Kinetic proofreading of gene activation by chromatin remodeling

Gene activation in eukaryotes involves the concerted action of histone tail modifiers, chromatin remodellers and transcription factors, whose precise coordination is currently unknown. We demonstrate that the experimentally observed interactions of the molecules are in accord with a kinetic proofreading scheme. Our finding could provide a basis for the development of quantitative models for gene regulation in eukaryotes based on the combinatorical interactions of chromatin modifiers.Comment: 8 pages, 2 Figures; application adde

Improved ChIP-chip analysis by a mixture model approach

<p>Abstract</p> <p>Background</p> <p>Microarray analysis of immunoprecipitated chromatin (ChIP-chip) has evolved from a novel technique to a standard approach for the systematic study of protein-DNA interactions. In ChIP-chip, sites of protein-DNA interactions are identified by signals from the hybridization of selected DNA to tiled oligomers and are graphically represented as peaks. Most existing methods were designed for the identification of relatively sparse peaks, in the presence of replicates.</p> <p>Results</p> <p>We propose a data normalization method and a statistical method for peak identification from ChIP-chip data based on a mixture model approach. In contrast to many existing methods, including methods that also employ mixture model approaches, our method is more flexible by imposing less restrictive assumptions and allowing a relatively large proportion of peak regions. In addition, our method does not require experimental replicates and is computationally efficient. We compared the performance of our method with several representative existing methods on three datasets, including a spike-in dataset. These comparisons demonstrate that our approach is more robust and has comparable or higher power than the other methods, especially in the context of abundant peak regions.</p> <p>Conclusion</p> <p>Our data normalization and peak detection methods have improved performance to detect peak regions in ChIP-chip data.</p

Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites.

Alterations in chromatin structure play a major role in the epigenetic regulation of gene expression. Here, we describe a step-by-step protocol for differential viral accessibility (DIVA), a method for identifying changes in chromatin accessibility genome-wide. Commonly used methods for mapping accessible genomic loci have strong preferences toward detecting 'open' chromatin found at regulatory regions but are not well suited to studying chromatin accessibility in gene bodies and intergenic regions. DIVA overcomes this limitation, enabling a broader range of sites to be interrogated. Conceptually, DIVA is similar to ATAC-seq in that it relies on the integration of exogenous DNA into the genome to map accessible chromatin, except that chromatin architecture is probed through mapping integration sites of exogenous lentiviruses. An isogenic pair of cell lines are transduced with a lentiviral vector, followed by PCR amplification and Illumina sequencing of virus-genome junctions; the resulting sequences define a set of unique lentiviral integration sites, which are compared to determine whether genomic loci exhibit significantly altered accessibility between experimental and control cells. Experienced researchers will take 6 d to generate lentiviral stocks and transduce the target cells, a further 5 d to prepare the Illumina sequencing libraries and a few hours to perform the bioinformatic analysis

Complex exon-intron marking by histone modifications is not determined solely by nucleosome distribution

It has recently been shown that nucleosome distribution, histone modifications and RNA polymerase II (Pol II) occupancy show preferential association with exons (“exon-intron marking”), linking chromatin structure and function to co-transcriptional splicing in a variety of eukaryotes. Previous ChIP-sequencing studies suggested that these marking patterns reflect the nucleosomal landscape. By analyzing ChIP-chip datasets across the human genome in three cell types, we have found that this marking system is far more complex than previously observed. We show here that a range of histone modifications and Pol II are preferentially associated with exons. However, there is noticeable cell-type specificity in the degree of exon marking by histone modifications and, surprisingly, this is also reflected in some histone modifications patterns showing biases towards introns. Exon-intron marking is laid down in the absence of transcription on silent genes, with some marking biases changing or becoming reversed for genes expressed at different levels. Furthermore, the relationship of this marking system with splicing is not simple, with only some histone modifications reflecting exon usage/inclusion, while others mirror patterns of exon exclusion. By examining nucleosomal distributions in all three cell types, we demonstrate that these histone modification patterns cannot solely be accounted for by differences in nucleosome levels between exons and introns. In addition, because of inherent differences between ChIP-chip array and ChIP-sequencing approaches, these platforms report different nucleosome distribution patterns across the human genome. Our findings confound existing views and point to active cellular mechanisms which dynamically regulate histone modification levels and account for exon-intron marking. We believe that these histone modification patterns provide links between chromatin accessibility, Pol II movement and co-transcriptional splicing

Maps of Open Chromatin Guide the Functional Follow-Up of Genome-Wide Association Signals: Application to Hematological Traits

Turning genetic discoveries identified in genome-wide association (GWA) studies into biological mechanisms is an important challenge in human genetics. Many GWA signals map outside exons, suggesting that the associated variants may lie within regulatory regions. We applied the formaldehyde-assisted isolation of regulatory elements (FAIRE) method in a megakaryocytic and an erythroblastoid cell line to map active regulatory elements at known loci associated with hematological quantitative traits, coronary artery disease, and myocardial infarction. We showed that the two cell types exhibit distinct patterns of open chromatin and that cell-specific open chromatin can guide the finding of functional variants. We identified an open chromatin region at chromosome 7q22.3 in megakaryocytes but not erythroblasts, which harbors the common non-coding sequence variant rs342293 known to be associated with platelet volume and function. Resequencing of this open chromatin region in 643 individuals provided strong evidence that rs342293 is the only putative causative variant in this region. We demonstrated that the C- and G-alleles differentially bind the transcription factor EVI1 affecting PIK3CG gene expression in platelets and macrophages. A protein–protein interaction network including up- and down-regulated genes in Pik3cg knockout mice indicated that PIK3CG is associated with gene pathways with an established role in platelet membrane biogenesis and thrombus formation. Thus, rs342293 is the functional common variant at this locus; to the best of our knowledge this is the first such variant to be elucidated among the known platelet quantitative trait loci (QTLs). Our data suggested a molecular mechanism by which a non-coding GWA index SNP modulates platelet phenotype

Genome-Scale Validation of Deep-Sequencing Libraries

Chromatin immunoprecipitation followed by high-throughput (HTP) sequencing (ChIP-seq) is a powerful tool to establish protein-DNA interactions genome-wide. The primary limitation of its broad application at present is the often-limited access to sequencers. Here we report a protocol, Mab-seq, that generates genome-scale quality evaluations for nucleic acid libraries intended for deep-sequencing. We show how commercially available genomic microarrays can be used to maximize the efficiency of library creation and quickly generate reliable preliminary data on a chromosomal scale in advance of deep sequencing. We also exploit this technique to compare enriched regions identified using microarrays with those identified by sequencing, demonstrating that they agree on a core set of clearly identified enriched regions, while characterizing the additional enriched regions identifiable using HTP sequencing

Heterarchy of Transcription Factors Driving Basal and Luminal Cell Phenotypes in Human Urothelium

Cell differentiation is effected by complex networks of transcription factors that co-ordinate re-organisation of the chromatin landscape. The hierarchies of these relationships can be difficult to dissect. During in vitro differentiation of normal human uro-epithelial cells, formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and RNA-seq were used to identify alterations in chromatin accessibility and gene expression changes following activation of the nuclear receptor PPARG as a differentiation-initiating event. Regions of chromatin identified by FAIRE-seq as having altered accessibility during differentiation were found to be enriched with sequence-specific binding motifs for transcription factors predicted to be involved in driving basal and differentiated urothelial cell phenotypes, including FOXA1, P63, GRHL2, CTCF and GATA3. In addition, co-occurrence of GATA3 motifs was observed within sub-sets of differentiation-specific peaks containing P63 or FOXA1 after induction of differentiation. Changes in abundance of GRHL2, GATA3, and P63 were observed in immunoblots of chromatin-enriched extracts. Transient siRNA knockdown of P63 revealed that P63 favoured a basal-like phenotype by inhibiting differentiation and promoting expression of basal marker genes. GATA3 siRNA prevented differentiation-associated downregulation of P63 protein and transcript, and demonstrated positive feedback of GATA3 on PPARG transcript, but showed no effect on FOXA1 transcript or protein expression. This approach indicates that as a transcriptionally-regulated programme, urothelial differentiation operates as a heterarchy wherein GATA3 is able to co-operate with FOXA1 to drive expression of luminal marker genes, but that P63 has potential to transrepress expression of the same genes

Interaction between genetic and epigenetic variation defines gene expression patterns at the asthma-associated locus 17q12-q21 in lymphoblastoid cell lines

Phenotypic variation results from variation in gene expression, which is modulated by genetic and/or epigenetic factors. To understand the molecular basis of human disease, interaction between genetic and epigenetic factors needs to be taken into account. The asthma-associated region 17q12-q21 harbors three genes, the zona pellucida binding protein 2 (ZPBP2), gasdermin B (GSDMB) and ORM1-like 3 (ORMDL3), that show allele-specific differences in expression levels in lymphoblastoid cell lines (LCLs) and CD4+ T cells. Here, we report a molecular dissection of allele-specific transcriptional regulation of the genes within the chromosomal region 17q12-q21 combining in vitro transfection, formaldehyde-assisted isolation of regulatory elements, chromatin immunoprecipitation and DNA methylation assays in LCLs. We found that a single nucleotide polymorphism rs4795397 influences the activity of ZPBP2 promoter in vitro in an allele-dependent fashion, and also leads to nucleosome repositioning on the asthma-associated allele. However, variable methylation of exon 1 of ZPBP2 masks the strong genetic effect on ZPBP2 promoter activity in LCLs. In contrast, the ORMDL3 promoter is fully unmethylated, which allows detection of genetic effects on its transcription. We conclude that the cis-regulatory effects on 17q12-q21 gene expression result from interaction between several regulatory polymorphisms and epigenetic factors within the cis-regulatory haplotype region

Global Mapping of Cell Type–Specific Open Chromatin by FAIRE-seq Reveals the Regulatory Role of the NFI Family in Adipocyte Differentiation

Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type–specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation) and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI) transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA–mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our study demonstrates the utility of FAIRE-seq in providing a global view of cell type–specific regulatory elements in the genome and in identifying transcriptional regulators of adipocyte differentiation