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Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells.
Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14+ monocytes, CD16+ neutrophils, and naive CD4+ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.
eFORGE: A Tool for Identifying Cell Type-Specific Signal in Epigenomic Data.
Epigenome-wide association studies (EWAS) provide an alternative approach for studying human disease through consideration of non-genetic variants such as altered DNA methylation. To advance the complex interpretation of EWAS, we developed eFORGE (http://eforge.cs.ucl.ac.uk/), a new standalone and web-based tool for the analysis and interpretation of EWAS data. eFORGE determines the cell type-specific regulatory component of a set of EWAS-identified differentially methylated positions. This is achieved by detecting enrichment of overlap with DNase I hypersensitive sites across 454 samples (tissues, primary cell types, and cell lines) from the ENCODE, Roadmap Epigenomics, and BLUEPRINT projects. Application of eFORGE to 20 publicly available EWAS datasets identified disease-relevant cell types for several common diseases, a stem cell-like signature in cancer, and demonstrated the ability to detect cell-composition effects for EWAS performed on heterogeneous tissues. Our approach bridges the gap between large-scale epigenomics data and EWAS-derived target selection to yield insight into disease etiology.
Discovery and refinement of genetic loci associated with cardiometabolic risk using dense imputation maps.
Large-scale whole-genome sequence data sets offer novel opportunities to identify genetic variation underlying human traits. Here we apply genotype imputation based on whole-genome sequence data from the UK10K and 1000 Genomes Project into 35,981 study participants of European ancestry, followed by association analysis with 20 quantitative cardiometabolic and hematological traits. We describe 17 new associations, including 6 rare (minor allele frequency (MAF) < 1%) or low-frequency (1% < MAF < 5%) variants with platelet count (PLT), red blood cell indices (MCH and MCV) and HDL cholesterol. Applying fine-mapping analysis to 233 known and new loci associated with the 20 traits, we resolve the associations of 59 loci to credible sets of 20 or fewer variants and describe trait enrichments within regions of predicted regulatory function. These findings improve understanding of the allelic architecture of risk factors for cardiometabolic and hematological diseases and provide additional functional insights with the identification of potentially novel biological targets.
The Allelic Landscape of Human Blood Cell Trait Variation and Links to Common Complex Disease.
Many common variants have been associated with hematological traits, but identification of causal genes and pathways has proven challenging. We performed a genome-wide association analysis in the UK Biobank and INTERVAL studies, testing 29.5 million genetic variants for association with 36 red cell, white cell, and platelet properties in 173,480 European-ancestry participants. This effort yielded hundreds of low frequency (<5%) and rare (<1%) variants with a strong impact on blood cell phenotypes. Our data highlight general properties of the allelic architecture of complex traits, including the proportion of the heritable component of each blood trait explained by the polygenic signal across different genome regulatory domains. Finally, through Mendelian randomization, we provide evidence of shared genetic pathways linking blood cell indices with complex pathologies, including autoimmune diseases, schizophrenia, and coronary heart disease and evidence suggesting previously reported population associations between blood cell indices and cardiovascular disease may be non-causal.
A rare variant in APOC3 is associated with plasma triglyceride and VLDL levels in Europeans.
The analysis of rich catalogues of genetic variation from population-based sequencing provides an opportunity to screen for functional effects. Here we report a rare variant in APOC3 (rs138326449-A, minor allele frequency ~0.25% (UK)) associated with plasma triglyceride (TG) levels (-1.43 s.d. (s.e.=0.27 per minor allele (P-value=8.0 × 10(-8))) discovered in 3,202 individuals with low read-depth, whole-genome sequence. We replicate this in 12,831 participants from five additional samples of Northern and Southern European origin (-1.0 s.d. (s.e.=0.173), P-value=7.32 × 10(-9)). This is consistent with an effect between 0.5 and 1.5 mmol l(-1) dependent on population. We show that a single predicted splice donor variant is responsible for association signals and is independent of known common variants. Analyses suggest an independent relationship between rs138326449 and high-density lipoprotein (HDL) levels. This represents one of the first examples of a rare, large effect variant identified from whole-genome sequencing at a population scale.
MultiMeta: an R package for meta-analyzing multi-phenotype genome-wide association studies.
UNLABELLED: As new methods for multivariate analysis of genome wide association studies become available, it is important to be able to combine results from different cohorts in a meta-analysis. The R package MultiMeta provides an implementation of the inverse-variance-based method for meta-analysis, generalized to an n-dimensional setting. AVAILABILITY AND IMPLEMENTATION: The R package MultiMeta can be downloaded from CRAN. CONTACT: dragana.vuckovic@burlo.trieste.it; vi1@sanger.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
The UK10K project identifies rare variants in health and disease.
The contribution of rare and low-frequency variants to human traits is largely unexplored. Here we describe insights from sequencing whole genomes (low read depth, 7×) or exomes (high read depth, 80×) of nearly 10,000 individuals from population-based and disease collections. In extensively phenotyped cohorts we characterize over 24 million novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with levels of triglycerides (APOB), adiponectin (ADIPOQ) and low-density lipoprotein cholesterol (LDLR and RGAG1) from single-marker and rare variant aggregation tests. We describe population structure and functional annotation of rare and low-frequency variants, use the data to estimate the benefits of sequencing for association studies, and summarize lessons from disease-specific collections. Finally, we make available an extensive resource, including individual-level genetic and phenotypic data and web-based tools to facilitate the exploration of association results.
Significant impact of miRNA-target gene networks on genetics of human complex traits.
The impact of microRNA (miRNA) on the genetics of human complex traits, especially in the context of miRNA-target gene networks, has not been fully assessed. Here, we developed a novel analytical method, MIGWAS, to comprehensively evaluate enrichment of genome-wide association study (GWAS) signals in miRNA-target gene networks. We applied the method to the GWAS results of the 18 human complex traits from >1.75 million subjects, and identified significant enrichment in rheumatoid arthritis (RA), kidney function, and adult height (P < 0.05/18 = 0.0028, most significant enrichment in RA with P = 1.7 × 10(-4)). Interestingly, these results were consistent with current literature-based knowledge of the traits on miRNA obtained through the NCBI PubMed database search (adjusted P = 0.024). Our method provided a list of miRNA and target gene pairs with excess genetic association signals, part of which included drug target genes. We identified a miRNA (miR-4728-5p) that downregulates PADI2, a novel RA risk gene considered as a promising therapeutic target (rs761426, adjusted P = 2.3 × 10(-9)). Our study indicated the significant impact of miRNA-target gene networks on the genetics of human complex traits, and provided resources which should contribute to drug discovery and nucleic acid medicine.
ATRX dysfunction induces replication defects in primary mouse cells.
The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells.
The chromatin remodeller ATRX: a repeat offender in human disease.
The regulation of chromatin structure is of paramount importance for a variety of fundamental nuclear processes, including gene expression, DNA repair, replication, and recombination. The ATP-dependent chromatin-remodelling factor ATRX (α thalassaemia/mental retardation X-linked) has emerged as a key player in each of these processes. Exciting recent developments suggest that ATRX plays a variety of key roles at tandem repeat sequences within the genome, including the deposition of a histone variant, prevention of replication fork stalling, and the suppression of a homologous recombination-based pathway of telomere maintenance. Here, we provide a mechanistic overview of the role of ATRX in each of these processes, and propose how they may be connected to give rise to seemingly disparate human diseases.
Nucleosome remodeling and transcriptional repression are distinct functions of Isw1 in Saccharomyces cerevisiae.
The SANT domain is a nucleosome recognition module found in transcriptional regulatory proteins, including chromatin-modifying enzymes. It shows high functional degeneracy between species, varying in sequence and copy number. Here, we investigate functions in vivo associated with two SANT motifs, SANT and SLIDE, in the Saccharomyces cerevisiae Isw1 chromatin-remodeling ATPase. We show that differences in the primary structures of the SANT and SLIDE domains in yeast and Drosophila melanogaster reflect their different functions. In yeast, the SLIDE domain is required for histone interactions, while this is a function of the SANT domain in flies. In yeast, both motifs are required for optimal association with chromatin and for formation of the Isw1b complex (Isw1, Ioc2, and Ioc4). Moreover, nucleosome remodeling at the MET16 locus is defective in strains lacking the SANT or SLIDE domain. In contrast, the SANT domain is dispensable for the interaction between Isw1 and Ioc3 in the Isw1a complex. We show that, although defective in nucleosome remodeling, Isw1 lacking the SANT domain is able to repress transcription initiation at the MET16 promoter. Thus, chromatin remodeling and transcriptional repression are distinct activities of Isw1.
14-3-3 interaction with histone H3 involves a dual modification pattern of phosphoacetylation.
Histone modifications occur in precise patterns and are proposed to signal the recruitment of effector molecules that profoundly impact chromatin structure, gene regulation, and cell cycle events. The linked modifications serine 10 phosphorylation and lysine 14 acetylation on histone H3 (H3S10phK14ac), modifications conserved from Saccharomyces cerevisiae to humans, are crucial for transcriptional activation of many genes. However, the mechanism of H3S10phK14ac involvement in these processes is unclear. To shed light on the role of this dual modification, we utilized H3 peptide affinity assays to identify H3S10phK14ac-interacting proteins. We found that the interaction of the known phospho-binding 14-3-3 proteins with H3 is dependent on the presence of both of these marks, not just phosphorylation alone. This is true of mammalian 14-3-3 proteins as well as the yeast homologues Bmh1 and Bmh2. The importance of acetylation in this interaction is also seen in vivo, where K14 acetylation is required for optimal Bmh1 recruitment to the GAL1 promoter during transcriptional activation.
A glimpse into the epigenetic landscape of gene regulation.
Post-translational modifications to histone proteins and methylation of DNA comprise the epigenome of a cell. The epigenome, which changes through development, controls access to our genes. Recent advances in DNA sequencing technology has led to genome-wide distribution data for a limited number of histone modifications in mammalian stem cells and some differentiated lineages. These studies reveal predictive correlations between histone modifications, different classes of gene and chromosomal features. Moreover, this glimpse into our epigenome challenges current ideas about regulation of gene expression. Many genes in stem cells are poised for expression with initiated RNA polymerase II at the promoter. This state is maintained by an epigenetic mark through multiple lineages until the gene is expressed.
Molecular basis for the recognition of phosphorylated and phosphoacetylated histone h3 by 14-3-3.
Phosphorylation of histone H3 is implicated in transcriptional activation and chromosome condensation, but its immediate molecular function has remained obscure. By affinity chromatography of nuclear extracts against modified H3 tail peptides, we identified 14-3-3 isoforms as proteins that bind these tails in a strictly phosphorylation-dependent manner. Acetylation of lysines 9 and 14 does not impede 14-3-3 binding to serine 10-phosphorylated H3 tails. In vivo, 14-3-3 is inducibly recruited to c-fos and c-jun nucleosomes upon gene activation, concomitant with H3 phosphoacetylation. We have determined the structures of 14-3-3zeta complexed with serine 10-phosphorylated or phosphoacetylated H3 peptides. These reveal a distinct mode of 14-3-3/phosphopeptide binding and provide a structural understanding for the lack of effect of acetylation at lysines 9 and 14 on this interaction. 14-3-3 isoforms thus represent a class of proteins that mediate the effect of histone phosphorylation at inducible genes.
JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response.
Cancers result from the accumulation of genetic lesions, but the cellular consequences of driver mutations remain unclear, especially during the earliest stages of malignancy. The V617F mutation in the JAK2 non-receptor tyrosine kinase (JAK2V617F) is present as an early somatic event in most patients with myeloproliferative neoplasms (MPNs), and the study of these chronic myeloid malignancies provides an experimentally tractable approach to understanding early tumorigenesis. Introduction of exogenous JAK2V617F impairs replication fork progression and is associated with activation of the intra-S checkpoint, with both effects mediated by phosphatidylinositide 3-kinase (PI3K) signaling. Analysis of clonally derived JAK2V617F-positive erythroblasts from MPN patients also demonstrated impaired replication fork progression accompanied by increased levels of replication protein A (RPA)-containing foci. However, the associated intra-S checkpoint response was impaired in erythroblasts from polycythemia vera (PV) patients, but not in those from essential thrombocythemia (ET) patients. Moreover, inhibition of p53 in PV erythroblasts resulted in more gamma-H2Ax (γ-H2Ax)-marked double-stranded breaks compared with in like-treated ET erythroblasts, suggesting the defective intra-S checkpoint function seen in PV increases DNA damage in the context of attenuated p53 signaling. These results demonstrate oncogene-induced impairment of replication fork progression in primary cells from MPN patients, reveal unexpected disease-restricted differences in activation of the intra-S checkpoint, and have potential implications for the clonal evolution of malignancies.
Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX.
Fifteen per cent of cancers maintain telomere length independently of telomerase by the homologous recombination (HR)-associated alternative lengthening of telomeres (ALT) pathway. A unifying feature of these tumours are mutations in ATRX. Here we show that expression of ectopic ATRX triggers a suppression of the pathway and telomere shortening. Importantly ATRX-mediated ALT suppression is dependent on the histone chaperone DAXX. Re-expression of ATRX is associated with a reduction in replication fork stalling, a known trigger for HR and loss of MRN from telomeres. A G-quadruplex stabilizer partially reverses the effect of ATRX, inferring ATRX may normally facilitate replication through these sequences that, if they persist, promote ALT. We propose that defective telomere chromatinization through loss of ATRX promotes the persistence of aberrant DNA secondary structures, which in turn present a barrier to DNA replication, leading to replication fork stalling, collapse, HR and subsequent recombination-mediated telomere synthesis in ALT cancers.