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The Polycomb repressive complex 2 (PRC2) is an essential chromatin regulatory complex involved in repressing the transcription of diverse developmental genes. PRC2 consists of a core complex; possessing H3K27 methyltransferase activity and various associated factors that are important to modulate its function. During evolution, the composition of PRC2 and the functionality of PRC2 components have changed considerably. Here, we compare the PRC2 complex members of Drosophila and mammals and describe their adaptation to altered biological needs. We also highlight how the PRC2.1 subcomplex has gained multiple novel functions and discuss the implications of these changes for the function of PRC2 in chromatin regulation.

HDGF-related protein 3 (HRP3, also known as HDGFL3) belongs to the family of HDGF-related proteins (HRPs) and plays an essential role in hepatocellular carcinoma pathogenesis. All HRPs have a PWWP domain at the N-terminus that binds both histone and DNA substrates. Despite previous advances in PWWP domains, the molecular basis by which HRP3 interacts with chromatin is unclear. In this study, we solved the crystal structures of the HRP3 PWWP domain in complex with various double-stranded DNAs with/without bound histone peptides. We found that HRP3 PWWP bound to the phosphate backbone of the DNA minor groove and showed a preference for DNA molecules bearing a narrow minor groove width. In addition, HRP3 PWWP preferentially bound to histone peptides bearing the H3K36me3/2 modification. HRP3 PWWP uses two adjacent surfaces to bind both DNA and histone substrates simultaneously, enabling us to generate a model illustrating the recruitment of PWWP to H3K36me3-containing nucleosomes. Cell-based analysis indicated that both DNA and histone binding by the HRP3 PWWP domain is important for HRP3 recruitment to chromatin in vivo. Our work establishes that HRP3 PWWP is a new family of minor groove-specific DNA-binding proteins, which improves our understanding of HRP3 and other PWWP domain-containing proteins.

SAMD1 is a newly identified repressive CpG island regulator., CpG islands (CGIs) are key regulatory DNA elements at most promoters, but how they influence the chromatin status and transcription remains elusive. Here, we identify and characterize SAMD1 (SAM domain-containing protein 1) as an unmethylated CGI-binding protein. SAMD1 has an atypical winged-helix domain that directly recognizes unmethylated CpG-containing DNA via simultaneous interactions with both the major and the minor groove. The SAM domain interacts with L3MBTL3, but it can also homopolymerize into a closed pentameric ring. At a genome-wide level, SAMD1 localizes to H3K4me3-decorated CGIs, where it acts as a repressor. SAMD1 tethers L3MBTL3 to chromatin and interacts with the KDM1A histone demethylase complex to modulate H3K4me2 and H3K4me3 levels at CGIs, thereby providing a mechanism for SAMD1-mediated transcriptional repression. The absence of SAMD1 impairs ES cell differentiation processes, leading to misregulation of key biological pathways. Together, our work establishes SAMD1 as a newly identified chromatin regulator acting at unmethylated CGIs.

Increasing evidence shows that many transcription factors execute important biologic functions independent from their DNA-binding capacity. The NF-κB p65 (RELA) subunit is a central regulator of innate immunity. Here, we investigated the relative functional contribution of p65 DNA-binding and dimerization in p65-deficient human and murine cells reconstituted with single amino acid mutants preventing either DNA-binding (p65 E/I) or dimerization (p65 FL/DD). DNA-binding of p65 was required for RelB-dependent stabilization of the NF-κB p100 protein. The antiapoptotic function of p65 and expression of the majority of TNF-α-induced genes were dependent on p65's ability to bind DNA and to dimerize. Chromatin immunoprecipitation with massively parallel DNA sequencing experiments revealed that impaired DNA-binding and dimerization strongly diminish the chromatin association of p65. However, there were also p65-independent TNF-α-inducible genes and a subgroup of p65 binding sites still allowed some residual chromatin association of the mutants. These sites were enriched in activator protein 1 (AP-1) binding motifs and showed increased chromatin accessibility and basal transcription. This suggests a mechanism of assisted p65 chromatin association that can be in part facilitated by chromatin priming and cooperativity with other transcription factors such as AP-1.-Riedlinger, T., Liefke, R., Meier-Soelch, J., Jurida, L., Nist, A., Stiewe, T., Kracht, M., Schmitz, M. L. NF-κB p65 dimerization and DNA-binding is important for inflammatory gene expression.

The Polycomb repressive complex 2 (PRC2) mainly mediates transcriptional repression and has essential roles in various biological processes including the maintenance of cell identity and proper differentiation. Polycomb-like (PCL) proteins, such as PHF1, MTF2 and PHF19, are PRC2-associated factors that form sub-complexes with PRC2 core components, and have been proposed to modulate the enzymatic activity of PRC2 or the recruitment of PRC2 to specific genomic loci. Mammalian PRC2-binding sites are enriched in CG content, which correlates with CpG islands that display a low level of DNA methylation14. However, the mechanism of PRC2 recruitment to CpG islands is not fully understood. Here we solve the crystal structures of the N-terminal domains of PHF1 and MTF2 with bound CpG-containing DNAs in the presence of H3K36me3-containing histone peptides. We show that the extended homologous regions of both proteins fold into a winged-helix structure, which specifically binds to the unmethylated CpG motif but in a completely different manner from the canonical winged-helix DNA recognition motif. We also show that the PCL extended homologous domains are required for efficient recruitment of PRC2 to CpG island-containing promoters in mouse embryonic stem cells. Our research provides the first, to our knowledge, direct evidence to demonstrate that PCL proteins are crucial for PRC2 recruitment to CpG islands, and further clarifies the roles of these proteins in transcriptional regulation in vivo.

Gene regulatory networks are pivotal for many biological processes. In mouse embryonic stem cells (mESCs), the transcriptional network can be divided into three functionally distinct modules: Polycomb, Core, and Myc. The Polycomb module represses developmental genes, while the Myc module is associated with proliferative functions, and its mis-regulation is linked to cancer development. Here, we show that, in mESCs, the Polycomb repressive complex 2 (PRC2)-associated protein EPOP (Elongin BC and Polycomb Repressive Complex 2-associated protein; a.k.a. C17orf96, esPRC2p48, and E130012A19Rik) co-localizes at chromatin with members of the Myc and Polycomb module. EPOP interacts with the transcription elongation factor Elongin BC and the H2B deubiquitinase USP7 to modulate transcriptional processes in mESCs similar to MYC. EPOP is commonly upregulated in human cancer, and its loss impairs the proliferation of several human cancer cell lines. Our findings establish EPOP as a transcriptional modulator, which impacts both Polycomb and active gene transcription in mammalian cells.

CpG islands (CGIs) are key DNA regulatory elements in the vertebrate genome and are often found at gene promoters. In mammalian embryonic stem (ES) cells, CGIs are decorated by either the active or repressive histone marks, H3K4me3 and H3K27me3, respectively, or by both modifications (‘bivalent domains’), but their precise regulation is incompletely understood. Remarkably, we find that the polycomb repressive complex 2 (PRC2)-associated protein C17orf96 (a.k.a. esPRC2p48 and E130012A19Rik) is present at most CGIs in mouse ES cells. At PRC2-rich CGIs, loss of C17orf96 results in an increased chromatin binding of Suz12 and elevated H3K27me3 levels concomitant with gene repression. In contrast, at PRC2-poor CGIs, located at actively transcribed genes, C17orf96 colocalizes with RNA polymerase II and its depletion leads to a focusing of H3K4me3 in the core of CGIs. Our findings thus identify C17orf96 as a novel context-dependent CGI regulator.

Chromatin-based mechanisms significantly contribute to the regulation of many developmentally regulated genes, including Notch target genes. After specific ligand binding, the intracellular part of the Notch receptor is cleaved off and translocates to the nucleus, where it binds to the transcription factor CSL (encoded by the RBPJ gene in mammals), in order to activate transcription. In the absence of a Notch signal, CSL represses Notch target genes by recruiting a co-repressor complex. Both NICD co-activator and CSL co-repressor complexes contain chromatin modifiers such as histone acetyltransferases and methyltransferases, which dynamically regulate chromatin marks at Notch target genes. Here we provide protocols for ChIP (chromatin immunoprecipitation) to analyze the chromatin status of dynamically regulated Notch target genes. Furthermore, an example is presented how to perform a primary analysis of ChIP-Seq data at Notch target genes using the Cistrome platform.

Chromatin-based mechanisms significantly contribute to the regulation of many developmentally regulated genes, including Notch target genes. After specific ligand binding, the intracellular part of the Notch receptor is cleaved off and translocates to the nucleus, where it binds to the transcription factor CSL (encoded by the RBPJ gene in mammals), in order to activate transcription. In the absence of a Notch signal, CSL represses Notch target genes by recruiting a co-repressor complex. Both NICD co-activator and CSL co-repressor complexes contain chromatin modifiers such as histone acetyltransferases and methyltransferases, which dynamically regulate chromatin marks at Notch target genes. Here we provide protocols for ChIP (chromatin immunoprecipitation) to analyze the chromatin status of dynamically regulated Notch target genes. Furthermore, an example is presented how to perform a primary analysis of ChIP-Seq data at Notch target genes using the Cistrome platform.