Literature context: 119ub Cell Signaling Cat# 8240; RRID:AB_10891618 Î±-H3K27me2 Diagenode Cat# C1541
To date, it remains largely unclear to what extent chromatin machinery contributes to the susceptibility and progression of complex diseases. Here, we combine deep epigenome mapping with single-cell transcriptomics to mine for evidence of chromatin dysregulation in type 2 diabetes. We find two chromatin-state signatures that track β cell dysfunction in mice and humans: ectopic activation of bivalent Polycomb-silenced domains and loss of expression at an epigenomically unique class of lineage-defining genes. β cell-specific Polycomb (Eed/PRC2) loss of function in mice triggers diabetes-mimicking transcriptional signatures and highly penetrant, hyperglycemia-independent dedifferentiation, indicating that PRC2 dysregulation contributes to disease. The work provides novel resources for exploring β cell transcriptional regulation and identifies PRC2 as necessary for long-term maintenance of β cell identity. Importantly, the data suggest a two-hit (chromatin and hyperglycemia) model for loss of β cell identity in diabetes.
Literature context: ology #8240 (D27C4); RRID:AB_10891618 Anti-H3K27me3 (Western) Active
The polycomb repressive complex 2 (PRC2) consists of core subunits SUZ12, EED, RBBP4/7, and EZH1/2 and is responsible for mono-, di-, and tri-methylation of lysine 27 on histone H3. Whereas two distinct forms exist, PRC2.1 (containing one polycomb-like protein) and PRC2.2 (containing AEBP2 and JARID2), little is known about their differential functions. Here, we report the discovery of a family of vertebrate-specific PRC2.1 proteins, "PRC2 associated LCOR isoform 1" (PALI1) and PALI2, encoded by the LCOR and LCORL gene loci, respectively. PALI1 promotes PRC2 methyltransferase activity in vitro and in vivo and is essential for mouse development. Pali1 and Aebp2 define mutually exclusive, antagonistic PRC2 subtypes that exhibit divergent H3K27-tri-methylation activities. The balance of these PRC2.1/PRC2.2 activities is required for the appropriate regulation of polycomb target genes during differentiation. PALI1/2 potentially link polycombs with transcriptional co-repressors in the regulation of cellular identity during development and in cancer.
Literature context: Signaling Technology Cat#8240; RRID:AB_10891618 Rabbit monoclonal anti-HA Cell
Polycomb group proteins regulate self-renewal and differentiation in many stem cell systems. When assembled into two canonical complexes, PRC1 and PRC2, they sequentially deposit H3K27me3 and H2AK119ub histone marks and establish repressive chromatin, referred to as Polycomb domains. Non-canonical PRC1 complexes retain RING1/RNF2 E3-ubiquitin ligases but have unique sets of accessory subunits. How these non-canonical complexes recognize and regulate their gene targets remains poorly understood. Here, we show that the BCL6 co-repressor (BCOR), a member of the PRC1.1 complex, is critical for maintaining primed pluripotency in human embryonic stem cells (ESCs). BCOR depletion leads to the erosion of Polycomb domains at key developmental loci and the initiation of differentiation along endoderm and mesoderm lineages. The C terminus of BCOR regulates the assembly and targeting of the PRC1.1 complex, while the N terminus contributes to BCOR-PRC1.1 repressor function. Our findings advance understanding of Polycomb targeting and repression in ESCs and could apply broadly across developmental systems.
Literature context: ignaling Technology Cat# 8240P, RRID:AB_10891618), H2A (Cell Signaling Technolog
Malignant mesothelioma (MM) is poorly responsive to systemic cytotoxic chemotherapy and invariably fatal. Here we describe a screen of 94 drugs in 15 exome-sequenced MM lines and the discovery of a subset defined by loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1) that demonstrate heightened sensitivity to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand). This association is observed across human early passage MM cultures, mouse xenografts and human tumour explants. We demonstrate that BAP1 deubiquitinase activity and its association with ASXL1 to form the Polycomb repressive deubiquitinase complex (PR-DUB) impacts TRAIL sensitivity implicating transcriptional modulation as an underlying mechanism. Death receptor agonists are well-tolerated anti-cancer agents demonstrating limited therapeutic benefit in trials without a targeting biomarker. We identify BAP1 loss-of-function mutations, which are frequent in MM, as a potential genomic stratification tool for TRAIL sensitivity with immediate and actionable therapeutic implications.
Literature context: (K119) Cell Signaling Cat#8240; RRID:AB_10891618 H2A Cell Signaling Cat#2578; RR
Double-strand breaks (DSBs) of DNA in eukaryotic cells are predominantly repaired by non-homologous end joining (NHEJ). The histone chaperone anti-silencing factor 1a (ASF1a) interacts with MDC1 and is recruited to sites of DSBs to facilitate the interaction of phospho-ATM with MDC1 and phosphorylation of MDC1, which are required for the recruitment of RNF8/RNF168 histone ubiquitin ligases. Thus, ASF1a deficiency reduces histone ubiquitination at DSBs, decreasing the recruitment of 53BP1, and decreases NHEJ, rendering cells more sensitive to DSBs. This role of ASF1a in DSB repair cannot be provided by the closely related ASF1b and does not require its histone chaperone activity. Homozygous deletion of ASF1A is seen in 10%-15% of certain cancers, suggesting that loss of NHEJ may be selected in some malignancies and that the deletion can be used as a molecular biomarker for cancers susceptible to radiotherapy or to DSB-inducing chemotherapy.
Literature context: l Signaling Technologies #8240, RRID:AB_10891618 Rabbit polyclonal anti-WDR40B (
Interaction networks between chromatin complexes and long noncoding RNAs have become a recurrent theme in epigenetic regulation. However, technical limitations have precluded identification of RNA binding motifs for chromatin-associated proteins. Here, we add a denaturation step to UV-crosslink RNA immunoprecipitation (dCLIP) and apply dCLIP to mouse and human chromobox homolog 7 (CBX7), an RNA binding subunit of Polycomb repressive complex 1 (PRC1). In both species, CBX7 predominantly binds 3' UTRs of messenger RNAs. CBX7 binds with a median RNA "footprint" of 171-183 nucleotides, the small size of which facilitates motif identification by bioinformatics. We find four families of consensus RNA motifs in mouse, and independent analysis of human CBX7 dCLIP data identifies similar motifs. Their mutation abolishes CBX7 binding in vitro. Pharmacological intervention with antisense oligonucleotides paradoxically increases CBX7 binding and enhances gene expression. These data support the utility of dCLIP and reveal an unexpected functional interaction between CBX7 and the 3' UTRs of mRNA.
Literature context: _331563Histone H4Millipore04-858Ub-H2ACell Signaling Technology8240Ub-H2BEMD Millipore17-650; RRID:
ADP-ribosylation of proteins is emerging as an important regulatory mechanism. Depending on the family member, ADP-ribosyltransferases either conjugate a single ADP-ribose to a target or generate ADP-ribose chains. Here we characterize Parp9, a mono-ADP-ribosyltransferase reported to be enzymatically inactive. Parp9 undergoes heterodimerization with Dtx3L, a histone E3 ligase involved in DNA damage repair. We show that the Dtx3L/Parp9 heterodimer mediates NAD+-dependent mono-ADP-ribosylation of ubiquitin, exclusively in the context of ubiquitin processing by E1 and E2 enzymes. Dtx3L/Parp9 ADP-ribosylates the carboxyl group of Ub Gly76. Because Gly76 is normally used for Ub conjugation to substrates, ADP-ribosylation of the Ub carboxyl terminus precludes ubiquitylation. Parp9 ADP-ribosylation activity therefore restrains the E3 function of Dtx3L. Mutation of the NAD+ binding site in Parp9 increases the DNA repair activity of the heterodimer. Moreover, poly(ADP-ribose) binding to the Parp9 macrodomains increases E3 activity. Dtx3L heterodimerization with Parp9 enables NAD+ and poly(ADP-ribose) regulation of E3 activity.
Literature context: ng, 8240, RRID:AB_10891618), mouse Ig
The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of Pcgf6 in ESCs leads to robust de-repression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells.
Literature context: ing 8240; RRID:AB_10891618 Mouse anti
Understanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.
Literature context: logy Cat# 8240; RRID:AB_10891618 H3K27me3 Millipore Cat# 07-449;
Master regulatory genes require stable silencing by the polycomb group (PcG) to prevent misexpression during differentiation and development. Some PcG proteins covalently modify histones, which contributes to heritable repression. The role for other effects on chromatin structure is less understood. We characterized the organization of PcG target genes in ESCs and neural progenitors using 5C and super-resolution microscopy. The genomic loci of repressed PcG targets formed discrete, small (20-140 Kb) domains of tight interaction that corresponded to locations bound by canonical polycomb repressive complex 1 (PRC1). These domains changed during differentiation as PRC1 binding changed. Their formation depended upon the Polyhomeotic component of canonical PRC1 and occurred independently of PRC1-catalyzed ubiquitylation. PRC1 domains differ from topologically associating domains in size and boundary characteristics. These domains have the potential to play a key role in transmitting epigenetic silencing of PcG targets by linking PRC1 to formation of a repressive higher-order structure.