Yin Shen

A map of the cis-regulatory sequences in the mouse genome

The laboratory mouse is the most widely used mammalian model organism in biomedical research. The 2.6 × 109 bases of the mouse genome possess a high degree of conservation with the human genome, so a thorough annotation of the mouse genome will be of significant value to understanding the function of the human genome. So far, most of the functional sequences in the mouse genome have yet to be found, and the cis-regulatory sequences in particular are still poorly annotated. Comparative genomics has been a powerful tool for the discovery of these sequences, but on its own it cannot resolve their temporal and spatial functions. Recently, ChIP-Seq has been developed to identify cis-regulatory elements in the genomes of several organisms including humans, Drosophila melanogaster and Caenorhabditis elegans. Here we apply the same experimental approach to a diverse set of 19 tissues and cell types in the mouse to produce a map of nearly 300,000 murine cis-regulatory sequences. The annotated sequences add up to 11% of the mouse genome, and include more than 70% of conserved non-coding sequences. We define tissue-specific enhancers and identify potential transcription factors regulating gene expression in each tissue or cell type. Finally, we show that much of the mouse genome is organized into domains of coordinately regulated enhancers and promoters. Our results provide a resource for the annotation of functional elements in the mammalian genome and for the study of mechanisms regulating tissue-specific gene expression.

Base-Resolution Analysis of 5-Hydroxymethylcytosine in the Mammalian Genome

The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), that when combined with traditional bisulfite sequencing can be used for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor-binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance.

Chromatin architecture reorganization during stem cell differentiation

Higher-order chromatin structure is emerging as an important regulator of gene expression. Although dynamic chromatin structures have been identified in the genome, the full scope of chromatin dynamics during mammalian development and lineage specification remains to be determined. By mapping genome-wide chromatin interactions in human embryonic stem (ES) cells and four human ES-cell-derived lineages, we uncover extensive chromatin reorganization during lineage specification. We observe that although self-associating chromatin domains are stable during differentiation, chromatin interactions both within and between domains change in a striking manner, altering 36% of active and inactive chromosomal compartments throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome data sets, we find widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results therefore provide a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages.

Promoter CpG Methylation Contributes to ES Cell Gene Regulation in Parallel with Oct4/Nanog, PcG Complex, and Histone H3 K4/K27 Trimethylation

We report here genome-wide mapping of DNA methylation patterns at proximal promoter regions in mouse embryonic stem (mES) cells. Most methylated genes are differentiation associated and repressed in mES cells. By contrast, the unmethylated gene set includes many housekeeping and pluripotency genes. By crossreferencing methylation patterns to genome-wide mapping of histone H3 lysine (K) 4/27 trimethylation and binding of Oct4, Nanog, and Polycomb proteins on gene promoters, we found that promoter DNA methylation is the only marker of this group present on approximately 30% of genes, many of which are silenced in mES cells. In demethylated mutant mES cells, we saw upregulation of a subset of X-linked genes and developmental genes that are methylated in wild-type mES cells, but lack either H3K4 and H3K27 trimethylation or association with Polycomb, Oct4, or Nanog. Our data suggest that in mES cells promoter methylation represents a unique epigenetic program that complements other regulatory mechanisms to ensure appropriate gene expression.

DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling

DNA methylation is a major epigenetic factor that has been postulated to regulate cell lineage differentiation. We report here that conditional gene deletion of the maintenance DNA methyltransferase I (Dnmt1) in neural progenitor cells (NPCs) results in DNA hypomethylation and precocious astroglial differentiation. The developmentally regulated demethylation of astrocyte marker genes as well as genes encoding the crucial components of the gliogenic JAK-STAT pathway is accelerated in Dnmt1–/– NPCs. Through a chromatin remodeling process, demethylation of genes in the JAK-STAT pathway leads to an enhanced activation of STATs, which in turn triggers astrocyte differentiation. Our study suggests that during the neurogenic period, DNA methylation inhibits not only astroglial marker genes but also genes that are essential for JAK-STAT signaling. Thus, demethylation of these two groups of genes and subsequent elevation of STAT activity are key mechanisms that control the timing and magnitude of astroglial differentiation.

An encyclopedia of mouse DNA elements (Mouse ENCODE)

To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

Epigenetic memory at embryonic enhancers identified in DNA methylation maps from adult mouse tissues.

Mammalian development requires cytosine methylation, a heritable epigenetic mark of cellular memory believed to maintain a cell’s unique gene expression pattern. However, it remains unclear how dynamic DNA methylation relates to cell-type specific gene expression and animal development. Here, by mapping base resolution methylomes in 17 adult mouse tissues at shallow coverage, we identify 302,864 tissue-specific differentially methylated regions (tsDMRs) and estimate that >6.7% of the mouse genome is variably methylated. Supporting a prominent role for DNA methylation in gene regulation, most tsDMRs occur at distal cis-regulatory elements. Surprisingly, some tsDMRs mark enhancers dormant in adult tissues but active in embryonic development. These “vestigial” enhancers are hypomethylated and lack active histone modifications in adult tissue, but nevertheless exhibit activity during embryonic development. Our results provide new insights into the role of DNA methylation at tissue-specific enhancers and suggest that epigenetic memory of embryonic development may be retained in adult tissues.

X-inactivation in female human embryonic stem cells is in a nonrandom pattern and prone to epigenetic alterations

X chromosome inactivation (XCI) is an essential mechanism for dosage compensation of X-linked genes in female cells. We report that subcultures from lines of female human embryonic stem cells (hESCs) exhibit variation (0–100%) for XCI markers, including XIST RNA expression and enrichment of histone H3 lysine 27 trimethylation (H3K27me3) on the inactive X chromosome (Xi). Surprisingly, regardless of the presence or absence of XCI markers in different cultures, all female hESCs we examined (H7, H9, and HSF6 cells) exhibit a monoallelic expression pattern for a majority of X-linked genes. Our results suggest that these established female hESCs have already completed XCI during the process of derivation and/or propagation, and the XCI pattern of lines we investigated is already not random. Moreover, XIST gene expression in subsets of cultured female hESCs is unstable and subject to stable epigenetic silencing by DNA methylation. In the absence of XIST expression, ≈12% of X-linked promoter CpG islands become hypomethylated and a portion of X-linked alleles on the Xi are reactivated. Because alterations in dosage compensation of X-linked genes could impair somatic cell function, we propose that XCI status should be routinely checked in subcultures of female hESCs, with cultures displaying XCI markers better suited for use in regenerative medicine.

Polycomb-Like 3 Promotes Polycomb Repressive Complex 2 Binding to CpG Islands and Embryonic Stem Cell Self-Renewal

Polycomb repressive complex 2 (PRC2) trimethylates lysine 27 of histone H3 (H3K27me3) to regulate gene expression during diverse biological transitions in development, embryonic stem cell (ESC) differentiation, and cancer. Here, we show that Polycomb-like 3 (Pcl3) is a component of PRC2 that promotes ESC self-renewal. Using mass spectrometry, we identified Pcl3 as a Suz12 binding partner and confirmed Pcl3 interactions with core PRC2 components by co-immunoprecipitation. Knockdown of Pcl3 in ESCs increases spontaneous differentiation, yet does not affect early differentiation decisions as assessed in teratomas and embryoid bodies, indicating that Pcl3 has a specific role in regulating ESC self-renewal. Consistent with Pcl3 promoting PRC2 function, decreasing Pcl3 levels reduces H3K27me3 levels while overexpressing Pcl3 increases H3K27me3 levels. Furthermore, chromatin immunoprecipitation and sequencing (ChIP-seq) reveal that Pcl3 co-localizes with PRC2 core component, Suz12, and depletion of Pcl3 decreases Suz12 binding at over 60% of PRC2 targets. Mutation of conserved residues within the Pcl3 Tudor domain, a domain implicated in recognizing methylated histones, compromises H3K27me3 formation, suggesting that the Tudor domain of Pcl3 is essential for function. We also show that Pcl3 and its paralog, Pcl2, exist in different PRC2 complexes but bind many of the same PRC2 targets, particularly CpG islands regulated by Pcl3. Thus, Pcl3 is a component of PRC2 critical for ESC self-renewal, histone methylation, and recruitment of PRC2 to a subset of its genomic sites.

A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening

With <2% of the human genome coding for proteins, a major challenge is to interpret the function of the noncoding DNA. Millions of regulatory sequences have been predicted in the human genome through analysis of DNA methylation, chromatin modification, hypersensitivity to nucleases, and transcription factor binding, but few have been shown to regulate transcription in their native contexts. We have developed a high-throughput CRISPR/Cas9-based genome-editing strategy and used it to interrogate 174 candidate regulatory sequences within the 1-Mbp POU5F1 locus in human embryonic stem cells (hESCs). We identified two classical regulatory elements, including a promoter and a proximal enhancer, that are essential for POU5F1 transcription in hESCs. Unexpectedly, we also discovered a new class of enhancers that contribute to POU5F1 transcription in an unusual way: Disruption of such sequences led to a temporary loss of POU5F1 transcription that is fully restored after a few rounds of cell division. These results demonstrate the utility of high-throughput screening for functional characterization of noncoding DNA and reveal a previously unrecognized layer of gene regulation in human cells.