Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H||IF, WB, ICC, ChIP-seq, DB, Mplex, ChIP||Rb||Culture Supernatant||Monoclonal Antibody|
|Presentation||Cultured supernantant in 0.05% sodium azide.|
|Safety Information according to GHS|
|Material Size||100 µL|
|Reference overview||Application||Pub Med ID|
|Genome accessibility is widely preserved and locally modulated during mitosis.|
Hsiung, CC; Morrissey, CS; Udugama, M; Frank, CL; Keller, CA; Baek, S; Giardine, B; Crawford, GE; Sung, MH; Hardison, RC; Blobel, GA
Genome research 25 213-25 2015
Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that such mitotically retained molecular signatures could provide transcriptional memory through mitosis. To understand the role of chromatin structure in mitotic memory, we performed the first genome-wide comparison of DNase I sensitivity of chromatin in mitosis and interphase, using a murine erythroblast model. Despite chromosome condensation during mitosis visible by microscopy, the landscape of chromatin accessibility at the macromolecular level is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly DNase hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility more strongly, whereas distal regulatory elements tend to lose accessibility. Large domains of DNA hypomethylation mark a subset of promoters that retain accessibility during mitosis and across many cell types in interphase. Erythroid transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but has little influence on mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis, but are modulated at the level of individual genes and regulatory elements.
|A mex3 homolog is required for differentiation during planarian stem cell lineage development.|
Zhu, SJ; Hallows, SE; Currie, KW; Xu, C; Pearson, BJ
eLife 4 2015
Neoblasts are adult stem cells (ASCs) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving postmitotic fates remain poorly defined. In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 was required for generating differentiated cells of multiple lineages, while restricting the size of the stem cell compartment. We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment.
|Picropodophyllin causes mitotic arrest and catastrophe by depolymerizing microtubules via insulin-like growth factor-1 receptor-independent mechanism.|
Waraky, A; Akopyan, K; Parrow, V; Strömberg, T; Axelson, M; Abrahmsén, L; Lindqvist, A; Larsson, O; Aleem, E
Oncotarget 5 8379-92 2014
Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest. The mitotic block was induced in human cancer cell lines and in an A549 xenograft mouse but did not occur in normal hepatocytes/mouse tissues. Cell cycle arrest by PPP occurred in vitro and in vivo accompanied by prominent CDK1 activation, and was IGF-1R-independent since it occurred also in IGF-1R-depleted and null cells. The tumor cells were not arrested in G2/M but in mitosis. Centrosome separation was prevented during mitotic entry, resulting in a monopolar mitotic spindle with subsequent prometaphase-arrest, independent of Plk1/Aurora A or Eg5, and leading to cell features of mitotic catastrophe. PPP also increased soluble tubulin and decreased spindle-associated tubulin within minutes, indicating that it interfered with microtubule dynamics. These results provide a novel IGF-1R-independent mechanism of antitumor effects of PPP.
|Heritable loss of replication control of a minichromosome derived from the B chromosome of maize.|
Masonbrink, RE; Fu, S; Han, F; Birchler, JA
Genetics 193 77-84 2013
During an accumulation regime of a small telomere-truncated B chromosome, a derivative with large variations in size and multiple punctate centromere loci exhibiting amplified copy numbers was discovered. Multiple centromere satellite loci or transgene signals were documented in amplified chromosomes, suggesting over-replication. Immunolocalization studies revealed multiple foci of biochemical markers characteristic of active centromeres such as CENP-C and phosphorylation of histones H3S10 and H2AThr133. The amplified chromosomes exhibit an absence of chromosome disjunction in meiosis I and an infrequent chromosome disjunction in meiosis II. Despite their unusual structure and behavior these chromosomes were observed in the lineage for seven generations during the course of this study. While severely truncated relative to a normal B chromosome, the progenitor minichromosome is estimated to be at least several megabases in size. Given that the centromere and transgene signals at opposite ends of the chromosome generally match in copy number, the replication control is apparently lost over several megabases.
|Transmembrane voltage potential is an essential cellular parameter for the detection and control of tumor development in a Xenopus model.|
Chernet, BT; Levin, M
Disease models & mechanisms 6 595-607 2013
Understanding mechanisms that orchestrate cell behavior into appropriately patterned tissues and organs within the organism is an essential element of preventing, detecting and treating cancer. Bioelectric signals (resting transmembrane voltage potential gradients in all cells) underlie an important and broadly conserved set of control mechanisms that regulate pattern formation. We tested the role of transmembrane potential in tumorigenesis mediated by canonical oncogenes in Xenopus laevis. Depolarized membrane potential (Vmem) was a characteristic of induced tumor-like structures (ITLSs) generated by overexpression of Gli1, Kras(G12D), Xrel3 or p53(Trp248). This bioelectric signature was also present in precursor ITLS sites. Vmem is a bioelectric marker that reveals ITLSs before they become histologically and morphologically apparent. Moreover, voltage was functionally important: overexpression of hyperpolarizing ion transporters caused a return to normal Vmem and significantly reduced ITLS formation in vivo. To characterize the molecular mechanism by which Vmem change regulates ITLS phenotypes, we performed a suppression screen. Vmem hyperpolarization was transduced into downstream events via Vmem-regulated activity of SLC5A8, a sodium-butyrate exchanger previously implicated in human cancer. These data indicate that butyrate, a histone deacetylase (HDAC) inhibitor, might be responsible for transcriptional events that mediate suppression of ITLSs by hyperpolarization. Vmem is a convenient cellular parameter by which tumors induced by human oncogenes can be detected in vivo and represents a new diagnostic modality. Moreover, control of resting membrane potential is functionally involved in the process by which oncogene-bearing cells depart from normal morphogenesis programs to form tumors. Modulation of Vmem levels is a novel and promising strategy for tumor normalization.
|The superhealing MRL background improves muscular dystrophy.|
Heydemann, A; Swaggart, KA; Kim, GH; Holley-Cuthrell, J; Hadhazy, M; McNally, EM
Skeletal muscle 2 26 2012
Mice from the MRL or "superhealing" strain have enhanced repair after acute injury to the skin, cornea, and heart. We now tested an admixture of the MRL genome and found that it altered the course of muscle pathology and cardiac function in a chronic disease model of skeletal and cardiac muscle. Mice lacking γ-sarcoglycan (Sgcg), a dystrophin-associated protein, develop muscular dystrophy and cardiomyopathy similar to their human counterparts with limb girdle muscular dystrophy. With disruption of the dystrophin complex, the muscle plasma membrane becomes leaky and muscles develop increased fibrosis.MRL/MpJ mice were bred with Sgcg mice, and cardiac function was measured. Muscles were assessed for fibrosis and membrane leak using measurements of hydroxyproline and Evans blue dye. Quantitative trait locus mapping was conducted using single nucleotide polymorphisms distinct between the two parental strains.Introduction of the MRL genome reduced fibrosis but did not alter membrane leak in skeletal muscle of the Sgcg model. The MRL genome was also associated with improved cardiac function with reversal of depressed fractional shortening and the left ventricular ejection fraction. We conducted a genome-wide analysis of genetic modifiers and found that a region on chromosome 2 was associated with cardiac, diaphragm muscle and abdominal muscle fibrosis.These data are consistent with a model where the MRL genome acts in a dominant manner to suppress fibrosis in this chronic disease setting of heart and muscle disease.
|Sirtuin 1 is a key regulator of the interleukin-12 p70/interleukin-23 balance in human dendritic cells.|
Alvarez, Y; Rodríguez, M; Municio, C; Hugo, E; Alonso, S; Ibarrola, N; Fernández, N; Crespo, MS
The Journal of biological chemistry 287 35689-701 2012
Stimulation of human dendritic cells with the fungal surrogate zymosan produces IL-23 and a low amount of IL-12 p70. Trans-repression of il12a transcription, which encodes IL-12 p35 chain, by proteins of the Notch family and lysine deacetylation reactions have been reported as the underlying mechanisms, but a number of questions remain to be addressed. Zymosan produced the location of sirtuin 1 (SIRT1) to the nucleus, enhanced its association with the il12a promoter, increased the nuclear concentration of the SIRT1 co-substrate NAD(+), and decreased chromatin accessibility in the nucleosome-1 of il12a, which contains a κB-site. The involvement of deacetylation reactions in the inhibition of il12a transcription was supported by the absence of Ac-Lys-14-histone H3 in dendritic cells treated with zymosan upon coimmunoprecipitation of transducin-like enhancer of split. In contrast, we did not obtain evidence of a possible effect of SIRT1 through the deacetylation of c-Rel, the central element of the NF-κB family involved in il12a regulation. These data indicate that an enhancement of SIRT1 activity in response to phagocytic stimuli may reduce the accessibility of c-Rel to the il12a promoter and its transcriptional activation, thus regulating the IL-12 p70/IL-23 balance and modulating the ongoing immune response.
|Systematic dissection of roles for chromatin regulators in a yeast stress response.|
Weiner, A; Chen, HV; Liu, CL; Rahat, A; Klien, A; Soares, L; Gudipati, M; Pfeffner, J; Regev, A; Buratowski, S; Pleiss, JA; Friedman, N; Rando, OJ
PLoS biology 10 e1001369 2012
Packaging of eukaryotic genomes into chromatin has wide-ranging effects on gene transcription. Curiously, it is commonly observed that deletion of a global chromatin regulator affects expression of only a limited subset of genes bound to or modified by the regulator in question. However, in many single-gene studies it has become clear that chromatin regulators often do not affect steady-state transcription, but instead are required for normal transcriptional reprogramming by environmental cues. We therefore have systematically investigated the effects of 83 histone mutants, and 119 gene deletion mutants, on induction/repression dynamics of 170 transcripts in response to diamide stress in yeast. Importantly, we find that chromatin regulators play far more pronounced roles during gene induction/repression than they do in steady-state expression. Furthermore, by jointly analyzing the substrates (histone mutants) and enzymes (chromatin modifier deletions) we identify specific interactions between histone modifications and their regulators. Combining these functional results with genome-wide mapping of several histone marks in the same time course, we systematically investigated the correspondence between histone modification occurrence and function. We followed up on one pathway, finding that Set1-dependent H3K4 methylation primarily acts as a gene repressor during multiple stresses, specifically at genes involved in ribosome biosynthesis. Set1-dependent repression of ribosomal genes occurs via distinct pathways for ribosomal protein genes and ribosomal biogenesis genes, which can be separated based on genetic requirements for repression and based on chromatin changes during gene repression. Together, our dynamic studies provide a rich resource for investigating chromatin regulation, and identify a significant role for the "activating" mark H3K4me3 in gene repression.
|Akt2 interacts with Snail1 in the E-cadherin promoter.|
Villagrasa, P, et al.
Oncogene, (2011) 2011
Snail1 is a transcriptional factor essential for triggering epithelial-to-mesenchymal transition. Moreover, Snail1 promotes resistance to apoptosis, an effect associated to PTEN gene repression and Akt stimulation. In this article we demonstrate that Snail1 activates Akt at an additional level, as it directly binds to and activates this protein kinase. The interaction is observed in the nucleus and increases the intrinsic Akt activity. We determined that Akt2 is the isoform interacting with Snail1, an association that requires the pleckstrin homology domain in Akt2 and the C-terminal half in Snail1. Snail1 enhances the binding of Akt2 to the E-cadherin (CDH1) promoter and Akt2 interference prevents Snail1 repression of CDH1 gene. We also show that Snail1 binding increases Akt2 intrinsic activity on histone H3 and have identified Thr45 as a residue modified on this protein. Phosphorylation of Thr45 in histone H3 is sensitive to Snail1 and Akt2 cellular levels; moreover, Snail1 upregulates the binding of phosphoThr45 histone H3 to the CDH1 promoter. These results uncover an unexpected role of Akt2 in transcriptional control and point out to phosphorylation of Thr45 in histone H3 as a new epigenetic mark related to Snail1 and Akt2 action.Oncogene advance online publication, 12 December 2011; doi:10.1038/onc.2011.562.
|Notch- and transducin-like enhancer of split (TLE)-dependent histone deacetylation explain interleukin 12 (IL-12) p70 inhibition by zymosan.|
Alvarez, Y; Municio, C; Hugo, E; Zhu, J; Alonso, S; Hu, X; Fernández, N; Sánchez Crespo, M
The Journal of biological chemistry 286 16583-95 2011
The fungal analog zymosan induces IL-23 and low amounts of IL-12 p70. This study addresses the molecular mechanisms underlying this cytokine pattern in human monocyte-derived dendritic cells. The transcriptional regulation of il23a, one of the chains of IL-23, depended on the activation of c-Rel and histone H3 phosphorylation, as judged from the association of c-Rel with the il23a promoter and the correlation between IL-23 production and Ser-10-histone H3 phosphorylation. Consistent with its reduced ability to produce IL-12 p70, zymosan induced a transient occupancy of the il12a promoter by c-Rel, blocked the production of IL-12 p70 and the transcription of il12a induced by other stimuli, and triggered the expression and nuclear translocation of the transcriptional repressors of the Notch family hairy and enhancer of split (Hes)-1, Hes5, hairy/enhancer-of-split related with YRPW motif protein (Hey)-1, and transducin-like enhancer of split (TLE). Zymosan also induced the interaction of Hes1 and TLE with histone H3 phosphorylated on Ser-10 and deacetylated on Lys-14. Inhibition of class III histone deacetylases increased the production of IL-12 p70 and partially blunted the inhibitory effect of zymosan on the production of IL-12 p70 elicited by LPS and IFN-γ. These results indicate that the selective induction of IL-23 by β-glucans is explained by the activation of c-Rel associated with Ser-10-histone H3 phosphorylation in the il23a promoter mediated by mitogen- and stress-activated kinase and/or protein kinase A and inhibition of il12a transcription by a mechanism involving activation of several corepressors with the ability to bind TLE and to promote histone deacetylation.