Takashi Shimbo

INO80 Facilitates Pluripotency Gene Activation in Embryonic Stem Cell Self-Renewal, Reprogramming, and Blastocyst Development

The master transcription factors play integral roles in the pluripotency transcription circuitry of embryonic stem cells (ESCs). How they selectively activate expression of the pluripotency network while simultaneously repressing genes involved in differentiation is not fully understood. Here, we define a requirement for the INO80 complex, a SWI/SNF family chromatin remodeler, in ESC self-renewal, somatic cell reprogramming, and blastocyst development. We show that Ino80, the chromatin remodeling ATPase, co-occupies pluripotency gene promoters with the master transcription factors, and its occupancy is dependent on OCT4 and WDR5. At the pluripotency genes, Ino80 maintains an open chromatin architecture and licenses recruitment of Mediator and RNA polymerase II for gene activation. Our data reveal an essential role for INO80 in the expression of the pluripotency network and illustrate the coordination among chromatin remodeler, transcription factor, and histone-modifying enzyme in the regulation of the pluripotent state.

MBD3 Localizes at Promoters, Gene Bodies and Enhancers of Active Genes

The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. Recent reports describing localization of NuRD provide new insights that question previous models on NuRD action, but are not in complete agreement. Here, we provide location analysis of endogenous MBD3, a component of NuRD complex, in two human breast cancer cell lines (MCF-7 and MDA-MB-231) using two independent genomic techniques: DNA adenine methyltransferase identification (DamID) and ChIP-seq. We observed concordance of the resulting genomic localization, suggesting that these studies are converging on a robust map for NuRD in the cancer cell genome. MBD3 preferentially associated with CpG rich promoters marked by H3K4me3 and showed cell-type specific localization across gene bodies, peaking around the transcription start site. A subset of sites bound by MBD3 was enriched in H3K27ac and was in physical proximity to promoters in three-dimensional space, suggesting function as enhancers. MBD3 enrichment was also noted at promoters modified by H3K27me3. Functional analysis of chromatin indicated that MBD3 regulates nucleosome occupancy near promoters and in gene bodies. These data suggest that MBD3, and by extension the NuRD complex, may have multiple roles in fine tuning expression for both active and silent genes, representing an important step in defining regulatory mechanisms by which NuRD complex controls chromatin structure and modification status.

Inhibition of histone binding by supramolecular hosts

The tandem PHD (plant homeodomain) fingers of the CHD4 (chromodomain helicase DNA-binding protein 4) ATPase are epigenetic readers that bind either unmodified histone H3 tails or H3K9me3 (histone H3 trimethylated at Lys⁹). This dual function is necessary for the transcriptional and chromatin remodelling activities of the NuRD (nucleosome remodelling and deacetylase) complex. In the present paper, we show that calixarene-based supramolecular hosts disrupt binding of the CHD4 PHD2 finger to H3K9me3, but do not affect the interaction of this protein with the H3K9me0 (unmodified histone H3) tail. A similar inhibitory effect, observed for the association of chromodomain of HP1γ (heterochromatin protein 1γ) with H3K9me3, points to a general mechanism of methyl-lysine caging by calixarenes and suggests a high potential for these compounds in biochemical applications. Immunofluorescence analysis reveals that the supramolecular agents induce changes in chromatin organization that are consistent with their binding to and disruption of H3K9me3 sites in living cells. The results of the present study suggest that the aromatic macrocyclic hosts can be used as a powerful new tool for characterizing methylation-driven epigenetic mechanisms.

Genome-Wide Binding of MBD2 Reveals Strong Preference for Highly Methylated Loci

MBD2 is a subunit of the NuRD complex that is postulated to mediate gene repression via recruitment of the complex to methylated DNA. In this study we adopted an MBD2 tagging-approach to study its genome wide binding characteristics. We show that in vivo MBD2 is mainly recruited to CpG island promoters that are highly methylated. Interestingly, MBD2 binds around 1 kb downstream of the transcription start site of a subset of ∼400 CpG island promoters that are characterized by the presence of active histone marks, RNA polymerase II (Pol2) and low to medium gene expression levels and H3K36me3 deposition. These tagged-MBD2 binding sites in MCF-7 show increased methylation in a cohort of primary breast cancers but not in normal breast samples, suggesting a putative role for MBD2 in breast cancer.

GATA3-dependent cellular reprogramming requires activation-domain dependent recruitment of a chromatin remodeler

BackgroundTranscription factor-dependent cellular reprogramming is integral to normal development and is central to production of induced pluripotent stem cells. This process typically requires pioneer transcription factors (TFs) to induce de novo formation of enhancers at previously closed chromatin. Mechanistic information on this process is currently sparse.ResultsHere we explore the mechanistic basis by which GATA3 functions as a pioneer TF in a cellular reprogramming event relevant to breast cancer, the mesenchymal to epithelial transition (MET). In some instances, GATA3 binds previously inaccessible chromatin, characterized by stable, positioned nucleosomes where it induces nucleosome eviction, alters local histone modifications, and remodels local chromatin architecture. At other loci, GATA3 binding induces nucleosome sliding without concomitant generation of accessible chromatin. Deletion of the transactivation domain retains the chromatin binding ability of GATA3 but cripples chromatin reprogramming ability, resulting in failure to induce MET.ConclusionsThese data provide mechanistic insights into GATA3-mediated chromatin reprogramming during MET, and suggest unexpected complexity to TF pioneering. Successful reprogramming requires stable binding to a nucleosomal site; activation domain-dependent recruitment of co-factors including BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex; and appropriate genomic context. The resulting model provides a new conceptual framework for de novo enhancer establishment by a pioneer TF.

DNA Methylation Changes in Tbx3 in a Mouse Model Exposed to Polybrominated Diphenyl Ethers

DE-71, a commercial mixture of polybrominated diphenyl ethers widely used in flame retardants, is a pervasive environmental contaminant due to its continuing release from waste material and its long half-life in humans. Although the genotoxic potential of DE-71 appears to be low based on bacterial mutagenicity, it remains a public health concern due to its reported involvement in tumor development. Molecular mechanisms by which DE-71 influences tumor incidence or progression remain understudied. We used liver carcinoma tissue from mice exposed to DE-71 to test the hypothesis that epigenetic alterations consistent with tumor development, specifically DNA methylation, result from long-term DE-71 exposure. We profiled DNA methylation status using the methylated-CpG island recovery assay coupled with microarray analysis of hepatocellular carcinoma DNA from animals exposed to DE-71. DE-71 exposure had little impact on global DNA methylation. However, we detected gene body–specific hypomethylation within the Tbx3 locus, a transcription factor important in liver tumorigenesis and in embryonic and cancer stem cell proliferation. This nonpromoter hypomethylation was accompanied by upregulation of Tbx3 mRNA and protein and by alterations in downstream cell cycle–associated marker expression. Thus, exposure to DE-71 may facilitate tumor development by inducing epigenetic programs that favor expansion of progenitor cell populations.

High-quality ChIP-seq analysis of MBD3 in human breast cancer cells.

Chromatin accessibility is tightly regulated by multiple factors/mechanisms to establish different cell type-specific gene expression programs from a single genome. Dysregulation of this process can lead to diseases including cancer. The Mi-2/nucleosome remodeling and deacetylase (NuRD) complex is thought to orchestrate chromatin structure using its intrinsic nucleosome remodeling and histone deacetylase activities. However, the detailed mechanisms by which the NuRD complex regulates chromatin structure in vivo are not yet known. To explore the regulatory mechanisms of the NuRD complex, we mapped genome-wide localization of MBD3, a structural component of NuRD, in a human breast cancer cell line (MDA-MB-231) using a modified ChIP-seq protocol. Our data showed high quality localization information (i.e., high mapping efficiency and low PCR duplication rate) and excellent consistency between biological replicates. The data are deposited in the Gene Expression Omnibus (GSE76116).

CHD4 and the NuRD complex directly control cardiac sarcomere formation

Significance Birth defects are the leading cause of infant mortality in the United States and Europe, with cardiac defects being the most prevalent. Here we define the requirement and mechanism of action of CHD4, the catalytic core component of the nucleosome remodeling and deacetylase (NuRD) complex, in embryonic heart development. CHD4 is essential from fly to human and mutations in CHD4 are causative to congenital heart disease, including atrial and ventricular septal defects. By generating a cardiac conditional null allele of CHD4, temporal transcriptional profiling, and systems-level analysis of CHD4 target genes and in utero echocardiography, we define molecular, biochemical, anatomical, and physiological mechanisms for CHD4 and the NuRD complex in repressing inappropriate expression of the skeletal and smooth muscle programs in the developing heart. Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac sarcomere. Control of this process involves not only promoting expression of cardiac sarcomere subunits but also repressing expression of noncardiac myofibril paralogs. This level of transcriptional control requires broadly expressed multiprotein machines that modify and remodel the chromatin landscape to restrict transcription machinery access. Prominent among these is the nucleosome remodeling and deacetylase (NuRD) complex, which includes the catalytic core subunit CHD4. Here, we demonstrate that direct CHD4-mediated repression of skeletal and smooth muscle myofibril isoforms is required for normal cardiac sarcomere formation, function, and embryonic survival early in gestation. Through transcriptomic and genome-wide analyses of CHD4 localization, we identified unique CHD4 binding sites in smooth muscle myosin heavy chain, fast skeletal α-actin, and the fast skeletal troponin complex genes. We further demonstrate that in the absence of CHD4, cardiomyocytes in the developing heart form a hybrid muscle cell that contains cardiac, skeletal, and smooth muscle myofibril components. These misexpressed paralogs intercalate into the nascent cardiac sarcomere to disrupt sarcomere formation and cause impaired cardiac function in utero. These results demonstrate the genomic and physiological requirements for CHD4 in mammalian cardiac development.

Abstract 964: GATA3 modulates chromatin structure to establish active enhancers in breast cancer cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Master transcription factors regulate cell-type-specific gene expression to define cellular identities. One such gene, GATA3, is a key regulator of multiple cellular programs, including T lymphocyte development, mammary luminal epithelial cell differentiation and trophoblast development. Recently, comprehensive genomic analysis has identified GATA3 as one of the most frequently mutated genes in breast cancer. It is also known that GATA3 expression levels directly correlate with favorable prognosis. These findings strongly suggest that GATA3 plays a critical role in tumorigenesis. However, the molecular mechanism(s) underlying GATA3-mediated gene regulation in breast cancer cells is not clearly defined. GATA3 participates in a complicated regulatory network with FOXA1 and ER-alpha, governing the transcriptional program in luminal tumors. Biochemical analyses indicate that: (1) GATA3 binds to chromatin in an estrogen-independent manner, (2) GATA3 acts upstream of FOXA1. These studies suggest GATA3 may act as a pioneer factor, which is capable of independently associating with closed chromatin and modulating chromatin structure to establish an active enhancer. In order to investigate GATA3 function as a pioneer transcription factor, we chose the MDA-MB-231 breast cancer cell line, which is GATA3, FOXA1 and ER-alpha negative, and established stable cell lines expressing wild-type GATA3 or GFP as a control. Consistent with previous results, GATA3-expressing cells represented an epithelial phenotype at the cellular and molecular level. To determine whether GATA3 can direct reprogramming of chromatin conformation, we performed genome-wide analyses of the chromatin binding activity of GATA3 and its impact on histone modifications and chromatin structure. We will present recent results describing how GATA3 licenses enhancer function to direct the luminal transcriptional program. Citation Format: Motoki Takaku, Sara A. Grimm, Takashi Shimbo, Lalith Perera, Shinichi Machida, Hitoshi Kurumizaka, Paul A. Wade. GATA3 modulates chromatin structure to establish active enhancers in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 964. doi:10.1158/1538-7445.AM2015-964

Abstract 2862: MBD3 regulates chromatin accessibility at active promoters

Chromatin structure is tightly regulated in cells and its dysregulation is associated with diseases such as cancer. The Mi-2/NuRD (Nucleosome Remodeling Deacetylase) complex is postulated to organize chromatin structure using its nucleosome remodeling and histone deacetylase activities. MBD3 is an integral component of NuRD, which potentially targets the complex to the specific sites of the genome. Recently, we have demonstrated that MBD3/NuRD targets regulatory elements of active genes, a finding diametrically opposed to historical models depicting NuRD as a co-repressor localized to transcriptionally non-permissive chromatin. Moreover, MBD3 co-localized with RNA polymerase II (pol II) at loci where promoter-enhancer loops are formed. Although our previous findings shed light on the role of NuRD in the regulation of active genes, the detailed mechanism of how NuRD is involved in transcriptional regulation is yet not fully understood. To investigate this question, we have developed a next generation, tagmentation based chromatin immunoprecipitation method followed by massively parallel sequencing (ChIP-nexo), which substantially increased the resolution of the MBD3/NuRD mapping. ChIP-nexo defined MBD3/NuRD localization at high resolution, showing accumulation of MBD3 at active promoters with a dip at transcription start site, suggesting an active role of NuRD in modulation of the cell-type specific transcriptional network. To interrogate the detailed functions of NuRD at active promoters, we conducted MNase-seq using conditions which measures both nucleosome positioning and sensitivity, in MBD3 depleted cells. Depletion of MBD3 changed the accessibility of nucleosomes at promoters, while maintaining overall nucleosome positioning. These data indicate an active regulatory function of NuRD in fine tuning the transcriptional network by modulating transcription rates of pol II. We propose that NuRD may be involved in establishing cell type specific gene expression patterns in diverse cell types by organizing promoter nucleoprotein architecture. Citation Format: Takashi Shimbo, Christopher Lavender, Sara A. Grimm, Makiko I. Doi, Telmo Henriques, Kimberly R. Cannady, Kevin J. Murphy, Daniel A. Gilchrist, Adam Burkholder, Jeffrey J. Hayes, Karen Adelman, Trevor K. Archer, Kenneth S. Zaret, Paul A. Wade. MBD3 regulates chromatin accessibility at active promoters. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2862. doi:10.1158/1538-7445.AM2015-2862