Gene enhancer elements are noncoding segments of DNA that play a central role in regulating transcriptional programs that control development, cell identity, and evolutionary processes. Recent ...studies have shown that noncoding single nucleotide polymorphisms (SNPs) that have been associated with risk for numerous common diseases through genome-wide association studies frequently lie in cell-type-specific enhancer elements. These enhancer variants probably influence transcriptional output, thereby offering a mechanistic basis to explain their association with risk for many common diseases. This review focuses on the identification and interpretation of disease-susceptibility variants that influence enhancer function. We discuss strategies for prioritizing the study of functional enhancer SNPs over those likely to be benign, review experimental and computational approaches to identifying the gene targets of enhancer variants, and highlight efforts to quantify the impact of enhancer variants on target transcript levels and cellular phenotypes. These studies are beginning to provide insights into the mechanistic basis of many common diseases, as well as into how we might translate this knowledge for improved disease diagnosis, prevention and treatments. Finally, we highlight five major challenges often associated with interpreting enhancer variants, and discuss recent technical advances that may help to surmount these challenges.
Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the ...boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.
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•Enhancers active in the cell of origin are co-amplified with oncogenes•Circular extrachromosomal amplicons are associated with enhancer rewiring•Endogenous and new enhancers on amplicons contribute to cell proliferation•Skewed co-amplification that selects enhancers is found across several tumor types
Extrachromosomal oncogene amplification is a common occurrence across a broad range of cancers, yet the chromatin landscape of these high-level amplifications is poorly understood. Using a combination of approaches to explore their chromatin topology and enhancer landscape, Morton et al. observed that these oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.
Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input “easy Hi-C” protocol to map the 3D genome ...architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.
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•HiCorr allows robust mapping of sub-TAD chromatin interactions with Hi-C•Low-input “easy Hi-C” protocol compatible with 50–100k cells•Enhancer loops and aggregates are better marks of cell identity than compartments•Chromatin loops outperform eQTLs in defining neurological GWAS target genes
Lu et al. developed a rigorous Hi-C bias-correction pipeline to significantly improve the robustness of high-resolution chromatin interaction maps. With a new low-input “easy Hi-C” protocol, they mapped chromatin interactions in neural samples, defined cell-type-specific enhancer loops and aggregates, and concluded that Hi-C outperforms eQTL in explaining GWAS results.
CHD7 is one of nine members of the chromodomain helicase DNA-binding domain family of ATP-dependent chromatin remodeling enzymes found in mammalian cells. De novo mutation of CHD7 is a major cause of ...CHARGE syndrome, a genetic condition characterized by multiple congenital anomalies. To gain insights to the function of CHD7, we used the technique of chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-Seq) to map CHD7 sites in mouse ES cells. We identified 10,483 sites on chromatin bound by CHD7 at high confidence. Most of the CHD7 sites show features of gene enhancer elements. Specifically, CHD7 sites are predominantly located distal to transcription start sites, contain high levels of H3K4 mono-methylation, found within open chromatin that is hypersensitive to DNase I digestion, and correlate with ES cell-specific gene expression. Moreover, CHD7 co-localizes with P300, a known enhancer-binding protein and strong predictor of enhancer activity. Correlations with 18 other factors mapped by ChIP-seq in mouse ES cells indicate that CHD7 also co-localizes with ES cell master regulators OCT4, SOX2, and NANOG. Correlations between CHD7 sites and global gene expression profiles obtained from Chd7(+/+), Chd7(+/-), and Chd7(-/-) ES cells indicate that CHD7 functions at enhancers as a transcriptional rheostat to modulate, or fine-tune the expression levels of ES-specific genes. CHD7 can modulate genes in either the positive or negative direction, although negative regulation appears to be the more direct effect of CHD7 binding. These data indicate that enhancer-binding proteins can limit gene expression and are not necessarily co-activators. Although ES cells are not likely to be affected in CHARGE syndrome, we propose that enhancer-mediated gene dysregulation contributes to disease pathogenesis and that the critical CHD7 target genes may be subject to positive or negative regulation.
DNA variants (SNPs) that predispose to common traits often localize within noncoding regulatory elements such as enhancers. Moreover, loci identified by genome-wide association studies (GWAS) often ...contain multiple SNPs in linkage disequilibrium (LD), any of which may be causal. Thus, determining the effect of these multiple variant SNPs on target transcript levels has been a major challenge. Here, we provide evidence that for six common autoimmune disorders (rheumatoid arthritis, Crohn's disease, celiac disease, multiple sclerosis, lupus, and ulcerative colitis), the GWAS association arises from multiple polymorphisms in LD that map to clusters of enhancer elements active in the same cell type. This finding suggests a "multiple enhancer variant" hypothesis for common traits, where several variants in LD impact multiple enhancers and cooperatively affect gene expression. Using a novel method to delineate enhancer-gene interactions, we show that multiple enhancer variants within a given locus typically target the same gene. Using available data from HapMap and B lymphoblasts as a model system, we provide evidence at numerous loci that multiple enhancer variants cooperatively contribute to altered expression of their gene targets. The effects on target transcript levels tend to be modest and can be either gain- or loss-of-function. Additionally, the genes associated with multiple enhancer variants encode proteins that are often functionally related and enriched in common pathways. Overall, the multiple enhancer variant hypothesis offers a new paradigm by which noncoding variants can confer susceptibility to common traits.
Naive mouse embryonic stem cells (mESCs) and primed epiblast stem cells (mEpiSCs) represent successive snapshots of pluripotency during embryogenesis. Using transcriptomic and epigenomic mapping we ...show that a small fraction of transcripts are differentially expressed between mESCs and mEpiSCs and that these genes show expected changes in chromatin at their promoters and enhancers. Unexpectedly, the cis-regulatory circuitry of genes that are expressed at identical levels between these cell states also differs dramatically. In mESCs, these genes are associated with dominant proximal enhancers and dormant distal enhancers, which we term seed enhancers. In mEpiSCs, the naive-dominant enhancers are lost, and the seed enhancers take up primary transcriptional control. Seed enhancers have increased sequence conservation and show preferential usage in downstream somatic tissues, often expanding into super enhancers. We propose that seed enhancers ensure proper enhancer utilization and transcriptional fidelity as mammalian cells transition from naive pluripotency to a somatic regulatory program.
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•Enhancer switching is a general mechanism of pluripotent gene regulation•Seed enhancers are dormant in naive cells but take up control in primed cells•Seed enhancers have increased sequence conservation relative to typical enhancers•Seed enhancers give rise to super enhancers in downstream somatic lineages
Factor et al. report that the transition from naive to primed pluripotency is regulated by activation of “seed” enhancers, which act to maintain pluripotency gene expression and prepare for transition to a somatic regulatory program.
Mutations in the noncoding genome Scacheri, Cheryl A; Scacheri, Peter C
Current opinion in pediatrics,
12/2015, Volume:
27, Issue:
6
Journal Article
Peer reviewed
Open access
Clinical diagnostic sequencing currently focuses on identifying causal mutations in the exome, wherein most disease-causing mutations are known to occur. The rest of the genome is mostly comprised of ...regulatory elements that control gene expression, but these have remained largely unexplored in clinical diagnostics due to the high cost of whole genome sequencing and interpretive challenges. The purpose of this review is to illustrate examples of diseases caused by mutations in regulatory elements and introduce the diagnostic potential for whole genome sequencing. Different classes of functional elements and chromatin structure are described to provide the clinician with a foundation for understanding the basis of these mutations.
The utilization of whole-genome sequence data, epigenomic maps and induced pluripotent stem (IPS) cell technologies facilitated the discovery that mutations in the pancreas-specific transcription factor 1a enhancer can cause isolated pancreatic agenesis. High resolution array comparative genomic hybridisation (CGH), whole-genome sequencing, maps of 3-D chromatin architecture, and mouse models generated using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas were used to show that disruption of topological-associated domain boundary elements cause limb defects. Structural variants that reposition enhancers in somatic cells have also been described in cancer.
Although not ready for diagnostics, new technologies, epigenomic maps, and improved knowledge of chromatin architecture will soon enable a better understanding and diagnostic solutions for currently unexplained genetic disorders.
Epigenetic regulation of gene enhancer elements is important for establishing and maintaining the identity of cells. Gene enhancer elements are thought to exist in either active or poised states ...distinguishable by chromatin features, but a complete understanding of the regulation of enhancers is lacking. Here, by using mouse embryonic stem cells and their differentiated derivatives, as well as terminally differentiated cells, we report the coexistence of multiple, defined classes of enhancers that serve distinct cellular functions. Specifically, we found that active enhancers can be subclassified based on varying levels of H3K4me1, H3K27ac, and H3K36me3 and the pSer2/5 forms of RNA polymerase II. The abundance of these histone modifications positively correlates with the expression of associated genes and cellular functions consistent with the identity of the cell type. Poised enhancers can also be subclassified based on presence or absence of H3K27me3 and H3K9me3, conservation, genomic location, expression levels of associated genes, and predicted function of associated genes. These findings not only refine the repertoire of histone modifications at both active and poised gene enhancer elements but also raise the possibility that enhancers associated with distinct cellular functions are partitioned based on specific combinations of histone modifications.
Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) ...has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. Here we show that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. We also show that TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. We propose that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing.
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