a set of other stimuli which include the nutrient availability of the plant. Here, we have extended these studies by analysing histone acetylation during the diurnal and circadian rhythm of the plant.We show that histone acetylation of individual lysine residues is removed from the core promoter before the end of the illumination period which is an indication that light is not the only factor influencing core promoter acetylation. Deacetylation is accompanied by a decrease in gene activity. Pharmacological inhibition of histone deacetylation is not sufficient to prevent transcriptional repression, indicating that deacetylation is not controlling diurnal gene regulation. Variation of the Pepc promoter activity during the day is controlled by the circadian oscillator as it is maintained under constant illumination for at least 3 days. During this period, light-induced changes in histone acetylation are completely removed from the core promoter, although the light stimulus is continuously applied. However, acetylation of most sites on upstream promoter elements follows the circadian rhythm.Our results suggest a central role of upstream promoter acetylation in the quantitative regulation of gene expression in this model gene. Induced core promoter acetylation is dispensable for the highest gene expression in the diurnal and circadian rhythm.
|A silencer promotes the assembly of silenced chromatin independently of recruitment. |
Lynch, PJ; Rusche, LN
Molecular and cellular biology 29 43-56 2009
In Saccharomyces cerevisiae, silenced chromatin occurs at telomeres and the silent mating-type loci HMR and HML. At these sites, the Sir proteins are recruited to a silencer and then associate with adjacent chromatin. We used chromatin immunoprecipitation to compare the rates of Sir protein assembly at different genomic locations and discovered that establishment of silenced chromatin was much more rapid at HMR than at the telomere VI-R. Silenced chromatin also assembled more quickly on one side of HMR-E than on the other. Despite differences in spreading, the Sir proteins were recruited to HMR-E and telomeric silencers at equivalent rates. Additionally, insertion of HMR-E adjacent to the telomere VI-R increased the rate of Sir2p association with the telomere. These data suggest that HMR-E functions to both recruit Sir proteins and promote their assembly across several kilobases. Observations that association of Sir2p occurs simultaneously throughout HMR and that silencing at HMR is insensitive to coexpression of catalytically inactive Sir2p suggest that HMR-E acts by enabling assembly to occur in a nonlinear fashion. The ability of silencers to promote assembly of silenced chromatin over several kilobases is likely an important mechanism for maintaining what would otherwise be unstable chromatin at the correct genomic locations.
|Drosophila MSL complex globally acetylates H4K16 on the male X chromosome for dosage compensation. |
Gelbart, ME; Larschan, E; Peng, S; Park, PJ; Kuroda, MI
Nature structural & molecular biology 16 825-32 2009
The Drosophila melanogaster male-specific lethal (MSL) complex binds the single male X chromosome to upregulate gene expression to equal that from the two female X chromosomes. However, it has been puzzling that approximately 25% of transcribed genes on the X chromosome do not stably recruit MSL complex. Here we find that almost all active genes on the X chromosome are associated with robust H4 Lys16 acetylation (H4K16ac), the histone modification catalyzed by the MSL complex. The distribution of H4K16ac is much broader than that of the MSL complex, and our results favor the idea that chromosome-wide H4K16ac reflects transient association of the MSL complex, occurring through spreading or chromosomal looping. Our results parallel those of localized Polycomb repressive complex and its more broadly distributed chromatin mark, trimethylated histone H3 Lys27 (H3K27me3), suggesting a common principle for the establishment of active and silenced chromatin domains.
|Long-range spreading of dosage compensation in Drosophila captures transcribed autosomal genes inserted on X. |
Gorchakov, AA; Alekseyenko, AA; Kharchenko, P; Park, PJ; Kuroda, MI
Genes & development 23 2266-71 2009
Dosage compensation in Drosophila melanogaster males is achieved via targeting of male-specific lethal (MSL) complex to X-linked genes. This is proposed to involve sequence-specific recognition of the X at approximately 150-300 chromatin entry sites, and subsequent spreading to active genes. Here we ask whether the spreading step requires transcription and is sequence-independent. We find that MSL complex binds, acetylates, and up-regulates autosomal genes inserted on X, but only if transcriptionally active. We conclude that a long-sought specific DNA sequence within X-linked genes is not obligatory for MSL binding. Instead, linkage and transcription play the pivotal roles in MSL targeting irrespective of gene origin and DNA sequence.
|Mof (MYST1 or KAT8) is essential for progression of embryonic development past the blastocyst stage and required for normal chromatin architecture. |
Thomas, T; Dixon, MP; Kueh, AJ; Voss, AK
Molecular and cellular biology 28 5093-105 2008
Acetylation of histone tails is a hallmark of transcriptionally active chromatin. Mof (males absent on the first; also called MYST1 or KAT8) is a member of the MYST family of histone acetyltransferases and was originally discovered as an essential component of the X chromosome dosage compensation system in Drosophila. In order to examine the role of Mof in mammals in vivo, we generated mice carrying a null mutation of the Mof gene. All Mof-deficient embryos fail to develop beyond the expanded blastocyst stage and die at implantation in vivo. Mof-deficient cell lines cannot be derived from Mof(-/-) embryos in vitro. Mof(-/-) embryos fail to acetylate histone 4 lysine 16 (H4K16) but have normal acetylation of other N-terminal histone lysine residues. Mof(-/-) cell nuclei exhibit abnormal chromatin aggregation preceding activation of caspase 3 and DNA fragmentation. We conclude that Mof is functionally nonredundant with the closely related MYST histone acetyltransferase Tip60. Our results show that Mof performs a different role in mammals from that in flies at the organism level, although the molecular function is conserved. We demonstrate that Mof is required specifically for the maintenance of H4K16 acetylation and normal chromatin architecture of all cells of early male and female embryos.Artículo Texto completo
|Estrogen receptor regulation of carbonic anhydrase XII through a distal enhancer in breast cancer. |
Barnett, DH; Sheng, S; Charn, TH; Waheed, A; Sly, WS; Lin, CY; Liu, ET; Katzenellenbogen, BS
Cancer research 68 3505-15 2008
The expression of carbonic anhydrase XII (CA12), a gene that encodes a zinc metalloenzyme responsible for acidification of the microenvironment of cancer cells, is highly correlated with estrogen receptor alpha (ER alpha) in human breast tumors. Here, we show that CA12 is robustly regulated by estrogen via ER alpha in breast cancer cells, and that this regulation involves a distal estrogen-responsive enhancer region. Upon the addition of estradiol, ER alpha binds directly to this distal enhancer in vivo, resulting in the recruitment of RNA polymerase II and steroid receptor coactivators SRC-2 and SRC-3, and changes in histone acetylation. Mutagenesis of an imperfect estrogen-responsive element within this enhancer region abolishes estrogen-dependent activity, and chromosome conformation capture and chromatin immunoprecipitation assays show that this distal enhancer communicates with the transcriptional start site of the CA12 gene via intrachromosomal looping upon hormone treatment. This distal enhancer element is observed in the homologous mouse genomic sequence, and the expression of the mouse homologue, Car12, is rapidly and robustly stimulated by estradiol in the mouse uterus in vivo, suggesting that the ER regulation of CA12 is mechanistically and evolutionarily conserved. Our findings highlight the crucial role of ER in the regulation of the CA12 gene, and provide insight into the transcriptional regulatory mechanism that accounts for the strong association of CA12 and ER in human breast cancers.
|HDAC inhibitors correct frataxin deficiency in a Friedreich ataxia mouse model. |
Rai, M; Soragni, E; Jenssen, K; Burnett, R; Herman, D; Coppola, G; Geschwind, DH; Gottesfeld, JM; Pandolfo, M
PloS one 3 e1958 2008
Friedreich ataxia, an autosomal recessive neurodegenerative and cardiac disease, is caused by abnormally low levels of frataxin, an essential mitochondrial protein. All Friedreich ataxia patients carry a GAATTC repeat expansion in the first intron of the frataxin gene, either in the homozygous state or in compound heterozygosity with other loss-of-function mutations. The GAA expansion inhibits frataxin expression through a heterochromatin-mediated repression mechanism. Histone modifications that are characteristic of silenced genes in heterochromatic regions occur at expanded alleles in cells from Friedreich ataxia patients, including increased trimethylation of histone H3 at lysine 9 and hypoacetylation of histones H3 and H4.By chromatin immunoprecipitation, we detected the same heterochromatin marks in homozygous mice carrying a (GAA)(230) repeat in the first intron of the mouse frataxin gene (KIKI mice). These animals have decreased frataxin levels and, by microarray analysis, show significant gene expression changes in several tissues. We treated KIKI mice with a novel histone deacetylase inhibitor, compound 106, which substantially increases frataxin mRNA levels in cells from Friedreich ataxia individuals. Treatment increased histone H3 and H4 acetylation in chromatin near the GAA repeat and restored wild-type frataxin levels in the nervous system and heart, as determined by quantitative RT-PCR and semiquantitative western blot analysis. No toxicity was observed. Furthermore, most of the differentially expressed genes in KIKI mice reverted towards wild-type levels.Lack of acute toxicity, normalization of frataxin levels and of the transcription profile changes resulting from frataxin deficiency provide strong support to a possible efficacy of this or related compounds in reverting the pathological process in Friedreich ataxia, a so far incurable neurodegenerative disease.Artículo Texto completo
|HST3/HST4-dependent deacetylation of lysine 56 of histone H3 in silent chromatin. |
Yang, B; Miller, A; Kirchmaier, AL
Molecular biology of the cell 19 4993-5005 2008
The composition of posttranslational modifications on newly synthesized histones must be altered upon their incorporation into chromatin. These changes are necessary to maintain the same gene expression state at individual chromosomal loci before and after DNA replication. We have examined how one modification that occurs on newly synthesized histone H3, acetylation of K56, influences gene expression at epigenetically regulated loci in Saccharomyces cerevisiae. H3 K56 is acetylated by Rtt109p before its incorporation into chromatin during S phase, and this modification is then removed by the NAD(+)-dependent deacetylases Hst3p and Hst4p during G2/M phase. We found silenced loci maintain H3 K56 in a hypoacetylated state, and the absence of this modification in rtt109 mutants was compatible with HM and telomeric silencing. In contrast, loss of HST3 and HST4 resulted in hyperacetylation of H3 K56 within silent loci and telomeric silencing defects, despite the continued presence of Sir2p throughout these loci. These silencing defects in hst3Delta hst4Delta mutants could be suppressed by deletion of RTT109. In contrast, overexpression of Sir2p could not restore silencing in hst3Delta hst4Delta mutants. Together, our findings argue that HST3 HST4 play critical roles in maintaining the hypoacetylated state of K56 on histone H3 within silent chromatin.Artículo Texto completo
|Transcription-coupled methylation of histone H3 at lysine 36 regulates dosage compensation by enhancing recruitment of the MSL complex in Drosophila melanogaster. |
Bell, O; Conrad, T; Kind, J; Wirbelauer, C; Akhtar, A; Schübeler, D
Molecular and cellular biology 28 3401-9 2008
In Drosophila melanogaster, dosage compensation relies on the targeting of the male-specific lethal (MSL) complex to hundreds of sites along the male X chromosome. Transcription-coupled methylation of histone H3 lysine 36 is enriched toward the 3' end of active genes, similar to the MSL proteins. Here, we have studied the link between histone H3 methylation and MSL complex targeting using RNA interference and chromatin immunoprecipitation. We show that trimethylation of histone H3 at lysine 36 (H3K36me3) relies on the histone methyltransferase Hypb and is localized promoter distal at dosage-compensated genes, similar to active genes on autosomes. However, H3K36me3 has an X-specific function, as reduction specifically decreases acetylation of histone H4 lysine 16 on the male X chromosome. This hypoacetylation is caused by compromised MSL binding and results in a failure to increase expression twofold. Thus, H3K36me3 marks the body of all active genes yet is utilized in a chromosome-specific manner to enhance histone acetylation at sites of dosage compensation.Artículo Texto completo
|Genomewide screen for negative regulators of sirtuin activity in Saccharomyces cerevisiae reveals 40 loci and links to metabolism. |
Raisner, RM; Madhani, HD
Genetics 179 1933-44 2008
Sirtuins are conserved proteins implicated in myriad key processes including gene control, aging, cell survival, metabolism, and DNA repair. In Saccharomyces cerevisiae, the sirtuin Silent information regulator 2 (Sir2) promotes silent chromatin formation, suppresses recombination between repeats, and inhibits senescence. We performed a genomewide screen for factors that negatively regulate Sir activity at a reporter gene placed immediately outside a silenced region. After linkage analysis, assessment of Sir dependency, and knockout tag verification, 40 loci were identified, including 20 that have not been previously described to regulate Sir. In addition to chromatin-associated factors known to prevent ectopic silencing (Bdf1, SAS-I complex, Rpd3L complex, Ku), we identified the Rtt109 DNA repair-associated histone H3 lysine 56 acetyltransferase as an anti-silencing factor. Our findings indicate that Rtt109 functions independently of its proposed effectors, the Rtt101 cullin, Mms1, and Mms22, and demonstrate unexpected interplay between H3K56 and H4K16 acetylation. The screen also identified subunits of mediator (Soh1, Srb2, and Srb5) and mRNA metabolism factors (Kem1, Ssd1), thus raising the possibility that weak silencing affects some aspect of mRNA structure. Finally, several factors connected to metabolism were identified. These include the PAS-domain metabolic sensor kinase Psk2, the mitochondrial homocysteine detoxification enzyme Lap3, and the Fe-S cluster protein maturase Isa2. We speculate that PAS kinase may integrate metabolic signals to control sirtuin activity.
|Developmental and environmental signals induce distinct histone acetylation profiles on distal and proximal promoter elements of the C4-Pepc gene in maize. |
Offermann, S; Dreesen, B; Horst, I; Danker, T; Jaskiewicz, M; Peterhansel, C
Genetics 179 1891-901 2008
The maize C(4)-Pepc gene is expressed in an organ- and cell-type-specific manner, inducible by light and modulated by nutrient availability and the metabolic state of the cell. We studied the contribution of histone acetylation at five lysine residues to the integration of these signals into a graduated promoter response. In roots and coleoptiles, where the gene is constitutively inactive, three of the five lysines were acetylated and the modifications showed unique patterns with respect to their distribution on the gene. A similar pattern was observed in etiolated leaves, where the gene is poised for activation by light. Here, illumination selectively induced the acetylation of histone H4 lysine 5 and histone H3 lysine 9 in both the promoter and the transcribed region, again with unique distribution patterns. Induction was independent of transcription and fully reversible in the dark. Nitrate and hexose availability modulated acetylation of all five lysines restricted to a distal promoter region, whereas proximal promoter acetylation was highly resistant to these stimuli. Our data suggest that light induction of acetylation is controlled by regulating HDAC activity, whereas metabolic signals regulate HAT activity. Acetylation turnover rates were high in the distal promoter and the transcribed regions, but low on the proximal promoter. On the basis of these results, we propose a model with three levels of stimulus-induced histone modifications that collectively adjust promoter activity. The results support a charge neutralization model for the distal promoter and a stimulus-mediated, but transcription-independent, histone acetylation pattern on the core promoter, which might be part of a more complex histone code.
|Double strand breaks can initiate gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous promoter CpG island. |
O'Hagan, HM; Mohammad, HP; Baylin, SB
PLoS genetics 4 e1000155 2008
Chronic exposure to inducers of DNA base oxidation and single and double strand breaks contribute to tumorigenesis. In addition to the genetic changes caused by this DNA damage, such tumors often contain epigenetically silenced genes with aberrant promoter region CpG island DNA hypermethylation. We herein explore the relationships between such DNA damage and epigenetic gene silencing using an experimental model in which we induce a defined double strand break in an exogenous promoter construct of the E-cadherin CpG island, which is frequently aberrantly DNA hypermethylated in epithelial cancers. Following the onset of repair of the break, we observe recruitment to the site of damage of key proteins involved in establishing and maintaining transcriptional repression, namely SIRT1, EZH2, DNMT1, and DNMT3B, and the appearance of the silencing histone modifications, hypoacetyl H4K16, H3K9me2 and me3, and H3K27me3. Although in most cells selected after the break, DNA repair occurs faithfully with preservation of activity of the promoter, a small percentage of the plated cells demonstrate induction of heritable silencing. The chromatin around the break site in such a silent clone is enriched for most of the above silent chromatin proteins and histone marks, and the region harbors the appearance of increasing DNA methylation in the CpG island of the promoter. During the acute break, SIRT1 appears to be required for the transient recruitment of DNMT3B and subsequent methylation of the promoter in the silent clones. Taken together, our data suggest that normal repair of a DNA break can occasionally cause heritable silencing of a CpG island-containing promoter by recruitment of proteins involved in silencing. Furthermore, with contribution of the stress-related protein SIRT1, the break can lead to the onset of aberrant CpG island DNA methylation, which is frequently associated with tight gene silencing in cancer.
|Bypassing Sir2 and O-acetyl-ADP-ribose in transcriptional silencing. |
Chou, CC; Li, YC; Gartenberg, MR
Molecular cell 31 650-9 2008
The yeast Sir2/3/4 complex forms a heterochromatin-like structure that represses transcription. The proteins nucleate at silencers and spread distally, utilizing the Sir2 NAD(+)-dependent histone deacetylase activity and the affinity of Sir3/4 for deacetylated histone tails. A by-product of the Sir2 reaction, O-acetyl-ADP-ribose (OAADPr), is thought to aid spreading by binding one of the Sir proteins. We developed a protein chimera approach to reexamine the contributions of Sir2. We show that a Sir3 chimera-bearing Hos3, an unrelated NAD(+)-independent histone deacetylase, substitutes for Sir2 in silencing. Sir3-Hos3 operates within the Sir pathway, spreading while deacetylating histones. Moreover, the chimera represses HM loci in strains lacking all five OAADPr-producing deacetylases, indicating that OAADPr is not necessary for silencing. Repression by a Hos3 hybrid bearing the targeting motifs of Sir2 shows that targeting doesn't require the Sir2 reaction. Together, these data demonstrate that protein deacetylation is the only essential function of Sir2 in creating silenced chromatin.
|Analysis of Myc-induced histone modifications on target chromatin. |
Martinato, F; Cesaroni, M; Amati, B; Guccione, E
PloS one 3 e3650 2008
The c-myc proto-oncogene is induced by mitogens and is a central regulator of cell growth and differentiation. The c-myc product, Myc, is a transcription factor that binds a multitude of genomic sites, estimated to be over 10-15% of all promoter regions. Target promoters generally pre-exist in an active or poised chromatin state that is further modified by Myc, contributing to fine transcriptional regulation (activation or repression) of the afferent gene. Among other mechanisms, Myc recruits histone acetyl-transferases to target chromatin and locally promotes hyper-acetylation of multiple lysines on histones H3 and H4, although the identity and combination of the modified lysines is unknown. Whether Myc dynamically regulates other histone modifications (or marks) at its binding sites also remains to be addressed. Here, we used quantitative chromatin immunoprecipitation (qChIP) to profile a total of 24 lysine-acetylation and -methylation marks modulated by Myc at target promoters in a human B-cell line with a regulatable c-myc transgene. Myc binding promoted acetylation of multiple lysines, primarily of H3K9, H3K14, H3K18, H4K5 and H4K12, but significantly also of H4K8, H4K91 and H2AK5. Dimethylation of H3K79 was also selectively induced at target promoters. A majority of target promoters showed co-induction of multiple marks - in various combinations - correlating with recruitment of the two HATs tested (Tip60 and HBO1), incorporation of the histone variant H2A.Z and transcriptional activation. Based on this and previous findings, we surmise that Myc recruits the Tip60/p400 complex to achieve a coordinated histone acetylation/exchange reaction at activated promoters. Our data are also consistent with the additive and redundant role of multiple acetylation events in transcriptional activation.
|Control of replication initiation by the Sum1/Rfm1/Hst1 histone deacetylase. |
Weber, JM; Irlbacher, H; Ehrenhofer-Murray, AE
BMC molecular biology 9 100 2008
Replication initiation at origins of replication in the yeast genome takes place on chromatin as a template, raising the question how histone modifications, for instance histone acetylation, influence origin firing. Initiation requires binding of the replication initiator, the Origin Recognition Complex (ORC), to a consensus sequence within origins. In addition, other proteins bind to recognition sites in the vicinity of ORC and support initiation. In previous work, we identified Sum1 as an origin-binding protein that contributes to efficient replication initiation. Sum1 is part of the Sum1/Rfm1/Hst1 complex that represses meiotic genes during vegetative growth via histone deacetylation by the histone deacetylase (HDAC) Hst1.In this study, we investigated how Sum1 affected replication initiation. We found that it functioned in initiation as a component of the Sum1/Rfm1/Hst1 complex, implying a role for histone deacetylation in origin activity. We identified several origins in the yeast genome whose activity depended on both Sum1 and Hst1. Importantly, sum1Delta or hst1Delta caused a significant increase in histone H4 lysine 5 (H4 K5) acetylation levels, but not other H4 acetylation sites, at those origins. Furthermore, mutation of lysines to glutamines in the H4 tail, which imitates the constantly acetylated state, resulted in a reduction of origin activity comparable to that in the absence of Hst1, showing that deacetylation of H4 was important for full initiation capacity of these origins.Taken together, our results demonstrate a role for histone deacetylation in origin activity and reveal a novel aspect of origin regulation by chromatin. These results suggest recruitment of the Sum1/Rfm1/Hst1 complex to a number of yeast origins, where Hst1 deacetylated H4 K5.
|The fungus Neurospora crassa displays telomeric silencing mediated by multiple sirtuins and by methylation of histone H3 lysine 9. |
Smith, KM; Kothe, GO; Matsen, CB; Khlafallah, TK; Adhvaryu, KK; Hemphill, M; Freitag, M; Motamedi, MR; Selker, EU
Epigenetics & chromatin 1 5 2008
Silencing of genes inserted near telomeres provides a model to investigate the function of heterochromatin. We initiated a study of telomeric silencing in Neurospora crassa, a fungus that sports DNA methylation, unlike most other organisms in which telomeric silencing has been characterized.The selectable marker, hph, was inserted at the subtelomere of Linkage Group VR in an nst-1 (neurospora sir two-1) mutant and was silenced when nst-1 function was restored. We show that NST-1 is an H4-specific histone deacetylase. A second marker, bar, tested at two other subtelomeres, was similarly sensitive to nst-1 function. Mutation of three additional SIR2 homologues, nst-2, nst-3 and nst-5, partially relieved silencing. Two genes showed stronger effects: dim-5, which encodes a histone H3 K9 methyltransferase and hpo, which encodes heterochromatin protein-1. Subtelomeres showed variable, but generally low, levels of DNA methylation. Elimination of DNA methylation caused partial derepression of one telomeric marker. Characterization of histone modifications at subtelomeric regions revealed H3 trimethyl-K9, H3 trimethyl-K27, and H4 trimethyl-K20 enrichment. These modifications were slightly reduced when telomeric silencing was compromised. In contrast, acetylation of histones H3 and H4 increased.We demonstrate the presence of telomeric silencing in Neurospora and show a dependence on histone deacetylases and methylation of histone H3 lysine 9. Our studies also reveal silencing functions for DIM-5 and HP1 that appear independent of their role in de novo DNA methylation.
|Transcription-coupled deposition of histone modifications during MHC class II gene activation. |
Rybtsova, N; Leimgruber, E; Seguin-Estévez, Q; Dunand-Sauthier, I; Krawczyk, M; Reith, W
Nucleic acids research 35 3431-41 2007
Posttranslational histone modifications associated with actively expressed genes are generally believed to be introduced primarily by histone-modifying enzymes that are recruited by transcription factors or their associated co-activators. We have performed a comprehensive spatial and temporal analyses of the histone modifications that are deposited upon activation of the MHC class II gene HLA-DRA by the co-activator CIITA. We find that transcription-associated histone modifications are introduced during two sequential phases. The first phase precedes transcription initiation and is characterized exclusively by a rapid increase in histone H4 acetylation over a large upstream domain. All other modifications examined, including the acetylation and methylation of several residues in histone H3, are restricted to short regions situated at or within the 5' end of the gene and are established during a second phase that is concomitant with ongoing transcription. This second phase is completely abrogated when elongation by RNA polymerase II is blocked. These results provide strong evidence that transcription elongation can play a decisive role in the deposition of histone modification patterns associated with inducible gene activation.Artículo Texto completo
|Chromium cross-links histone deacetylase 1-DNA methyltransferase 1 complexes to chromatin, inhibiting histone-remodeling marks critical for transcriptional activation. |
Schnekenburger, M; Talaska, G; Puga, A
Molecular and cellular biology 27 7089-101 2007
Transcriptional regulation of gene expression requires posttranslational modification of histone proteins, which, in concert with chromatin-remodeling factors, modulate chromatin structure. Exposure to environmental agents may interfere with specific histone modifications and derail normal patterns of gene expression. To test this hypothesis, we coexposed cells to binary mixtures of benzo[a]pyrene (B[a]P), an environmental procarcinogen that activates Cyp1a1 transcriptional responses mediated by the aryl hydrocarbon receptor (AHR), and chromium, a carcinogenic heavy metal that represses B[a]P-inducible AHR-mediated gene expression. We show that chromium cross-links histone deacetylase 1-DNA methyltransferase 1 (HDAC1-DNMT1) complexes to Cyp1a1 promoter chromatin and inhibits histone marks induced by AHR-mediated gene transactivation, including phosphorylation of histone H3 Ser-10, trimethylation of H3 Lys-4, and various acetylation marks in histones H3 and H4. These changes inhibit RNA polymerase II recruitment without affecting the kinetics of AHR DNA binding. HDAC1 and DNMT1 inhibitors or depletion of HDAC1 or DNMT1 with siRNAs blocks chromium-induced transcriptional repression by decreasing the interaction of these proteins with the Cyp1a1 promoter and allowing histone acetylation to proceed. By inhibiting Cyp1a1 expression, chromium stimulates the formation of B[a]P DNA adducts. Epigenetic modification of gene expression patterns may be a key element of the developmental and carcinogenic outcomes of exposure to chromium and to other environmental agents.Artículo Texto completo
|The triploid endosperm genome of Arabidopsis adopts a peculiar, parental-dosage-dependent chromatin organization. |
Baroux, C; Pecinka, A; Fuchs, J; Schubert, I; Grossniklaus, U
The Plant cell 19 1782-94 2007
The endosperm is a seed tissue unique to flowering plants. Due to its central role in nourishing and protecting the embryo, endosperm development is subject to parental conflicts and adaptive processes, which led to the evolution of parent-of-origin-dependent gene regulation. The role of higher-order chromatin organization in regulating the endosperm genome was long ignored due to technical hindrance. We developed a combination of approaches to analyze nuclear structure and chromatin organization in Arabidopsis thaliana endosperm. Endosperm nuclei showed a less condensed chromatin than other types of nuclei and a peculiar heterochromatin organization, with smaller chromocenters and additional heterochromatic foci interspersed in euchromatin. This is accompanied by a redistribution of the heterochromatin mark H3K9me1 from chromocenters toward euchromatin and interspersed heterochromatin. Thus, endosperm nuclei have a specific nuclear architecture and organization, which we interpret as a relaxed chromocenter-loop model. The analysis of endosperm with altered parental genome dosage indicated that the additional heterochromatin may be predominantly of maternal origin, suggesting differential regulation of maternal and paternal genomes, possibly linked to genome dosage regulation.
|Characterization of BEAF mutations isolated by homologous recombination in Drosophila. |
Roy, S; Gilbert, MK; Hart, CM
Genetics 176 801-13 2007
The Drosophila BEAF-32A and BEAF-32B proteins bind to the scs' insulator and to hundreds of other sites on Drosophila chromosomes. These two proteins are encoded by the same gene. We used ends-in homologous recombination to generate the null BEAF(AB-KO) allele and also isolated the BEAF(A-KO) allele that eliminates production of only the BEAF-32A protein. We find that the BEAF proteins together are essential, but BEAF-32B alone is sufficient to obtain viable flies. Our results show that BEAF is important for both oogenesis and development. Maternal or zygotic BEAF is sufficient to obtain adults, although having only maternal BEAF impairs female fertility. In the absence of all BEAF, a few fertile but sickly males are obtained. Using both a chromosomal position-effect assay and an enhancer-blocking assay, we find that BEAF is necessary for scs' insulator function. Lack of BEAF causes a disruption of male X polytene chromosome morphology. However, we did not find evidence that dosage compensation was affected. Position-effect variegation of the w(m4h) allele and different variegating y transgenes was enhanced by the knockout mutation. Combined with the effects on male X polytene chromosomes, we conclude that BEAF function affects chromatin structure or dynamics.
|Inhibition of SIRT1 reactivates silenced cancer genes without loss of promoter DNA hypermethylation. |
Pruitt, K; Zinn, RL; Ohm, JE; McGarvey, KM; Kang, SH; Watkins, DN; Herman, JG; Baylin, SB
PLoS genetics 2 e40 2006
The class III histone deactylase (HDAC), SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs) in which 5' CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively), had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA)-mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression.
|The expression of endothelial nitric-oxide synthase is controlled by a cell-specific histone code. |
Fish, JE; Matouk, CC; Rachlis, A; Lin, S; Tai, SC; D'Abreo, C; Marsden, PA
The Journal of biological chemistry 280 24824-38 2005
Expression of endothelial nitric-oxide synthase (eNOS) mRNA is highly restricted to the endothelial cell layer of medium to large sized arterial blood vessels. Here we assessed the chromatin environment of the eNOS gene in expressing and nonexpressing cell types. Within endothelial cells, but not a variety of nonendothelial cells, the nucleosomes that encompassed the eNOS core promoter and proximal downstream coding regions were highly enriched in acetylated histones H3 and H4 and methylated lysine 4 of histone H3. This differentially modified chromatin domain was selectively associated with functionally competent RNA polymerase II complexes. Endothelial cells were particularly enriched in acetylated histone H3 lysine 9, histone H4 lysine 12, and di- and tri-methylated lysine 4 of histone H3 at the core promoter. Histone modifications at this region, which we have previously demonstrated to exhibit cell-specific DNA methylation, were functionally relevant to eNOS expression. Inhibition of histone deacetylase activity by trichostatin A increased acetylation of histones H3 and H4 at the eNOS proximal promoter in nonexpressing cell types and led to increased steady-state eNOS mRNA transcript levels. H3 lysine 4 methylation was also essential for eNOS expression, since treatment of endothelial cells with methylthioadenosine, a known lysine 4 methylation inhibitor, decreased eNOS RNA levels, H3 lysine 4 methylation, and RNA polymerase II loading at the eNOS proximal promoter. Importantly, methylthioadenosine also prevented the trichostatin A-mediated increase in eNOS mRNA transcript levels in nonendothelial cells. Taken together, these findings provide strong evidence that the endothelial cell-specific expression of eNOS is controlled by cell-specific histone modifications.
|BAF53/Arp4 homolog Alp5 in fission yeast is required for histone H4 acetylation, kinetochore-spindle attachment, and gene silencing at centromere. |
Minoda, A; Saitoh, S; Takahashi, K; Toda, T
Molecular biology of the cell 16 316-27 2005
Nuclear actin-related proteins play vital roles in transcriptional regulation; however, their biological roles remain elusive. Here, we characterize Alp5, fission yeast homolog of Arp4/BAF53. The temperature-sensitive mutant alp5-1134 contains a single amino acid substitution in the conserved C-terminal domain (S402N) and displays mitotic phenotypes, including chromosome condensation and missegregation. Alp5 forms a complex with Mst1-HAT (histone acetyltransferase). Consistently, inhibition of histone deacetylases (HDACs), by either addition of a specific inhibitor or a mutation in HDAC-encoding clr6+ gene, rescues alp5-1134. Immunoblotting with specific antibodies against acetylated histones shows that Alp5 is required for histone H4 acetylation at lysines 5, 8, and 12, but not histone H3 lysines 9 or 14, and furthermore Clr6 plays an opposing role. Mitotic arrest is ascribable to activation of the Mad2/Bub1 spindle checkpoint, in which both proteins localize to the mitotic kinetochores in alp5-1134. Intriguingly, alp5-1134 displays transcriptional desilencing at the core centromere without altering the overall chromatin structure, which also is suppressed by a simultaneous mutation in clr6+. This result shows that Alp5 is essential for histone H4 acetylation, and its crucial role lies in the establishment of bipolar attachment of the kinetochore to the spindle and transcriptional silencing at the centromere.
|Regulation of Swi6/HP1-dependent heterochromatin assembly by cooperation of components of the mitogen-activated protein kinase pathway and a histone deacetylase Clr6. |
Kim, HS; Choi, ES; Shin, JA; Jang, YK; Park, SD
The Journal of biological chemistry 279 42850-9 2004
A study of gene silencing within the mating-type region of fission yeast defines two distinct pathways responsible for the establishment of heterochromatin assembly. One is RNA interference-dependent and acts on centromere-homologous repeats (cenH). The other is a stochastic Swi6 (the fission yeast HP1 homolog)-dependent mechanism that is not fully understood. Here we find that activating transcription factor (Atf1) and Pcr1, the fission yeast bZIP transcription factors homologous to human ATF-2, are crucial for proper histone deacetylation of both H3 and H4. This deacetylation is a prerequisite for subsequent H3 lysine 9 methylation and Swi6-dependent heterochromatin assembly across the rest of the silent mating-type (mat) region lacking the RNA interference-dependent cenH repeat. Moreover, Atf1 and Pcr1 can form complexes with both a histone deacetylase, Clr6, and Swi6, and clr6 mutations affected the H3/H4 acetylation patterns, similar to the atf1 and pcr1 deletion mutant phenotypes at the endogenous mat loci and at the ctt1+ promoter region surrounding ATF/CRE-binding site. These data suggest that Atf1 and Pcr1 participate in an early step essential for heterochromatin assembly at the mat locus and silencing of transcriptional targets of Atf1. Furthermore, a phosphorylation event catalyzed by the conserved mitogen-activated protein kinase pathway is important for regulation of heterochromatin silencing by Atf1 and Pcr1. These findings suggest a role for the mitogen-activated protein kinase pathway and histone deacetylase in Swi6-based heterochromatin assembly.
|Highly specific antibodies determine histone acetylation site usage in yeast heterochromatin and euchromatin. |
Suka, N, et al.
Mol. Cell, 8: 473-9 (2001) 2001
We have developed a highly specific antibody set for acetylation sites in yeast histones H4 (K5, K8, K12, and K16); H3 (K9, K14, K18, K23, and K27); H2A (K7); and H2B (K11 and K16). Since ELISA does not assure antibody specificity in chromatin immunoprecipitation, we have employed additional screens against the respective histone mutations. We now show that telomeric and silent mating locus heterochromatin is hypoacetylated at all histone sites. At the INO1 promoter, RPD3 is required for strongly deacetylating all sites except H4 K16, ESA1 for acetylating H2A, H2B, and H4 sites except H4 K16, and GCN5 for acetylating H2B and H3 sites except H3 K14. These data uncover the in vivo usage of acetylation sites in heterochromatin and euchromatin.
|Chromatin Immunoprecipitation (ChIP)||11545749|
|Barrier-to-Autointegration Factor influences specific histone modifications. |
Montes de Oca, R; Andreassen, PR; Wilson, KL
Nucleus (Austin, Tex.) 2 580-90 2001
Defects in the nuclear envelope or nuclear 'lamina' networks cause disease and can perturb histone posttranslational (epigenetic) regulation. Barrier-to-Autointegration Factor (BAF) is an essential but enigmatic lamina component that binds lamins, LEM-domain proteins, DNA and histone H3 directly. We report that BAF copurified with nuclease-digested mononucleosomes and associated with modified histones in vivo. BAF overexpression significantly reduced global histone H3 acetylation by 18%. In cells that stably overexpressed BAF 3-fold, silencing mark H3-K27-Me1/3 and active marks H4-K16-Ac and H4-Ac5 decreased significantly. Significant increases were also seen for silencing mark H3-K9-Me3, active marks H3-K4-Me2, H3-K9/K14-Ac and H4-K5-Ac and a mark (H3-K79-Me2) associated with both active and silent chromatin. Other increases (H3-S10-P, H3-S28-P and silencing mark H3-K9-Me2) did not reach statistical significance. BAF overexpression also significantly influenced cell cycle distribution. Moreover, BAF associated in vivo with SET/I2PP2A (protein phosphatase 2A inhibitor; blocks H3 dephosphorylation) and G9a (H3-K9 methyltransferase), but showed no detectable association with HDAC1 or HATs. These findings reveal BAF as a novel epigenetic regulator and are discussed in relation to BAF deficiency phenotypes, which include a hereditary progeria syndrome and loss of pluripotency in embryonic stem cells.
|What is the concentration of this antibody?||We are not able to quantitate antiserum.|