06-866 Sigma-AldrichAnti-acetyl-Histone H4 Antibody

BRCA1 and 53BP1 antagonistically regulate homology-directed repair (HDR) and non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB). The histone deacetylase (HDAC) inhibitor trichostatin A directly inhibits the retention of 53BP1 at DSB sites by acetylating histone H4 (H4ac), which interferes with 53BP1 binding to dimethylated histone H4 Lys20 (H4K20me2). Conversely, we recently found that the retention of the BRCA1/BARD1 complex is also affected by another methylated histone residue, H3K9me2, which can be suppressed by the histone lysine methyltransferase (HKMT) inhibitor UNC0638. Here, we investigate the effects of the class I HDAC inhibitors MS-275 and FK228 compared to UNC0638 on histone modifications and the DNA damage response. In addition to H4ac, the HDAC inhibitors induce H3K9ac and inhibit H3K9me2 at doses that do not affect the expression levels of DNA repair genes. By contrast, UNC0638 selectively inhibits H3K9me2 without affecting the levels of H3K9ac, H3K56ac or H4ac. Reflecting their effects on histone modifications, the HDAC inhibitors inhibit ionizing radiation-induced foci (IRIF) formation of BRCA1 and BARD1 as well as 53BP1 and RIF1, whereas UNC0638 suppresses IRIF formation of BRCA1 and BARD1 but not 53BP1 and RIF1. Although HDAC inhibitors suppressed HDR, they did not cooperate with the poly(ADP-ribose) polymerase inhibitor olaparib to block cancer cell growth, possibly due to simultaneous suppression of NHEJ pathway components. Collectively, these results suggest the mechanism by that HDAC inhibitors inhibit both the HDR and NHEJ pathways, whereas HKMT inhibitor inhibits only the HDR pathway; this finding may affect the chemosensitizing effects of the inhibitors.

Mesenchymal stem cells (MSCs) are highly plastic cells that are able to transdifferentiate or dedifferentiate under appropriate conditions. In the present study, we reported here that after in vitro induction of osteogenic differentiation, MSCs could be reverted to a primitive stem cell population (dedifferentiated osteogenic MSCs, De-Os-MSCs) with improved cell survival, colony formation, osteogenic potential, migratory capacity and increased expression of Nanog, Oct4 and Sox2. Most importantly, our results showed great superiority of the De-Os-MSCs over untreated MSCs in ectopic bone formation in vivo. Furthermore, Nanog-knockdown in MSCs could reverse these enhanced properties in De-Os-MSCs in vitro, indicating a central role of Nanog in the transcriptional network. In addition, epigenetic regulations including DNA methylation and histone modifications may play important roles in regulating the de-osteogenic differentiation process. And we found decreased methylation and promoter accrual of activating histone marks, such as H3K4me3 and H4ac on both Nanog and Oct4 gene promoters. Taken together, our study demonstrated that epigenetic memory in De-Os-MSCs gained by priming with osteogenic induction medium favored their differentiation along osteoblastic lineage with improved cell survival and migratory abilities, which may have application potential in enhancing their regenerative capacity in mammals.

Epigenetic regulation of gene expression is critical for controlling embryonic properties during the embryo-to-seedling phase transition. Here we report that a histone deacetylase19 (HDA19)-associated regulator, scarecrow-like15 (SCL15), is essential for repressing the seed maturation programme in vegetative tissues. SCL15 is expressed in and GFP-tagged SCL15 predominantly localizes to, the vascular bundles particularly in the phloem companion cells and neighbouring specialized cells. Mutation of SCL15 leads to a global shift in gene expression in seedlings to a profile resembling late embryogenesis in seeds. In scl15 seedlings, many genes involved in seed maturation are markedly derepressed with concomitant accumulation of seed 12S globulin; this is correlated with elevated levels of histone acetylation at a subset of seed-specific loci. SCL15 physically interacts with HDA19 and direct targets of HDA19-SCL15 association are identified. These studies reveal that SCL15 acts as an HDA19-associated regulator to repress embryonic traits in seedlings.

HDAC isoform-specific inhibitors may improve the therapeutic window while limiting toxicities. Developing inhibitors against class I isoforms poses difficulties as they share high homology among their catalytic sites; however, HDAC8 is structurally unique compared to other class I isoforms. HDAC8 inhibitors are novel compounds and have affinity for class I HDAC isoforms demonstrating anti-cancer effects; little is known about their activity in malignant peripheral nerve sheath tumors (MPNST). Recently, we demonstrated anti-MPNST efficacy of HDAC8i in human and murine-derived MPNST pre-clinical models; we now seek to consider the potential therapeutic inhibition of HDAC8 in MPNST.Four Human MPNST cell lines, a murine-derived MPNST cell line, and two HDAC8 inhibitors (PCI-34051, PCI-48012; Pharmacyclics, Inc. Sunnyvale, CA) were studied. Proliferation was determined using MTS and clonogenic assays. Effects on cell cycle were determined via PI FACS analysis; effects on apoptosis were determined using Annexin V-PI FACS analysis and cleaved caspase 3 expression. In vivo growth effects of HDAC8i were evaluated using MPNST xenograft models. 2D gel electrophoresis and mass spectrometry were used to identify potential HDAC8 deacetylation substrates.HDAC8i induced cell growth inhibition and marked S-phase cell cycle arrest in human and murine-derived MPNST cells. Relative to control, HDAC8i induced apoptosis in both human and murine-derived MPNST cells. HDAC8i exhibited significant effects on MPNST xenograft growth (p=0.001) and tumor weight (p=0.02). Four potential HDAC8 substrate targets were identified using a proteomic approach: PARK7, HMGB1, PGAM1, PRDX6.MPNST is an aggressive sarcoma that is notoriously therapy-resistant, hence the urgent need for improved anti-MPNST therapies. HDAC8 inhibition may be useful for MPNST by improving efficacy while limiting toxicities as compared to pan-HDACis.

In addition to osteoblast lineage cells, the TNF-like factor receptor activator of NF-κB ligand (RANKL) is expressed in both B and T cells and may play a role in bone resorption. Rankl gene (Tnfsf11) expression in mouse T cells is mediated through multiple distal elements marked by increased transcription factor occupancy, histone tail acetylation, and RNA polymerase II recruitment. Little is known, however, of the regulation of human TNFSF11 in T cells. Accordingly, we examined the consequence of T cell activation on the expression of this factor both in Jurkat cells and in primary human T cells. We then explored the mechanism of this regulation by scanning over 400 kb of DNA surrounding the TNFSF11 locus for regulatory enhancers using ChIP-chip analysis. Histone H3/H4 acetylation enrichment identified putative regulatory regions located between -170 and -220 kb upstream of the human TNFSF11 TSS that we designated the human T cell control region (hTCCR). This region showed high sequence conservation with the mouse TCCR. Inhibition of MEK1/2 by U0126 resulted in decreased RANKL expression suggesting that stimulation through MEK1/2 was a prerequisite. ChIP-chip analysis also revealed that c-FOS was recruited to the hTCCR as well. Importantly, both the human TNFSF11 D5a/b (RLD5a/b) enhancer and segments of the hTCCR mediated robust inducible reporter activity following TCR activation. Finally, SNPs implicated in diseases characterized by dysregulated BMD co-localized to the hTCCR region. We conclude that the hTCCR region contains a cell-selective set of enhancers that plays an integral role in the transcriptional regulation of the TNFSF11 gene in human T cells.

NUT midline carcinoma (NMC) is a rare but highly aggressive cancer typically caused by the translocation t(15;19), which results in the formation of the BRD4-NUT fusion oncoprotein. Previous studies have demonstrated that fusion of the NUT protein with the double bromodomains of BRD4 may significantly alter the cellular gene expression profile to contribute to NMC tumorigenesis. However, the mechanistic details of this BRD4-NUT function remain poorly understood. In this study, we examined the NUT function in transcriptional regulation by targeting it to a LacO transgene array integrated in U2OS 2-6-3 cells, which allow us to visualize how NUT alters the in situ gene transcription dynamic. Using this system, we demonstrated that the NUT protein tethered to the LacO locus recruits p300/CREB-binding protein (CBP), induces histone hyperacetylation, and enriches BRD4 to the transgene array chromatin foci. We also discovered that, in BRD4-NUT expressed in NMC cells, the NUT moiety of the fusion protein anchored to chromatin by the double bromodomains also stimulates histone hyperacetylation, which causes BRD4 to bind tighter to chromatin. Consequently, multiple BRD4-interacting factors are recruited to the NUT-associated chromatin locus to activate in situ transgene expression. This gene transcription function was repressed by either expression of a dominant negative inhibitor of the p300-NUT interaction or treatment with (+)-JQ1, which dissociates BRD4 from the LacO chromatin locus. Our data support a model in which BRD4-NUT-stimulated histone hyperacetylation recruits additional BRD4 and interacting partners to support transcriptional activation, which underlies the BRD4-NUT oncogenic mechanism in NMC.

Aberrant Notch activity is oncogenic in several malignancies, but it is unclear how expression or function of downstream elements in the Notch pathway affects tumor growth. Transcriptional regulation by Notch is dependent on interaction with the DNA-binding transcriptional repressor, RBPJ, and consequent derepression or activation of associated gene promoters. We show here that RBPJ is frequently depleted in human tumors. Depletion of RBPJ in human cancer cell lines xenografted into immunodeficient mice resulted in activation of canonical Notch target genes, and accelerated tumor growth secondary to reduced cell death. Global analysis of activated regions of the genome, as defined by differential acetylation of histone H4 (H4ac), revealed that the cell death pathway was significantly dysregulated in RBPJ-depleted tumors. Analysis of transcription factor binding data identified several transcriptional activators that bind promoters with differential H4ac in RBPJ-depleted cells. Functional studies demonstrated that NF-κB and MYC were essential for survival of RBPJ-depleted cells. Thus, loss of RBPJ derepresses target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumorigenesis.

During spermiogenesis, the postmeiotic phase of mammalian spermatogenesis, transcription is progressively repressed as nuclei of haploid spermatids are compacted through a dramatic chromatin reorganization involving hyperacetylation and replacement of most histones with protamines. Although BRDT functions in transcription and histone removal in spermatids, it is unknown whether other BET family proteins play a role. Immunofluorescence of spermatogenic cells revealed BRD4 in a ring around the nuclei of spermatids containing hyperacetylated histones. The ring lies directly adjacent to the acroplaxome, the cytoskeletal base of the acrosome, previously linked to chromatin reorganization. The BRD4 ring does not form in acrosomal mutant mice. Chromatin immunoprecipitation followed by sequencing in spermatids revealed enrichment of BRD4 and acetylated histones at the promoters of active genes. BRD4 and BRDT show distinct and synergistic binding patterns, with a pronounced enrichment of BRD4 at spermatogenesis-specific genes. Direct association of BRD4 with acetylated H4 decreases in late spermatids as acetylated histones are removed from the condensing nucleus in a wave following the progressing acrosome. These data provide evidence of a prominent transcriptional role for BRD4 and suggest a possible removal mechanism for chromatin components from the genome via the progressing acrosome as transcription is repressed and chromatin is compacted during spermiogenesis.

Recent evidence in adipocytes points to a role for synuclein-γ in metabolism and lipid droplet dynamics, but interestingly this factor is also robustly expressed in peripheral neurons. Specific regulation of the synuclein-γ gene (Sncg) by PPARγ requires further evaluation, especially in peripheral neurons, prompting us to test if Sncg is a bona fide PPARγ target in murine adipocytes and peripheral somatosensory neurons derived from the dorsal root ganglia (DRG). Sncg mRNA was decreased in 3T3-L1 adipocytes (~68%) by rosiglitazone, and this effect was diminished by the PPARγ antagonist T0070907. Chromatin immunoprecipitation experiments confirmed PPARγ protein binding at two promoter sequences of Sncg during 3T3-L1 adipogenesis. Rosiglitazone did not affect Sncg mRNA expression in murine cultured DRG neurons. In subcutaneous human WAT samples from two cohorts treated with pioglitazone (greater than 11 wks), SNCG mRNA expression was reduced, albeit highly variable and most evident in type 2 diabetes. Leptin (Lep) expression, thought to be coordinately-regulated with Sncg based on correlations in human adipose tissue, was also reduced in 3T3-L1 adipocytes by rosiglitazone. However, Lep was unaffected by PPARγ antagonist, and the LXR agonist T0901317 significantly reduced Lep expression (~64%) while not impacting Sncg. The results support the concept that synuclein-γ shares some, but not all, gene regulators with leptin and is a PPARγ target in adipocytes but not DRG neurons. Regulation of synuclein-γ by cues such as PPARγ agonism in adipocytes is logical based on recent evidence for an important role for synuclein-γ in the maintenance and dynamics of adipocyte lipid droplets.

Signal transducer and activator of transcription STAT5 is essential for the regulation of proliferation and survival genes. Its activity is tightly regulated through cytokine signaling and is often upregulated in cancer. We showed previously that the deacetylase inhibitor trichostatin A (TSA) inhibits STAT5-mediated transcription by preventing recruitment of the transcriptional machinery at a step following STAT5 binding to DNA. The mechanism and factors involved in this inhibition remain unknown. We now show that deacetylase inhibitors do not target STAT5 acetylation, as we initially hypothesized. Instead, they induce a rapid increase in global histone acetylation apparently resulting in the delocalization of the bromodomain and extra-terminal (BET) protein Brd2 and of the Brd2-associated factor TBP to hyperacetylated chromatin. Treatment with the BET inhibitor (+)-JQ1 inhibited expression of STAT5 target genes, supporting a role of BET proteins in the regulation of STAT5 activity. Accordingly, chromatin immunoprecipitation demonstrated that Brd2 is associated with the transcriptionally active STAT5 target gene Cis and is displaced upon TSA treatment. Our data therefore indicate that Brd2 is required for the proper recruitment of the transcriptional machinery at STAT5 target genes and that deacetylase inhibitors suppress STAT5-mediated transcription by interfering with Brd2 function.