07-360 Sigma-AldrichAnti-acetyl-Histone H3 (Lys27) Antibody

MAF1 represses Pol III-mediated transcription by interfering with TFIIIB and Pol III. Herein, we found that MAF1 knockdown induced CDKN1A transcription and chromatin looping concurrently with Pol III recruitment. Simultaneous knockdown of MAF1 with Pol III or BRF1 (subunit of TFIIIB) diminished the activation and looping effect, which indicates that recruiting Pol III was required for activation of Pol II-mediated transcription and chromatin looping. Chromatin-immunoprecipitation analysis after MAF1 knockdown indicated enhanced binding of Pol III and BRF1, as well as of CFP1, p300, and PCAF, which are factors that mediate active histone marks, along with the binding of TATA binding protein (TBP) and POLR2E to the CDKN1A promoter. Simultaneous knockdown with Pol III abolished these regulatory events. Similar results were obtained for GDF15. Our results reveal a novel mechanism by which MAF1 and Pol III regulate the activity of a protein-coding gene transcribed by Pol II.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGF-R) are central regulators of vasculogenesis, angiogenesis, and lymphangiogenesis. They contribute to many vascular-related pathologies, and hence VEGF-targeted therapies have been widely sought after. In this study, the authors investigated the ability of promoter-targeted small hairpin RNAs (shRNAs) to regulate VEGF-A, VEGF-C and VEGF-R1 in different cell lines. The authors identified shRNAs that can upregulate hVEGF-C at both the mRNA and protein levels, and differentially regulate hVEGF-A depending on the cell type. Likewise, the authors identified shRNA that downregulated VEGF-R1 gene expression. Hence, promoter-targeted shRNAs can affect endogenous gene expression not only bimodally, but also differentially in a cell-type specific manner. Importantly, all three genes tested were regulated by at least one shRNA, supporting the idea that nuclear RNA interference is a widespread phenomenon. The level of regulation across the panel of shRNAs varied maximally from a 2.2-fold increase to a 4-fold decrease. This level of change should be useful in fine-tuning and modulating target gene expression, which for potent molecules, such as VEGF-A and VEGF-C, can be very beneficial. These promoter-targeted shRNAs may facilitate the design and development of targeted, context-dependent strategies for both pro- and antiangiogenic therapies for the treatment of vascular-related pathologies.

The class II transactivator (CIITA) is essential for the expression of major histocompatibility complex class II (MHC-II) genes; however, the role of CIITA in gene regulation outside of MHC-II biology is not fully understood. To comprehensively map CIITA-bound loci, ChIP-seq was performed in the human B lymphoblastoma cell line Raji. CIITA bound 480 sites, and was significantly enriched at active promoters and enhancers. The complexity of CIITA transcriptional regulation of target genes was analyzed using a combination of CIITA-null cells, including a novel cell line created using CRISPR/Cas9 tools. MHC-II genes and a few novel genes were regulated by CIITA; however, most other genes demonstrated either diminished or no changes in the absence of CIITA. Nearly all CIITA-bound sites were within regions containing accessible chromatin, and CIITA's presence at these sites was associated with increased histone H3K27 acetylation, suggesting that CIITA's role at these non-regulated loci may be to poise the region for subsequent regulation. Computational genome-wide modeling of the CIITA bound XY box motifs provided constraints for sequences associated with CIITA-mediated gene regulation versus binding. These data therefore define the CIITA regulome in B cells and establish sequence specificities that predict activity for an essential regulator of the adaptive immune response.

CCCTC-binding factor (CTCF) is a key regulator of nuclear chromatin structure and gene regulation. The impact of CTCF on transcriptional output is highly varied, ranging from repression to transcriptional pausing and transactivation. The multifunctional nature of CTCF may be directed solely through remodeling chromatin architecture. However, another hypothesis is that the multifunctional nature of CTCF is mediated, in part, through differential association with protein partners having unique functions. Consistent with this hypothesis, our mass spectrometry analyses of CTCF interacting partners reveal a previously undefined association with the transcription factor general transcription factor II-I (TFII-I). Biochemical fractionation of CTCF indicates that a distinct CTCF complex incorporating TFII-I is assembled on DNA. Unexpectedly, we found that the interaction between CTCF and TFII-I is essential for directing CTCF to the promoter proximal regulatory regions of target genes across the genome, particularly at genes involved in metabolism. At genes coregulated by CTCF and TFII-I, we find knockdown of TFII-I results in diminished CTCF binding, lack of cyclin-dependent kinase 8 (CDK8) recruitment, and an attenuation of RNA polymerase II phosphorylation at serine 5. Phenotypically, knockdown of TFII-I alters the cellular response to metabolic stress. Our data indicate that TFII-I directs CTCF binding to target genes, and in turn the two proteins cooperate to recruit CDK8 and enhance transcription initiation.

The transcription factor Pax6 is comprised of the paired domain (PD) and homeodomain (HD). In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific subpopulations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification. Pax6 also regulates the entire lens developmental program. To reconstruct Pax6-dependent gene regulatory networks (GRNs), ChIP-seq studies were performed using forebrain and lens chromatin from mice. A total of 3514 (forebrain) and 3723 (lens) Pax6-containing peaks were identified, with ∼70% of them found in both tissues and thereafter called 'common' peaks. Analysis of Pax6-bound peaks identified motifs that closely resemble Pax6-PD, Pax6-PD/HD and Pax6-HD established binding sequences. Mapping of H3K4me1, H3K4me3, H3K27ac, H3K27me3 and RNA polymerase II revealed distinct types of tissue-specific enhancers bound by Pax6. Pax6 directly regulates cortical neurogenesis through activation (e.g. Dmrta1 and Ngn2) and repression (e.g. Ascl1, Fezf2, and Gsx2) of transcription factors. In lens, Pax6 directly regulates cell cycle exit via components of FGF (Fgfr2, Prox1 and Ccnd1) and Wnt (Dkk3, Wnt7a, Lrp6, Bcl9l, and Ccnd1) signaling pathways. Collectively, these studies provide genome-wide analysis of Pax6-dependent GRNs in lens and forebrain and establish novel roles of Pax6 in organogenesis.

Emerging evidence has led to considerable interest in the role of Traf2- and Nck-interacting kinase (TNIK) in polycystic ovary syndrome (PCOS) development. However, the epigenetic mechanism regulating TNIK transcription remains largely unknown. Here, we show that (i) TNIK mRNA expression is significantly increased in PCOS ovarian tissues, compared to normal ovarian tissues; (ii) PCOS ovarian tissues exhibit a hypermethylation pattern at the cg10180092 site, (iii) and cg10180092 is the critical site for the transcriptional regulation of TNIK. Mechanistically, hypermethylated cg10180092 site-mediated loss of holocarboxylase synthetase (HLCS)-related H3K9me enrichment activated TNIK transcription in PCOS ovarian tissues. Notably, a substantial body of evidence indicates that DNA hypermethylation is an alternative mechanism for gene inactivation, and a new role for DNA hypermethylationmediated TNIK activating was observed in this study. This may improve our understanding of divergent transcriptional regulation in the initiation and progression of TNIK-related PCOS.

KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. Here, we demonstrate that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. Our results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification.

The molecular pathogenesis of autism is complex and involves numerous genomic, epigenomic, proteomic, metabolic, and physiological alterations. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. The goal of this study is to investigate key molecular alterations postulated to play a role in autism and their role in the pathophysiology of autism. In this study we demonstrate that DNA isolated from the cerebellum of BTBR T+tf/J mice, a relevant mouse model of autism, and from human post-mortem cerebellum of individuals with autism, are both characterized by an increased levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC). The increase in 8-oxodG and 5mC content was associated with a markedly reduced expression of the 8-oxoguanine DNA-glycosylase 1 (Ogg1) and increased expression of de novo DNA methyltransferases 3a and 3b (Dnmt3a and Dnmt3b). Interestingly, a rise in the level of 5hmC occurred without changes in the expression of ten-eleven translocation expression 1 (Tet1) and Tet2 genes, but significantly correlated with the presence of 8-oxodG in DNA. This finding and similar elevation in 8-oxodG in cerebellum of individuals with autism and in the BTBR T+tf/J mouse model warrant future large-scale studies to specifically address the role of OGG1 alterations in pathogenesis of autism.

We have utilized retinoic acid receptor γ (gamma) knockout (RARγ(-/-)) embryonic stem (ES) cells as a model system to analyze RARγ mediated transcriptional regulation of stem cell differentiation. Most of the transcripts regulated by all-trans retinoic acid (RA) in ES cells are dependent upon functional RARγ signaling. Notably, many of these RA-RARγ target genes are implicated in retinoid uptake and metabolism. For instance, Lrat (lecithin:retinol acyltransferase), Stra6 (stimulated by retinoic acid 6), Crabp2 (cellular retinoic acid binding protein 2), and Cyp26a1 (cytochrome p450 26a1) transcripts are induced in wild type (WT), but not in RARγ(-/-) cells. Transcripts for the transcription factors Pbx1 (pre-B cell leukemia homeobox-1), Wt1 (Wilm's tumor gene-1), and Meis1 (myeloid ecotropic viral integration site-1) increase upon RA treatment of WT, but not RARγ(-/-) cells. In contrast, Stra8, Dleu7, Leftb, Pitx2, and Cdx1 mRNAs are induced by RA even in the absence of RARγ. Mapping of the epigenetic signature of Meis1 revealed that RA induces a rapid increase in the H3K9/K14ac epigenetic mark at the proximal promoter and at two sites downstream of the transcription start site in WT, but not in RARγ(-/-) cells. Thus, RA-associated increases in H3K9/K14ac epigenetic marks require RARγ and are associated with increased Meis1 transcript levels, whereas H3K4me3 is present at the Meis1 proximal promoter even in the absence of RARγ. In contrast, at the Lrat proximal promoter primarily the H3K4me3 mark, and not the H3K9/K14ac mark, increases in response to RA, independently of the presence of RARγ. Our data show major epigenetic changes associated with addition of the RARγ agonist RA in ES cells.

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.