cis-Regulatory Complexity within a Large Non-Coding Region in the Drosophila Genome

Analysis of cis-regulatory enhancers has revealed that they consist of clustered blocks of highly conserved sequences. Although most characterized enhancers reside near their target genes, a growing number of studies have shown that enhancers located over 50 kb from their minimal promoter(s) are required for appropriate gene expression and many of these ‘long-range’ enhancers are found in genomic regions that are devoid of identified exons. To gain insight into the complexity of Drosophila cis-regulatory sequences within exon-poor regions, we have undertaken an evolutionary analysis of 39 of these regions located throughout the genome. This survey revealed that within these genomic expanses, clusters of conserved sequence blocks (CSBs) are positioned once every 1.1 kb, on average, and that a typical cluster contains multiple (5 to 30 or more) CSBs that have been maintained for at least 190 My of evolutionary divergence. As an initial step toward assessing the cis-regulatory activity of conserved clusters within gene-free genomic expanses, we have tested the in-vivo enhancer activity of 19 consecutive CSB clusters located in the middle of a 115 kb gene-poor region on the 3^rd chromosome. Our studies revealed that each cluster functions independently as a specific spatial/temporal enhancer. In total, the enhancers possess a diversity of regulatory functions, including dynamically activating expression in defined patterns within subsets of cells in discrete regions of the embryo, larvae and/or adult. We also observed that many of the enhancers are multifunctional–that is, they activate expression during multiple developmental stages. By extending these results to the rest of the Drosophila genome, which contains over 70,000 non-coding CSB clusters, we suggest that most function as enhancers.     

Genome-wide enhancer prediction from epigenetic signatures using genetic algorithm-optimized support vector machines

The chemical modification of histones at specific DNA regulatory elements is linked to the activation, inactivation and poising of genes. A number of tools exist to predict enhancers from chromatin modification maps, but their practical application is limited because they either (i) consider a smaller number of marks than those necessary to define the various enhancer classes or (ii) work with an excessive number of marks, which is experimentally unviable. We have developed a method for chromatin state detection using support vector machines in combination with genetic algorithm optimization, called ChromaGenSVM. ChromaGenSVM selects optimum combinations of specific histone epigenetic marks to predict enhancers. In an independent test, ChromaGenSVM recovered 88% of the experimentally supported enhancers in the pilot ENCODE region of interferon gamma-treated HeLa cells. Furthermore, ChromaGenSVM successfully combined the profiles of only five distinct methylation and acetylation marks from ChIP-seq libraries done in human CD4⁺ T cells to predict ∼21 000 experimentally supported enhancers within 1.0 kb regions and with a precision of ∼90%, thereby improving previous predictions on the same dataset by 21%. The combined results indicate that ChromaGenSVM comfortably outperforms previously published methods and that enhancers are best predicted by specific combinations of histone methylation and acetylation marks.     

Assessing genome-wide dynamic changes in enhancer activity during early mESC differentiation by FAIRE-STARR-seq

Embryonic stem cells (ESCs) can differentiate into any given cell type and therefore represent a versatile model to study the link between gene regulation and differentiation. To quantitatively assess the dynamics of enhancer activity during the early stages of murine ESC differentiation, we analyzed accessible genomic regions using STARR-seq, a massively parallel reporter assay. This resulted in a genome-wide quantitative map of active mESC enhancers, in pluripotency and during the early stages of differentiation. We find that only a minority of accessible regions is active and that such regions are enriched near promoters, characterized by specific chromatin marks, enriched for distinct sequence motifs, and modeling shows that active regions can be predicted from sequence alone. Regions that change their activity upon retinoic acid-induced differentiation are more prevalent at distal intergenic regions when compared to constitutively active enhancers. Further, analysis of differentially active enhancers verified the contribution of individual TF motifs toward activity and inducibility as well as their role in regulating endogenous genes. Notably, the activity of retinoic acid receptor alpha (RARα) occupied regions can either increase or decrease upon the addition of its ligand, retinoic acid, with the direction of the change correlating with spacing and orientation of the RARα consensus motif and the co-occurrence of additional sequence motifs. Together, our genome-wide enhancer activity map elucidates features associated with enhancer activity levels, identifies regulatory regions disregarded by computational prediction tools, and provides a resource for future studies into regulatory elements in mESCs.     

原钙粘蛋白基因簇调控区域中成簇的CTCF结合位点分析

哺乳动物中原钙粘蛋白(Protocadherin, Pcdh)基因簇包含50多个串联排列的基因,这些基因形成3个紧密相连的基因簇(Pcdhα、Pcdhβ和Pcdhγ),所编码的原钙粘蛋白质群在神经元多样性(Neuronal diversity)和单细胞特异性(Single cell identity)以及神经突触信号转导中发挥重要作用。前期的工作已证实转录因子CTCF(CCCTC-binding factor)与CTCF结合位点(CTCF-binding site, CBS)的方向性结合能够决定增强子和启动子环化的方向以及其远距离交互作用的特异性,并进一步在Pcdh基因座(Locus)形成两个(Pcdhα和Pcdhγ)染色质拓扑结构域(CTCF/cohesin- mediated chromatin domain, CCD),而且染色质拓扑结构域对于控制基因表达调控至关重要。本文通过生物信息学方法对比人类和小鼠序列,发现Pcdhβγ染色质拓扑结构域调控区域中的DNase I超敏位点(DNase I hypersensitive sites, HSs)较为保守。染色质免疫沉淀及大规模测序实验(Chromatin immunoprecipitation and massive parallel sequencing, ChIP-Seq)揭示CBS位点在Pcdhβγ调控区域中成簇分布并且具有相同的方向。凝胶电泳迁移实验(Electrophoresis mobility shift assay, EMSA)确定Pcdhβγ调控区域内具体的42 bp CBS位点并且发现一个CTCF峰包含两个CBS位点。在全基因组范围内,运用计算生物学方法分析CTCF和增强子、启动子等调控元件的关系,发现CBS位点在调控元件附近有较多分布,推测CTCF通过介导增强子和启动子的特异性交互作用,在细胞核三维基因组内形成活性转录枢纽调控基因精准表达。     

Enrichment of Genetic Variants for Rheumatoid Arthritis within T-Cell and NK-Cell Enhancer Regions

To identify disease-causative variants, we intersected the published results of a metaanalysis of genome-wide association studies (GWAS) for rheumatoid arthritis (RA) with the set of enhancer regions for 71 primary cell types that was provided by the FANTOM consortium. We first retrieved all single nucleotide polymorphisms (SNPs) that are associated (P < 5 × 10⁸) with RA in the GWAS meta-analysis and that are located in any of these enhancer regions. After excluding the major histocompatibility complex (MHC) region, we identified 50 such RA-associated SNPs that are located in enhancer regions. Enhancer sets from different cell types were then compared with each other for their number of RA-associated SNPs by permutation analysis. This analysis showed that RA-associated SNPs are preferentially located in enhancers from several immunological cell types. In particular, we see a strong relative enrichment in enhancer regions that are active in T cells (P < 0.001) and NK cells (P < 0.001). Several loci display multiple RA-associated SNPs in tight linkage disequilibrium that are located within the same or neighboring enhancers. These haplotypes may have a greater likelihood to influence enhancer activity than any SNP on its own. Taken together, these results support the hypothesis that RA-causative variants often act through altering the activity of immune cell enhancers. The enrichment in T-cell and NK-cell enhancer regions indicates that expression changes in these cell types are particularly relevant for the pathogenesis of RA. The specific SNPs that account for this enrichment can be used as a basis for focused genotype-phenotype studies of these cell types.