Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|M, H, Ch, Dr, Vrt, Yeast (S. cerevisiae)||ChIP, WB, DB||Rb||Serum||Polyclonal Antibody|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at -20°C from date of receipt. For maximum recovery of product, centrifuge the vial prior to removing the cap.|
|Material Size||200 µL|
|Reference overview||Application||Species||Pub Med ID|
|Impaired p32 regulation caused by the lymphoma-prone RECQ4 mutation drives mitochondrial dysfunction.|
Wang, JT; Xu, X; Alontaga, AY; Chen, Y; Liu, Y
Cell reports 7 848-58 2014
Mitochondrial DNA (mtDNA) encodes proteins that are important for ATP biogenesis. Therefore, changes in mtDNA copy number will have profound consequences on cell survival and proliferation. RECQ4 DNA helicase participates in both nuclear DNA and mtDNA synthesis. However, the mechanism that balances the distribution of RECQ4 in the nucleus and mitochondria is unknown. Here, we show that RECQ4 forms protein complexes with Protein Phosphatase 2A (PP2A), nucleophosmin (NPM), and mitochondrial p32 in different cellular compartments. Critically, the interaction with p32 negatively controls the transport of both RECQ4 and its chromatin-associated replication factor, MCM10, from the nucleus to mitochondria. Amino acids that are deleted in the most common cancer-associated RECQ4 mutation are required for the interaction with p32. Hence, this RECQ4 mutant, which is no longer regulated by p32 and is enriched in the mitochondria, interacts with the mitochondrial replication helicase PEO1 and induces abnormally high levels of mtDNA synthesis.
|Snf2h-mediated chromatin organization and histone H1 dynamics govern cerebellar morphogenesis and neural maturation.|
Alvarez-Saavedra, M; De Repentigny, Y; Lagali, PS; Raghu Ram, EV; Yan, K; Hashem, E; Ivanochko, D; Huh, MS; Yang, D; Mears, AJ; Todd, MA; Corcoran, CP; Bassett, EA; Tokarew, NJ; Kokavec, J; Majumder, R; Ioshikhes, I; Wallace, VA; Kothary, R; Meshorer, E; Stopka, T; Skoultchi, AI; Picketts, DJ
Nature communications 5 4181 2014
Chromatin compaction mediates progenitor to post-mitotic cell transitions and modulates gene expression programs, yet the mechanisms are poorly defined. Snf2h and Snf2l are ATP-dependent chromatin remodelling proteins that assemble, reposition and space nucleosomes, and are robustly expressed in the brain. Here we show that mice conditionally inactivated for Snf2h in neural progenitors have reduced levels of histone H1 and H2A variants that compromise chromatin fluidity and transcriptional programs within the developing cerebellum. Disorganized chromatin limits Purkinje and granule neuron progenitor expansion, resulting in abnormal post-natal foliation, while deregulated transcriptional programs contribute to altered neural maturation, motor dysfunction and death. However, mice survive to young adulthood, in part from Snf2l compensation that restores Engrailed-1 expression. Similarly, Purkinje-specific Snf2h ablation affects chromatin ultrastructure and dendritic arborization, but alters cognitive skills rather than motor control. Our studies reveal that Snf2h controls chromatin organization and histone H1 dynamics for the establishment of gene expression programs underlying cerebellar morphogenesis and neural maturation.
|New insights into somatic embryogenesis: leafy cotyledon1, baby boom1 and WUSCHEL-related homeobox4 are epigenetically regulated in Coffea canephora.|
Nic-Can, GI; López-Torres, A; Barredo-Pool, F; Wrobel, K; Loyola-Vargas, VM; Rojas-Herrera, R; De-la-Peña, C
PloS one 8 e72160 2013
Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY cotyledon1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-related homeobox4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed.
|The chromatin remodeling and mRNA splicing functions of the Brahma (SWI/SNF) complex are mediated by the SNR1/SNF5 regulatory subunit.|
Zraly, CB; Dingwall, AK
Nucleic acids research 40 5975-87 2012
Nucleosome remodeling catalyzed by the ATP-dependent SWI/SNF complex is essential for regulated gene expression. Transcriptome profiling studies in flies and mammals identified cell cycle and hormone responsive genes as important targets of remodeling complex activities. Loss of chromatin remodeling function has been linked to developmental abnormalities and aggressive cancers. The Drosophila Brahma (Brm) SWI/SNF complex assists in reprogramming and coordinating gene expression in response to ecdysone hormone signaling at critical points during development. We used RNAi knockdown in cultured cells and transgenic flies, and conditional mutant alleles to identify unique and important functions of two conserved Brm complex core subunits, SNR1/SNF5 and BRM/SNF2-SWI2, on target gene regulation. Unexpectedly, we found that incorporation of a loss of function SNR1 subunit led to alterations in RNA polymerase elongation, pre-mRNA splicing regulation and chromatin accessibility of ecdysone hormone regulated genes, revealing that SNR1 functions to restrict BRM-dependent nucleosome remodeling activities downstream of the promoter region. Our results reveal critically important roles of the SNR1/SNF5 subunit and the Brm chromatin remodeling complex in transcription regulation during elongation by RNA Polymerase II and completion of pre-mRNA transcripts that are dependent on hormone signaling in late development.
|KDM3B is the H3K9 demethylase involved in transcriptional activation of lmo2 in leukemia.|
Kim, JY; Kim, KB; Eom, GH; Choe, N; Kee, HJ; Son, HJ; Oh, ST; Kim, DW; Pak, JH; Baek, HJ; Kook, H; Hahn, Y; Kook, H; Chakravarti, D; Seo, SB
Molecular and cellular biology 32 2917-33 2012
Histone lysine methylation and demethylation are considered critical steps in transcriptional regulation. In this report, we performed chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis to examine the genome-wide occupancy of H3K9-me2 during all-trans-retinoic acid (ATRA)-induced differentiation of HL-60 promyelocytic leukemia cells. Using this approach, we found that KDM3B, which contains a JmjC domain, was downregulated during differentiation through the recruitment of a corepressor complex. Furthermore, KDM3B displayed histone H3K9-me1/2 demethylase activity and induced leukemogenic oncogene lmo2 expression via a synergistic interaction with CBP. Here, we found that KDM3B repressed leukemia cell differentiation and was upregulated in blood cells from acute lymphoblastic leukemia (ALL)-type leukemia patients. The combined results of this study provide evidence that the H3K9-me1/2 demethylase KDM3B might play a role in leukemogenesis via activation of lmo2 through interdependent actions with the histone acetyltransferase (HAT) complex containing CBP.
|The H3K4me3 histone demethylase Fbxl10 is a regulator of chemokine expression, cellular morphology, and the metabolome of fibroblasts.|
Janzer, A; Stamm, K; Becker, A; Zimmer, A; Buettner, R; Kirfel, J
The Journal of biological chemistry 287 30984-92 2012
Fbxl10 (Jhdm1b/Kdm2b) is a conserved and ubiquitously expressed member of the JHDM (JmjC domain-containing histone demethylase) family. Fbxl10 was implicated in the demethylation of H3K4me3 or H3K36me2 thereby removing active chromatin marks and inhibiting gene transcription. Apart from the JmjC domain, Fbxl10 consists of a CxxC domain, a PHD domain, and an Fbox domain. By purifying the JmjC and the PHD domain of Fbxl10 and using different approaches we were able to characterize the properties of these domains in vitro. Our results suggest that Fbxl10 is rather a H3K4me3 than a H3K36me2 histone demethylase. The PHD domain exerts a dual function in binding H3K4me3 and H3K36me2 and exhibiting E3 ubiquitin ligase activity. We generated mouse embryonic fibroblasts stably overexpressing Fbxl10. These cells reveal an increase in cell size but no changes in proliferation, mitosis, or apoptosis. Using a microarray approach we were able to identify potentially new target genes for Fbxl10 including chemokines, the noncoding RNA Xist, and proteins involved in metabolic processes. Additionally, we found that Fbxl10 is recruited to the promoters of Ccl7, Xist, Crabp2, and RipK3. Promoter occupancy by Fbxl10 was accompanied by reduced levels of H3K4me3 but unchanged levels of H3K36me2. Furthermore, knockdown of Fbxl10 using small interfering RNA approaches showed inverse regulation of Fbxl10 target genes. In summary, our data reveal a regulatory role of Fbxl10 in cell morphology, chemokine expression, and the metabolic control of fibroblasts.
|JMJD5, a Jumonji C (JmjC) domain-containing protein, negatively regulates osteoclastogenesis by facilitating NFATc1 protein degradation.|
Youn, MY; Yokoyama, A; Fujiyama-Nakamura, S; Ohtake, F; Minehata, K; Yasuda, H; Suzuki, T; Kato, S; Imai, Y
The Journal of biological chemistry 287 12994-3004 2012
Osteoclastogenesis is a highly regulated process governed by diverse classes of regulators. Among them, nuclear factor of activated T-cells calcineurin-dependent 1 (NFATc1) is the primary osteoclastogenic transcription factor, and its expression is transcriptionally induced during early osteoclastogenesis by receptor activation of nuclear factor κB ligand (RANKL), an osteoclastogenic cytokine. Here, we report the novel enzymatic function of JMJD5, which regulates NFATc1 protein stability. Among the tested Jumonji C (JmjC) domain-containing proteins, decreased mRNA expression levels during osteoclastogenesis were found for JMJD5 in RAW264 cells stimulated by RANKL. To examine the functional role of JMJD5 in osteoclast differentiation, we established stable JMJD5 knockdown cells, and osteoclast formation was assessed. Down-regulated expression of JMJD5 led to accelerated osteoclast formation together with induction of several osteoclast-specific genes such as Ctsk and DC-STAMP, suggesting that JMJD5 is a negative regulator in osteoclast differentiation. Although JMJD5 was recently reported as a histone demethylase for histone H3K36me2, no histone demethylase activity was detected in JMJD5 in vitro or in living cells, even for other methylated histone residues. Instead, JMJD5 co-repressed transcriptional activity by destabilizing NFATc1 protein. Protein hydroxylase activity mediated by the JmjC domain in JMJD5 was required for the observed functions of JMJD5. JMJD5 induced the association of hydroxylated NFATc1 with the E3 ubiquitin ligase Von Hippel-Lindau tumor suppressor (VHL), thereby presumably facilitating proteasomal degradation of NFATc1 via ubiquitination. Taken together, the present study demonstrated that JMJD5 is a post-translational co-repressor for NFATc1 that attenuates osteoclastogenesis.
|KNOX1 is expressed and epigenetically regulated during in vitro conditions in Agave spp.|
De-la-Peña, C; Nic-Can, G; Ojeda, G; Herrera-Herrera, JL; López-Torres, A; Wrobel, K; Robert-Díaz, ML
BMC plant biology 12 203 2012
The micropropagation is a powerful tool to scale up plants of economical and agronomical importance, enhancing crop productivity. However, a small but growing body of evidence suggests that epigenetic mechanisms, such as DNA methylation and histone modifications, can be affected under the in vitro conditions characteristic of micropropagation. Here, we tested whether the adaptation to different in vitro systems (Magenta boxes and Bioreactors) modified epigenetically different clones of Agave fourcroydes and A. angustifolia. Furthermore, we assessed whether these epigenetic changes affect the regulatory expression of KNOTTED1-like HOMEOBOX (KNOX) transcription factors.To gain a better understanding of epigenetic changes during in vitro and ex vitro conditions in Agave fourcroydes and A. angustifolia, we analyzed global DNA methylation, as well as different histone modification marks, in two different systems: semisolid in Magenta boxes (M) and temporary immersion in modular Bioreactors (B). No significant difference was found in DNA methylation in A. fourcroydes grown in either M or B. However, when A. fourcroydes was compared with A. angustifolia, there was a two-fold difference in DNA methylation between the species, independent of the in vitro system used. Furthermore, we detected an absence or a low amount of the repressive mark H3K9me2 in ex vitro conditions in plants that were cultured earlier either in M or B. Moreover, the expression of AtqKNOX1 and AtqKNOX2, on A. fourcroydes and A. angustifolia clones, is affected during in vitro conditions. Therefore, we used Chromatin ImmunoPrecipitation (ChIP) to know whether these genes were epigenetically regulated. In the case of AtqKNOX1, the H3K4me3 and H3K9me2 were affected during in vitro conditions in comparison with AtqKNOX2.Agave clones plants with higher DNA methylation during in vitro conditions were better adapted to ex vitro conditions. In addition, A. fourcroydes and A. angustifolia clones displayed differential expression of the KNOX1 gene during in vitro conditions, which is epigenetically regulated by the H3K4me3 and H3K9me2 marks. The finding of an epigenetic regulation in key developmental genes will make it important in future studies to identify factors that help to find climate-resistant micropropagated plants.
|The histone demethylase Jhdm1a regulates hepatic gluconeogenesis.|
Pan, D; Mao, C; Zou, T; Yao, AY; Cooper, MP; Boyartchuk, V; Wang, YX
PLoS genetics 8 e1002761 2012
Hepatic gluconeogenesis is required for maintaining blood glucose homeostasis; yet, in diabetes mellitus, this process is unrestrained and is a major contributor to fasting hyperglycemia. To date, the impacts of chromatin modifying enzymes and chromatin landscape on gluconeogenesis are poorly understood. Through catalyzing the removal of methyl groups from specific lysine residues in the histone tail, histone demethylases modulate chromatin structure and, hence, gene expression. Here we perform an RNA interference screen against the known histone demethylases and identify a histone H3 lysine 36 (H3K36) demethylase, Jhdm1a, as a key negative regulator of gluconeogenic gene expression. In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level. Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity. Mechanistically, we find that Jhdm1a regulates the expression of a major gluconeogenic regulator, C/EBPα. This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus. Our work provides compelling evidence that links histone demethylation to transcriptional regulation of gluconeogenesis and has important implications for the treatment of diabetes.
|Arabidopsis HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1.|
To, TK; Kim, JM; Matsui, A; Kurihara, Y; Morosawa, T; Ishida, J; Tanaka, M; Endo, T; Kakutani, T; Toyoda, T; Kimura, H; Yokoyama, S; Shinozaki, K; Seki, M
PLoS genetics 7 e1002055 2011
Heterochromatin silencing is pivotal for genome stability in eukaryotes. In Arabidopsis, a plant-specific mechanism called RNA-directed DNA methylation (RdDM) is involved in heterochromatin silencing. Histone deacetylase HDA6 has been identified as a component of such machineries; however, its endogenous targets and the silencing mechanisms have not been analyzed globally. In this study, we investigated the silencing mechanism mediated by HDA6. Genome-wide transcript profiling revealed that the loci silenced by HDA6 carried sequences corresponding to the RDR2-dependent 24-nt siRNAs, however their transcript levels were mostly unaffected in the rdr2 mutant. Strikingly, we observed significant overlap of genes silenced by HDA6 to those by the CG DNA methyltransferase MET1. Furthermore, regardless of dependence on RdDM pathway, HDA6 deficiency resulted in loss of heterochromatic epigenetic marks and aberrant enrichment for euchromatic marks at HDA6 direct targets, along with ectopic expression of these loci. Acetylation levels increased significantly in the hda6 mutant at all of the lysine residues in the H3 and H4 N-tails, except H4K16. Interestingly, we observed two different CG methylation statuses in the hda6 mutant. CG methylation was sustained in the hda6 mutant at some HDA6 target loci that were surrounded by flanking DNA-methylated regions. In contrast, complete loss of CG methylation occurred in the hda6 mutant at the HDA6 target loci that were isolated from flanking DNA methylation. Regardless of CG methylation status, CHG and CHH methylation were lost and transcriptional derepression occurred in the hda6 mutant. Furthermore, we show that HDA6 binds only to its target loci, not the flanking methylated DNA, indicating the profound target specificity of HDA6. We propose that HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1, possibly recruiting MET1 to specific loci, thus forming the foundation of silent chromatin structure for subsequent non-CG methylation.