Predicting cell types and genetic variations contributing to disease by combining GWAS and epigenetic data

Genome-wide association studies (GWASs) identify single nucleotide polymorphisms (SNPs) that are enriched in individuals suffering from a given disease. Most disease-associated SNPs fall into non-coding regions, so that it is not straightforward to infer phenotype or function; moreover, many SNPs are in tight genetic linkage, so that a SNP identified as associated with a particular disease may not itself be causal, but rather signify the presence of a linked SNP that is functionally relevant to disease pathogenesis. Here, we present an analysis method that takes advantage of the recent rapid accumulation of epigenomics data to address these problems for some SNPs. Using asthma as a prototypic example; we show that non-coding disease-associated SNPs are enriched in genomic regions that function as regulators of transcription, such as enhancers and promoters. Identifying enhancers based on the presence of the histone modification marks such as H3K4me1 in different cell types, we show that the location of enhancers is highly cell-type specific. We use these findings to predict which SNPs are likely to be directly contributing to disease based on their presence in regulatory regions, and in which cell types their effect is expected to be detectable. Moreover, we can also predict which cell types contribute to a disease based on overlap of the disease-associated SNPs with the locations of enhancers present in a given cell type. Finally, we suggest that it will be possible to re-analyze GWAS studies with much higher power by limiting the SNPs considered to those in coding or regulatory regions of cell types relevant to a given disease

Glycogen Synthase Kinase (GSK) 3β phosphorylates and protects nuclear myosin 1c from proteasome-mediated degradation to activate rDNA transcription in early G1 cells

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3β phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3β selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3β-/- mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3β directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3β-mediated phosphorylation of NM1 is required for pol I transcription activation

Otitis Media in a New Mouse Model for CHARGE Syndrome with a Deletion in the Chd7 Gene

Otitis media is a middle ear disease common in children under three years old. Otitis media can occur in normal individuals with no other symptoms or syndromes, but it is often seen in individuals clinically diagnosed with genetic diseases such as CHARGE syndrome, a complex genetic disease caused by mutation in the Chd7 gene and characterized by multiple birth defects. Although otitis media is common in human CHARGE syndrome patients, it has not been reported in mouse models of CHARGE syndrome. In this study, we report a mouse model with a spontaneous deletion mutation in the Chd7 gene and with chronic otitis media of early onset age accompanied by hearing loss. These mice also exhibit morphological alteration in the Eustachian tubes, dysregulation of epithelial proliferation, and decreased density of middle ear cilia. Gene expression profiling revealed up-regulation of Muc5ac, Muc5b and Tgf-β1 transcripts, the products of which are involved in mucin production and TGF pathway regulation. This is the first mouse model of CHARGE syndrome reported to show otitis media with effusion and it will be valuable for studying the etiology of otitis media and other symptoms in CHARGE syndrome

CHD7 Mutational Analysis and Clinical Considerations for Auditory Rehabilitation in Deaf Patients with CHARGE Syndrome

BACKGROUND: Otologic manifestations are one of the most consistent findings of CHARGE syndrome found in more than 90%. Since genetic analysis of the CHD7 gene has rarely been performed in previous reports dealing with ear abnormalities, the genotypic spectrum of CHD7 mutations was analyzed in deaf patients with CHARGE syndrome, and the clinical considerations concerning auditory rehabilitation were investigated. METHODS: Nine Korean patients with CHARGE syndrome showing profound hearing loss and semicircular canal aplasia were included. All 38 exons of CHD7 were analyzed by direct sequencing. For splice site variations, in silico and exon-trapping analyses were performed to verify the pathogenicity of nucleotide variations. Clinical features and the outcome of auditory rehabilitation were also analyzed. RESULTS: Eight of 9 patients revealed alterations of the CHD7 gene including 3 frameshift, 2 nonsense, 2 splice site, and 1 missense mutations. Five of 9 patients were clinically diagnosed as atypical CHARGE syndrome but demonstrated various mutations of the CHD7 gene. One familial case showed intra-familial variability. Radiologic findings suggesting cochleovestibular nerve deficiency were identified in most of the patients. Of the 8 patients who underwent cochlear implantation, 5 patients demonstrated favorable outcome. Larger diameter of the cochleovestibular nerve on imaging and absence of severe mental retardation were factors related to better outcome after cochlear implantation rather than the type of CHD7 mutations. Auditory brainstem implantation was performed in two patients who did not benefit from cochlear implantation. CONCLUSIONS: Genetic analysis of the CHD7 gene should be performed in cases with semicircular canal aplasia even when other typical features of CHARGE syndrome are absent. For auditory rehabilitation in CHARGE syndrome, cochlear implantation should be strongly recommended in selected cases with favorable prognostic factors. Auditory brainstem implantation may be a viable option in patients with CHARGE syndrome who have failed to benefit from cochlear implantation

Histone H3 globular domain acetylation identifies a new class of enhancers

Histone acetylation is generally associated with active chromatin, but most studies have focused on the acetylation of histone tails. Various histone H3 and H4 tail acetylations mark the promoters of active genes. These modifications include acetylation of histone H3 at lysine 27 (H3K27ac), which blocks Polycomb-mediated trimethylation of H3K27 (H3K27me3). H3K27ac is also widely used to identify active enhancers, and the assumption has been that profiling H3K27ac is a comprehensive way of cataloguing the set of active enhancers in mammalian cell types. Here we show that acetylation of lysine residues in the globular domain of histone H3 (lysine 64 (H3K64ac) and lysine 122 (H3K122ac)) marks active gene promoters and also a subset of active enhancers. Moreover, we find a new class of active functional enhancers that is marked by H3K122ac but lacks H3K27ac. This work suggests that, to identify enhancers, a more comprehensive analysis of histone acetylation is required than has previously been considered

Clustered ChIP-Seq-defined transcription factor binding sites and histone modifications map distinct classes of regulatory elements

<p>Abstract</p> <p>Background</p> <p>Transcription factor binding to DNA requires both an appropriate binding element and suitably open chromatin, which together help to define regulatory elements within the genome. Current methods of identifying regulatory elements, such as promoters or enhancers, typically rely on sequence conservation, existing gene annotations or specific marks, such as histone modifications and p300 binding methods, each of which has its own biases.</p> <p>Results</p> <p>Herein we show that an approach based on clustering of transcription factor peaks from high-throughput sequencing coupled with chromatin immunoprecipitation (Chip-Seq) can be used to evaluate markers for regulatory elements. We used 67 data sets for 54 unique transcription factors distributed over two cell lines to create regulatory element clusters. By integrating the clusters from our approach with histone modifications and data for open chromatin, we identified general methylation of lysine 4 on histone H3 (H3K4me) as the most specific marker for transcription factor clusters. Clusters mapping to annotated genes showed distinct patterns in cluster composition related to gene expression and histone modifications. Clusters mapping to intergenic regions fall into two groups either directly involved in transcription, including miRNAs and long noncoding RNAs, or facilitating transcription by long-range interactions. The latter clusters were specifically enriched with H3K4me1, but less with acetylation of lysine 27 on histone 3 or p300 binding.</p> <p>Conclusion</p> <p>By integrating genomewide data of transcription factor binding and chromatin structure and using our data-driven approach, we pinpointed the chromatin marks that best explain transcription factor association with different regulatory elements. Our results also indicate that a modest selection of transcription factors may be sufficient to map most regulatory elements in the human genome.</p

IL-10 transcription is negatively regulated by BAF180, a component of the SWI/SNF chromatin remodeling enzyme

<p>Abstract</p> <p>Background</p> <p>SWI/SNF chromatin remodeling enzymes play a critical role in the development of T helper lymphocytes, including Th2 cells, and directly program chromatin structure at Th2 cytokine genes. Different versions of SWI/SNF complexes, including BAF and PBAF, have been described based on unique subunit composition. However, the relative role of BAF and PBAF in Th cell function and cytokine expression has not been reported.</p> <p>Results</p> <p>Here we examine the role of the PBAF SWI/SNF complex in Th cell development and gene expression using mice deficient for a PBAF-specific component, BAF180. We find that T cell development in the thymus and lymphoid periphery is largely normal when the BAF180 gene is deleted late in thymic development. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus.</p> <p>Conclusions</p> <p>These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription.</p

Integrating Diverse Datasets Improves Developmental Enhancer Prediction

Gene-regulatory enhancers have been identified using various approaches, including evolutionary conservation, regulatory protein binding, chromatin modifications, and DNA sequence motifs. To integrate these different approaches, we developed EnhancerFinder, a two-step method for distinguishing developmental enhancers from the genomic background and then predicting their tissue specificity. EnhancerFinder uses a multiple kernel learning approach to integrate DNA sequence motifs, evolutionary patterns, and diverse functional genomics datasets from a variety of cell types. In contrast with prediction approaches that define enhancers based on histone marks or p300 sites from a single cell line, we trained EnhancerFinder on hundreds of experimentally verified human developmental enhancers from the VISTA Enhancer Browser. We comprehensively evaluated EnhancerFinder using cross validation and found that our integrative method improves the identification of enhancers over approaches that consider a single type of data, such as sequence motifs, evolutionary conservation, or the binding of enhancer-associated proteins. We find that VISTA enhancers active in embryonic heart are easier to identify than enhancers active in several other embryonic tissues, likely due to their uniquely high GC content. We applied EnhancerFinder to the entire human genome and predicted 84,301 developmental enhancers and their tissue specificity. These predictions provide specific functional annotations for large amounts of human non-coding DNA, and are significantly enriched near genes with annotated roles in their predicted tissues and lead SNPs from genome-wide association studies. We demonstrate the utility of EnhancerFinder predictions through in vivo validation of novel embryonic gene regulatory enhancers from three developmental transcription factor loci. Our genome-wide developmental enhancer predictions are freely available as a UCSC Genome Browser track, which we hope will enable researchers to further investigate questions in developmental biology. © 2014 Erwin et al

SOX2 Co-Occupies Distal Enhancer Elements with Distinct POU Factors in ESCs and NPCs to Specify Cell State

SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors