Instruct: Given a scientific claim, retrieve relevant passages that support or refute the claim.
Query: Pioneer factor OCT3/4 interacts with major chromatin remodeling factors.
Oct4 is a well-known transcription factor that plays fundamental roles in stem cell self-renewal, pluripotency, and somatic cell reprogramming. However, limited information is available on Oct4-associated protein complexes and their intrinsic protein-protein interactions that dictate Oct4's critical regulatory activities. Here we employed an improved affinity purification approach combined with mass spectrometry to purify Oct4 protein complexes in mouse embryonic stem cells (mESCs), and discovered many novel Oct4 partners important for self-renewal and pluripotency of mESCs. Notably, we found that Oct4 is associated with multiple chromatin-modifying complexes with documented as well as newly proved functional significance in stem cell maintenance and somatic cell reprogramming. Our study establishes a solid biochemical basis for genetic and epigenetic regulation of stem cell pluripotency and provides a framework for exploring alternative factor-based reprogramming strategies.
In this study, we describe the 3D chromosome regulatory landscape of human naive and primed embryonic stem cells. To devise this map, we identified transcriptional enhancers and insulators in these cells and placed them within the context of cohesin-associated CTCF-CTCF loops using cohesin ChIA-PET data. The CTCF-CTCF loops we identified form a chromosomal framework of insulated neighborhoods, which in turn form topologically associating domains (TADs) that are largely preserved during the transition between the naive and primed states. Regulatory changes in enhancer-promoter interactions occur within insulated neighborhoods during cell state transition. The CTCF anchor regions we identified are conserved across species, influence gene expression, and are a frequent site of mutations in cancer cells, underscoring their functional importance in cellular regulation. These 3D regulatory maps of human pluripotent cells therefore provide a foundation for future interrogation of the relationships between chromosome structure and gene control in development and disease.
The tumor suppressor protein, p53, plays a critical role in mediating cellular response to stress signals by regulating genes involved in cell cycle arrest and apoptosis. p53 is believed to be inactive for DNA binding unless its C terminus is modified or structurally altered. We show that unmodified p53 actively binds to two sites at -1.4 and -2.3 kb within the chromatin-assembled p21 promoter and requires the C terminus and the histone acetyltransferase, p300, for transcription. Acetylation of the C terminus by p300 is not necessary for binding or promoter activation. Instead, p300 acetylates p53-bound nucleosomes in the p21 promoter with spreading to the TATA box. Thus, p53 is an active DNA and chromatin binding protein that may selectively regulate its target genes by recruitment of specific cofactors to structurally distinct binding sites.
Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here, we reconstitute four stages of nucleosome architecture using purified components: yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific, and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization.
Using mouse skin, where bountiful reservoirs of synchronized hair follicle stem cells (HF-SCs) fuel cycles of regeneration, we explore how adult SCs remodel chromatin in response to activating cues. By profiling global mRNA and chromatin changes in quiescent and activated HF-SCs and their committed, transit-amplifying (TA) progeny, we show that polycomb-group (PcG)-mediated H3K27-trimethylation features prominently in HF-lineage progression by mechanisms distinct from embryonic-SCs. In HF-SCs, PcG represses nonskin lineages and HF differentiation. In TA progeny, nonskin regulators remain PcG-repressed, HF-SC regulators acquire H3K27me3-marks, and HF-lineage regulators lose them. Interestingly, genes poised in embryonic stem cells, active in HF-SCs, and PcG-repressed in TA progeny encode not only key transcription factors, but also signaling regulators. We document their importance in balancing HF-SC quiescence, underscoring the power of chromatin mapping in dissecting SC behavior. Our findings explain how HF-SCs cycle through quiescent and activated states without losing stemness and define roles for PcG-mediated repression in governing a fate switch irreversibly.
Polycomb Group (PcG) proteins mediate heritable gene silencing by modifying chromatin structure. An essential PcG complex, PRC1, compacts chromatin and inhibits chromatin remodeling. In Drosophila melanogaster, the intrinsically disordered C-terminal region of PSC (PSC-CTR) mediates these noncovalent effects on chromatin, and is essential for viability. Because the PSC-CTR sequence is poorly conserved, the significance of its effects on chromatin outside of Drosophila was unclear. The absence of folded domains also made it difficult to understand how the sequence of PSC-CTR encodes its function. To determine the mechanistic basis and extent of conservation of PSC-CTR activity, we identified 17 metazoan PSC-CTRs spanning chordates to arthropods, and examined their sequence features and biochemical properties. PSC-CTR sequences are poorly conserved, but are all highly charged and structurally disordered. We show that active PSC-CTRs--which bind DNA tightly and inhibit chromatin remodeling efficiently--are distinguished from less active ones by the absence of extended negatively charged stretches. PSC-CTR activity can be increased by dispersing its contiguous negative charge, confirming the importance of this property. Using the sequence properties defined as important for PSC-CTR activity, we predicted the presence of active PSC-CTRs in additional diverse genomes. Our analysis reveals broad conservation of PSC-CTR activity across metazoans. This conclusion could not have been determined from sequence alignments. We further find that plants that lack active PSC-CTRs instead possess a functionally analogous PcG protein, EMF1. Thus, our study suggests that a disordered domain with dispersed negative charges underlies PRC1 activity, and is conserved across metazoans and plants.
Transcription factors (TFs) bind specifically to discrete regions of mammalian genomes called cis-regulatory elements. Among those are enhancers, which play key roles in regulation of gene expression during development and differentiation. Despite the recognized central regulatory role exerted by chromatin in control of TF functions, much remains to be learned regarding the chromatin structure of enhancers and how it is established. Here, we have analyzed on a genomic-scale enhancers that recruit FOXA1, a pioneer transcription factor that triggers transcriptional competency of these cis-regulatory sites. Importantly, we found that FOXA1 binds to genomic regions showing local DNA hypomethylation and that its cell-type-specific recruitment to chromatin is linked to differential DNA methylation levels of its binding sites. Using neural differentiation as a model, we showed that induction of FOXA1 expression and its subsequent recruitment to enhancers is associated with DNA demethylation. Concomitantly, histone H3 lysine 4 methylation is induced at these enhancers. These epigenetic changes may both stabilize FOXA1 binding and allow for subsequent recruitment of transcriptional regulatory effectors. Interestingly, when cloned into reporter constructs, FOXA1-dependent enhancers were able to recapitulate their cell type specificity. However, their activities were inhibited by DNA methylation. Hence, these enhancers are intrinsic cell-type-specific regulatory regions of which activities have to be potentiated by FOXA1 through induction of an epigenetic switch that includes notably DNA demethylation.
Both signal transducer and activator of transcription 3 (STAT3) and SALL4 are important in maintaining the pluripotent and self-renewal state of embryonic stem cells. We hypothesized that STAT3, a latent transcriptional factor, may regulate the gene expression of SALL4. In support of this hypothesis, DNA sequence analysis of the SALL4 gene promoter revealed four putative STAT3-binding sites. Using a SALL4-luciferase reporter gene assay, we found that modulation of the STAT3 activity significantly up-regulated the luciferase activity. By chromatin immunoprecipitation, the segment of the SALL4 promoter showing the highest affinity to STAT3 was localized to -366 to -163, in which there was only one putative STAT3 binding site starting at -199. Site-directed mutagenesis of all four putative STAT3-binding sites in the SALL4 promoter significantly reduced its responsiveness to STAT3, although the most dramatic effect was seen at the binding site starting at -199. We further tested the functional relationship between STAT3 and SALL4 using MDA-MB-231, a breast cell line carrying constitutive SALL4 expression and STAT3 activity. Down-regulation of the STAT3 activity using a dominant-negative construct resulted in a significant decrease in the expression of SALL4. To conclude, our data suggest that STAT3 and SALL4 probably cooperate in both physiological and pathological states.
Recent structures of the nucleosome core particle reveal details of histone-histone and histone-DNA interactions. These structures have now set the stage for understanding chromatin assembly and dynamics during replication and transcription. Histone chaperones and chromatin remodeling complexes are important in both of these processes. The nucleosome and its protein core, the histone octamer, have twofold symmetry, which histone chaperones may use to bind core histones. Recent studies suggest that the nucleoplasmin pentamer may mediate histone storage, sperm chromatin decondensation and nucleosome assembly, by dimerizing to form a decamer. In this model, histone binding on the lateral surface of the chaperone involves stereospecific interactions and a shared twofold axis.
In yeast, remodeling of PHO5 promoter chromatin upon activation is accompanied by transient hyperacetylation and subsequent eviction of histones from the promoter in trans. In the course of rerepression, nucleosomes have to be reassembled on the promoter. We have analyzed where the histones for reassembly of the inactive promoter chromatin come from. The use of a strain with two differently tagged and differently regulated versions of histone H3 allowed us to discriminate between histones originating from the chromatin fraction and histones arising from the soluble histone pool. In this way, we show that the incorporated histones originate from a source in trans. Promoter closure occurs very rapidly, and the histone chaperones Asf1 and Hir1 as well as the SWI/SNF nucleosome remodeling complex appear to be important for rapid reassembly of nucleosomes at the PHO5 promoter.
Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) involves a marked reorganization of chromatin. To identify post-translational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared with both the starting fibroblasts and a late reprogramming intermediate (pre-iPSCs), are enriched for histone modifications associated with active chromatin, and depleted for marks of transcriptional elongation and a subset of repressive modifications including H3K9me2/me3. Dissecting the contribution of H3K9 methylation to reprogramming, we show that the H3K9 methyltransferases Ehmt1, Ehmt2 and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, we find that inhibition of heterochromatin protein-1γ (Cbx3), a protein known to recognize H3K9 methylation, enhances reprogramming. Genome-wide location analysis revealed that Cbx3 predominantly binds active genes in both pre-iPSCs and pluripotent cells but with a strikingly different distribution: in pre-iPSCs, but not in embryonic stem cells, Cbx3 associates with active transcriptional start sites, suggesting a developmentally regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the predominant association of Cbx3 with active transcription, the H3K9 methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9 methylation restrict late reprogramming events, and suggest that a marked change in global chromatin character constitutes an epigenetic roadblock for reprogramming.
Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.
Polyhomeotic-like 3 (PHC3) is a ubiquitously expressed member of the polycomb gene family and part of the human polycomb complex hPRC-H. We found that in normal cells PHC3 associated with both hPRC-H complex components and with the transcription factor E2F6. In differentiating and confluent cells, PHC3 and E2F6 showed nuclear colocalization in a punctate pattern that resembled the binding of polycomb bodies to heterochromatin. This punctate pattern was not seen in proliferating cells suggesting that PHC3 may be part of an E2F6-polycomb complex that has been shown to occupy and silence target promoters in G0. Previous loss of heterozygosity (LoH) analyses had shown that the region containing PHC3 underwent frequent LoH in primary human osteosarcoma tumors. When we examined normal bone and human osteosarcoma tumors, we found loss of PHC3 expression in 36 of 56 osteosarcoma tumors. Sequence analysis revealed that PHC3 was mutated in nine of 15 primary osteosarcoma tumors. These findings suggest that loss of PHC3 may favor tumorigenesis by potentially disrupting the ability of cells to remain in G0.
The sequence specificity of DNA-binding proteins is the primary mechanism by which the cell recognizes genomic features. Here, we describe systematic determination of yeast transcription factor DNA-binding specificities. We obtained binding specificities for 112 DNA-binding proteins representing 19 distinct structural classes. One-third of the binding specificities have not been previously reported. Several binding sequences have striking genomic distributions relative to transcription start sites, supporting their biological relevance and suggesting a role in promoter architecture. Among these are Rsc3 binding sequences, containing the core CGCG, which are found preferentially approximately 100 bp upstream of transcription start sites. Mutation of RSC3 results in a dramatic increase in nucleosome occupancy in hundreds of proximal promoters containing a Rsc3 binding element, but has little impact on promoters lacking Rsc3 binding sequences, indicating that Rsc3 plays a broad role in targeting nucleosome exclusion at yeast promoters.
BACKGROUND Open chromatin regions are correlated with active regulatory elements in development and are dysregulated in diseases. The BAF (SWI/SNF) complex is essential for development, and has been demonstrated to remodel reconstituted chromatin in vitro and to control the accessibility of a few individual regions in vivo. However, it remains unclear where and how BAF controls the open chromatin landscape to regulate developmental processes, such as human epidermal differentiation. RESULTS Using a novel "on-plate" ATAC-sequencing approach for profiling open chromatin landscapes with a low number of adherent cells, we demonstrate that the BAF complex is essential for maintaining 11.6 % of open chromatin regions in epidermal differentiation. These BAF-dependent open chromatin regions are highly cell-type-specific and are strongly enriched for binding sites for p63, a master epidermal transcription factor. The DNA sequences of p63 binding sites intrinsically favor nucleosome formation and are inaccessible in other cell types without p63 to prevent ectopic activation. In epidermal cells, BAF and p63 mutually recruit each other to maintain 14,853 open chromatin regions. We further demonstrate that BAF and p63 cooperatively position nucleosomes away from p63 binding sites and recruit transcriptional machinery to control tissue differentiation. CONCLUSIONS BAF displays high specificity in controlling the open chromatin landscape during epidermal differentiation by cooperating with the master transcription factor p63 to maintain lineage-specific open chromatin regions.
Detection of new genomic control elements is critical in understanding transcriptional regulatory networks in their entirety. We studied the genome-wide binding locations of three key regulatory proteins (POU5F1, also known as OCT4; NANOG; and CTCF) in human and mouse embryonic stem cells. In contrast to CTCF, we found that the binding profiles of OCT4 and NANOG are markedly different, with only ∼5% of the regions being homologously occupied. We show that transposable elements contributed up to 25% of the bound sites in humans and mice and have wired new genes into the core regulatory network of embryonic stem cells. These data indicate that species-specific transposable elements have substantially altered the transcriptional circuitry of pluripotent stem cells.
FACT, a heterodimer of Spt16 and Pob3, is an essential histone chaperone. We show that the H2A-H2B binding activity that is central to FACT function resides in short acidic regions near the C termini of each subunit. Mutations throughout these regions affect binding and cause correlated phenotypes that range from mild to lethal, with the largest individual contributions unexpectedly coming from an aromatic residue and a nearby carboxylate residue within each domain. Spt16 and Pob3 bind overlapping sites on H2A-H2B, and Spt16-Pob3 heterodimers simultaneously bind two H2A-H2B dimers, the same stoichiometry as the components of a nucleosome. An Spt16:H2A-H2B crystal structure explains the biochemical and genetic data, provides a model for Pob3 binding, and implies a mechanism for FACT reorganization that we confirm biochemically. Moreover, unexpected similarity to binding of ANP32E and Swr1 with H2A.Z-H2B reveals that diverse H2A-H2B chaperones use common mechanisms of histone binding and regulating nucleosome functions.
The sequence-specific binding to DNA is crucial for the p53 tumor suppressor function. To investigate the constraints imposed on p53-DNA recognition by nucleosomal organization, we studied binding of the p53 DNA binding domain (p53DBD) and full-length wild-type p53 protein to a single p53 response element (p53RE) placed near the nucleosomal dyad in six rotational settings. We demonstrate that the strongest p53 binding occurs when the p53RE in the nucleosome is bent in the same direction as observed for the p53-DNA complexes in solution and in co-crystals. The p53RE becomes inaccessible, however, if its orientation in the core particle is changed by approximately 180 degrees. Our observations indicate that the orientation of the binding sites on a nucleosome may play a significant role in the initial p53-DNA recognition and subsequent cofactor recruitment.
Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells. Here we report on a strategy to accomplish this replacement. Through a combination of transcriptome and bioinformatic analysis we have identified factors previously characterized as being lineage specifiers that are able to replace OCT4 and SOX2 in the reprogramming of human fibroblasts. Our results show that it is possible to replace OCT4 and SOX2 simultaneously with alternative lineage specifiers in the reprogramming of human cells. At a broader level, they also support a model in which counteracting lineage specification networks underlies the induction of pluripotency.
The eukaryotic genome consists of DNA molecules far longer than the cells that contain them. They reach their greatest compaction during chromosome condensation in mitosis. This process is aided by condensin, a structural maintenance of chromosomes (SMC) family member. The spatial organization of mitotic chromosomes and how condensin shapes chromatin architecture are not yet fully understood. Here we use chromosome conformation capture (Hi-C) to study mitotic chromosome condensation in the fission yeast Schizosaccharomyces pombe. This showed that the interphase landscape characterized by small chromatin domains is replaced by fewer but larger domains in mitosis. Condensin achieves this by setting up longer-range, intrachromosomal DNA interactions, which compact and individualize chromosomes. At the same time, local chromatin contacts are constrained by condensin, with profound implications for local chromatin function during mitosis. Our results highlight condensin as a major determinant that changes the chromatin landscape as cells prepare their genomes for cell division.
The embryonic stem (ES) cell transcriptional and chromatin-modifying networks are critical for self-renewal maintenance. However, it remains unclear whether these networks functionally interact and, if so, what factors mediate such interactions. Here, we show that WD repeat domain 5 (Wdr5), a core member of the mammalian Trithorax (trxG) complex, positively correlates with the undifferentiated state and is a regulator of ES cell self-renewal. We demonstrate that Wdr5, an "effector" of H3K4 methylation, interacts with the pluripotency transcription factor Oct4. Genome-wide protein localization and transcriptome analyses demonstrate overlapping gene regulatory functions between Oct4 and Wdr5. The Oct4-Sox2-Nanog circuitry and trxG cooperate in activating transcription of key self-renewal regulators, and furthermore, Wdr5 expression is required for the efficient formation of induced pluripotent stem (iPS) cells. We propose an integrated model of transcriptional and epigenetic control, mediated by select trxG members, for the maintenance of ES cell self-renewal and somatic cell reprogramming.
Posttranslational modifications mediate important regulatory functions in biology. The acetylation of the p53 transcription factor, for example, promotes transcriptional activation of target genes including p21. Here we show that the acetylation of two lysine residues in p53 promotes recruitment of the TFIID subunit TAF1 to the p21 promoter through its bromodomains. UV irradiation of cells diacetylates p53 at lysines 373 and 382, which in turn recruits TAF1 to a distal p53-binding site on the p21 promoter prior to looping to the core promoter. Disruption of acetyl-p53/bromodomain interaction inhibits TAF1 recruitment to both the distal p53-binding site and the core promoter. Further, the TFIID subunits TAF4, TAF5, and TBP are detected on the core promoter prior to TAF1, suggesting that, upon DNA damage, distinct subunits of TFIID may be recruited separately to the p21 promoter and that the transcriptional activation depends on posttranslational modification of the p53 transcription factor.
IL-6 is an immunoregulatory cytokine with multiple functions in hemopoiesis, proliferation, and tumorigenesis. IL-6 triggers phosphorylation, dimerization, and nuclear translocation of STAT3, which binds to target promoters and activates transcription. Brahma-related gene 1 (BRG1), the enzymatic engine of the yeast-mating type-switching and sucrose-nonfermenting chromatin-remodeling complex, is essential for recruitment of STAT1 or STAT1/STAT2-containing complexes to IFN targets. We hypothesized that BRG1 might also be required for STAT3 recruitment. In this study, we show that induction of a subset of human IL-6-responsive genes is BRG1 dependent. BRG1 is constitutively present at these targets and is required for STAT3 recruitment, downstream histone modifications, and IL-6-induced chromatin remodeling. IL-6-induced recruitment of STAT3 to the IFN regulatory factor 1 promoter and subsequent mRNA synthesis is BRG1 dependent, even though IFN-gamma-mediated STAT1 recruitment to this locus is BRG1 independent. BRG1 also increased basal expression of IFN-induced transmembrane protein 3 and IFN-gamma-induced protein 16, and the basal chromatin accessibility at the promoter of IFN regulatory factor 1. The effect on basal expression was STAT3 independent, as revealed by small interfering RNA knockdown. Together with prior observations, these data reveal that BRG1 has a broad role in mediating STAT accessibility at multiple cytokine-responsive promoters and exposes promoter specific differences in both the effect of BRG1 on basal chromatin accessibility and on access of different STAT proteins to the same target.
Histone proteins play essential structural and functional roles in the transition between active and inactive chromatin states. Although histones have a high degree of conservation due to constraints to maintain the overall structure of the nucleosomal octameric core, variants have evolved to assume diverse roles in gene regulation and epigenetic silencing. Histone variants, post-translational modifications and interactions with chromatin remodeling complexes influence DNA replication, transcription, repair and recombination. The authors review recent findings on the structure of chromatin that confirm previous interparticle interactions observed in crystal structures.
Super-enhancers (SEs) are large clusters of transcriptional enhancers that are co-occupied by multiple lineage-specific transcription factors driving expression of genes that define cell identity. In embryonic stem cells (ESCs), SEs are highly enriched for the core pluripotency factors Oct4, Sox2, and Nanog. In this study, we sought to dissect the molecular control mechanism of SE activity in pluripotency and reprogramming. Starting from a protein interaction network surrounding Sox2, we identified Tex10 as a key pluripotency factor that plays a functionally significant role in ESC self-renewal, early embryo development, and reprogramming. Tex10 is enriched at SEs in a Sox2-dependent manner and coordinates histone acetylation and DNA demethylation at SEs. Tex10 activity is also important for pluripotency and reprogramming in human cells. Our study therefore highlights Tex10 as a core component of the pluripotency network and sheds light on its role in epigenetic control of SE activity for cell fate determination.