Person:

Bernstein, Bradley

Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3), respectively. Compared to H3K27me3, localization and the role of H2AK119u1 are not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation.

Background: The histone variant H2A.Z has been implicated in nucleosome exchange, transcriptional activation and Polycomb repression. However, the relationships among these seemingly disparate functions remain obscure. Results: We mapped H2A.Z genome-wide in mammalian ES cells and neural progenitors. H2A.Z is deposited promiscuously at promoters and enhancers, and correlates strongly with H3K4 methylation. Accordingly, H2A.Z is present at poised promoters with bivalent chromatin and at active promoters with H3K4 methylation, but is absent from stably repressed promoters that are specifically enriched for H3K27 trimethylation. We also characterized post-translational modification states of H2A.Z, including a novel species dually-modified by ubiquitination and acetylation that is enriched at bivalent chromatin. Conclusions: Our findings associate H2A.Z with functionally distinct genomic elements, and suggest that post-translational modifications may reconcile its contrasting locations and roles.

Polycomb proteins are epigenetic regulators that localize to developmental loci in the early embryo where they mediate lineage-specific gene repression. In Drosophila, these repressors are recruited to sequence elements by DNA binding proteins associated with Polycomb repressive complex 2 (PRC2). However, the sequences that recruit PRC2 in mammalian cells have remained obscure. To address this, we integrated a series of engineered bacterial artificial chromosomes into embryonic stem (ES) cells and examined their chromatin. We found that a 44 kb region corresponding to the Zfpm2 locus initiates de novo recruitment of PRC2. We then pinpointed a CpG island within this locus as both necessary and sufficient for PRC2 recruitment. Based on this causal demonstration and prior genomic analyses, we hypothesized that large GC-rich elements depleted of activating transcription factor motifs mediate PRC2 recruitment in mammals. We validated this model in two ways. First, we showed that a constitutively active CpG island is able to recruit PRC2 after excision of a cluster of activating motifs. Second, we showed that two 1 kb sequence intervals from the Escherichia coli genome with GC-contents comparable to a mammalian CpG island are both capable of recruiting PRC2 when integrated into the ES cell genome. Our findings demonstrate a causal role for GC-rich sequences in PRC2 recruitment and implicate a specific subset of CpG islands depleted of activating motifs as instrumental for the initial localization of this key regulator in mammalian genomes.

A report on the genomics workshop 'Identification of Functional Elements in Mammalian Genomes', Cold Spring Harbor, New York, 11-13 November 2004.

Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing, chromatin immunoprecipitation sequencing, and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition, we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches, leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions.

Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described–one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.

Summary Genome-wide association studies have identified loci underlying human diseases, but the causal nucleotide changes and mechanisms remain largely unknown. Here we developed a fine-mapping algorithm to identify candidate causal variants for 21 autoimmune diseases from genotyping data. We integrated these predictions with transcription and cis-regulatory element annotations, derived by mapping RNA and chromatin in primary immune cells, including resting and stimulated CD4+ T-cell subsets, regulatory T-cells, CD8+ T-cells, B-cells, and monocytes. We find that ~90% of causal variants are noncoding, with ~60% mapping to immune-cell enhancers, many of which gain histone acetylation and transcribe enhancer-associated RNA upon immune stimulation. Causal variants tend to occur near binding sites for master regulators of immune differentiation and stimulus-dependent gene activation, but only 10–20% directly alter recognizable transcription factor binding motifs. Rather, most noncoding risk variants, including those that alter gene expression, affect non-canonical sequence determinants not well-explained by current gene regulatory models.

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but a similar reference has lacked for epigenomic studies. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection to-date of human epigenomes for primary cells and tissues. Here, we describe the integrative analysis of 111 reference human epigenomes generated as part of the program, profiled for histone modification patterns, DNA accessibility, DNA methylation, and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically-relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation, and human disease.

Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.