Differential contribution of cis -regulatory elements to higher order chromatin structure and expression of the CFTR locus

Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTR TAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure

Transcriptome-wide association study of schizophrenia and chromatin activity yields mechanistic disease insights

Genome-wide association studies (GWAS) have identified over 100 risk loci for schizophrenia, but the causal mechanisms remain largely unknown. We performed a transcriptome-wide association study (TWAS) integrating a schizophrenia GWAS of 79,845 individuals from the Psychiatric Genomics Consortium with expression data from brain, blood, and adipose tissues across 3,693 primarily control individuals. We identified 157 TWAS-significant genes, of which 35 did not overlap a known GWAS locus. Of these 157 genes, 42 were associated with specific chromatin features measured in independent samples, thus highlighting potential regulatory targets for follow-up. Suppression of one identified susceptibility gene, mapk3, in zebrafish showed a significant effect on neurodevelopmental phenotypes. Expression and splicing from the brain captured most of the TWAS effect across all genes. This large-scale connection of associations to target genes, tissues, and regulatory features is an essential step in moving toward a mechanistic understanding of GWAS

The accessible chromatin landscape of the human genome

DNaseI hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers, and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ~2.9 million DHSs that encompass virtually all known experimentally-validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation, and regulatory factor occupancy patterns. We connect ~580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is choreographed with dozens to hundreds of co-activated elements, and the trans-cellular DNaseI sensitivity pattern at a given region can predict cell type-specific functional behaviors. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation

Evolution of New Function through a Single Amino Acid Change in the Yeast Repressor Sum1p▿

The SUM1-1 mutation is an example of a single amino acid change that results in new function. Wild-type Sum1p in Saccharomyces cerevisiae is a DNA-binding repressor that acts locally, whereas mutant Sum1-1p forms an extended repressive chromatin structure. By characterizing a panel of mutations in which various amino acids replaced the critical residue, threonine 988, we found that threonine was required for wild-type function and that in the absence of threonine the association of Sum1p with DNA was reduced. Isoleucine, the amino acid in mutant Sum1-1p, was required for the novel spreading property. Thus, the SUM1-1 mutation results in both a loss and a gain of function. The presence of isoleucine caused Sum1-1p to self-associate, a property that may promote spreading. In addition, isoleucine enabled Sum1-1p to associate with the origin recognition complex (ORC) and accumulate near ORC binding sites. Thus, both threonine and isoleucine at position 988 enable Sum1p to form intermolecular interactions. We propose that interaction domains may be hotspots for gain-of-function mutations because alterations in such domains have the potential to redirect a protein to new sets of binding partners. In addition, self-association of chromatin proteins may promote the formation of extended chromatin structures

Dynamic change in histone H3K4me3 is correlated with stress-induced gene expression change.

<p>(A) Black bars (NT) display linear trend for increasing mean gene expression values with increasing H3K4me3 decile. Grey and open bars show gene expression change is greatest among genes that lack H3K4me3 at baseline. (B) Positive correlation (r² = 0.41, <i>p</i> <0.00001) of DXR-induced change in H3K4me3 modification at the TSS with the DXR-induced change in gene expression. (C) Overrepresentation of CTCF, Polycomb, heterochromatic marks among up-regulated genes relative to repressed genes. p53-bound genes were grouped into quintiles based on levels of H3K4me3 at TSS. The frequency of CTCF, polycomb and heterochromatic marks in up-regulated genes relative to their frequencies in down-regulated genes was calculated as enrichment ratio and the enrichment ratio for each quintile is graphed (Y-axis).</p

Distribution of p53 peaks among ENCODE combined chromatin states.

<p>(A) Distribution of p53 ChIP-seq peaks among chromatin states (ChromHMM7) and the median distance (kb) of the p53-binding locations to TSS of nearby genes. (B) Distribution of chromatin states at TSS of genes nearby p53 peaks.</p

Dynamic changes in chromatin accessibility accompany gene expression change.

<p>(A, B) Genes (<i>RRAD</i> and <i>SULF2</i>) with poised promoters display H3K27me3 and H3K4me2 marks (purple boxes), CTCF (blue arrows), no H3K4me3 marks present at the TSS, and low gene expression levels. DNase I-seq experiments were carried out in duplicate and DHS data for each replicate sample is displayed (lower tracks, orange boxes). Following DXR treatment, H3K4me3 marks appear (red box), mRNA expression increases, and increases in DHS occur at 4 and 18 hrs (orange boxes). (C) <i>TGFA</i> with heterochromatin marks at the location of its p53 binding peak (green box). <i>TGFA</i> has similar H3K4me2, H3K27me3, and CTCF marks indicative of a poised promoter at baseline and also displays a large p53 peak, however, little change was observed for DHS, H3K4me3 or gene expression.</p

Chromatin state characteristics of some known p53 regulated genes.

<p>* Two large p53 binding peaks, listed relative to size.</p><p>Chromatin state characteristics of some known p53 regulated genes.</p