We developed a quantitative methodology, digital analysis of chromatin structure (DACS), for high-throughput, automated mapping of DNase I-hypersensitive sites and associated cis-regulatory sequences in the human and other complex genomes. We used 19/20-bp genomic DNA tags to localize individual DNase I cutting events in nuclear chromatin and produced approximately 257,000 tags from erythroid cells. Tags were mapped to the human genome, and a quantitative algorithm was applied to discriminate statistically significant clusters of independent DNase I cutting events. We show that such clusters identify both known regulatory sequences and previously unrecognized functional elements across the genome. We used in silico simulation to demonstrate that DACS is capable of efficient and accurate localization of the majority of DNase I-hypersensitive sites in the human genome without requiring an independent validation step. A unique feature of DACS is that it permits unbiased evaluation of the chromatin state of regulatory sequences from widely separated genomic loci. We found surprisingly large differences in the accessibility of distant regulatory sequences, suggesting the existence of a hierarchy of nuclear organization that escapes detection by conventional chromatin assays.
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"In fact, 526 distal DNA elements were significantly enriched by both p53 and JMJD3. By including genome-wide data on DNase I hypersensitivity and H3K4me3 from BJ cells , ,  we observed that while p53 bound promoters were both DNase I hypersensitive and associated with H3K4me3, the p53 bound distal elements were largely depleted from H3K4me3 but still DNase I hypersensitive (Fig. 4a). Thus, we found that 86% of the distal DNA elements, which were co-bound by JMJD3 and p53, overlap with a DNase I hypersensitive site. "
[Show abstract][Hide abstract] ABSTRACT: The JmjC domain-containing protein JMJD3/KDM6B catalyses the demethylation of H3K27me3 and H3K27me2. JMJD3 appears to be highly regulated at the transcriptional level and is upregulated in response to diverse stimuli such as differentiation inducers and stress signals. Accordingly, JMJD3 has been linked to the regulation of different biological processes such as differentiation of embryonic stem cells, inflammatory responses in macrophages, and induction of cellular senescence via regulation of the INK4A-ARF locus. Here we show here that JMJD3 interacts with the tumour suppressor protein p53. We find that the interaction is dependent on the p53 tetramerization domain. Following DNA damage, JMJD3 is transcriptionally upregulated and by performing genome-wide mapping of JMJD3, we demonstrate that it binds genes involved in basic cellular processes, as well as genes regulating cell cycle, response to stress and apoptosis. Moreover, we find that JMJD3 binding sites show significant overlap with p53 bound promoters and enhancer elements. The binding of JMJD3 to p53 target sites is increased in response to DNA damage, and we demonstrate that the recruitment of JMJD3 to these sites is dependent on p53 expression. Therefore, we propose a model in which JMJD3 is recruited to p53 responsive elements via its interaction with p53 and speculate that JMJD3 could act as a fail-safe mechanism to remove low levels of H3K27me3 and H3K27me2 to allow for efficient acetylation of H3K27.
PLoS ONE 05/2014; 9(5):e96545. DOI:10.1371/journal.pone.0096545 · 3.23 Impact Factor
"We utilized data on genome-wide, high-resolution chromatin accessibility measurements for 20 distinct human primary and culture cell lines that were obtained by an established sequencing-based method . In principle, accessible " open " chromatin is cleaved by the non-specific endonuclease DNaseI, and the cleaved fragments are sequenced to provide a high-resolution, genome-wide map of DNaseI hypersensitivity (DHS) for every cell-type (SI, Table S2). "
[Show abstract][Hide abstract] ABSTRACT: A problem of substantial interest is to systematically map variation in chromatin structure to gene-expression regulation across conditions, environments, or differentiated cell types. We developed and applied a quantitative framework for determining the existence, strength, and type of relationship between high-resolution chromatin structure in terms of DNaseI hypersensitivity and genome-wide gene-expression levels in 20 diverse human cell types. We show that ∼25% of genes show cell-type-specific expression explained by alterations in chromatin structure. We find that distal regions of chromatin structure (e.g., ±200 kb) capture more genes with this relationship than local regions (e.g., ±2.5 kb), yet the local regions show a more pronounced effect. By exploiting variation across cell types, we were capable of pinpointing the most likely hypersensitive sites related to cell-type-specific expression, which we show have a range of contextual uses. This quantitative framework is likely applicable to other settings aimed at relating continuous genomic measurements to gene-expression variation.
Proceedings of the National Academy of Sciences 01/2014; 111(6). DOI:10.1073/pnas.1312523111 · 9.67 Impact Factor
"Genome-wide locations of eleven histone modifications (H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3, H3K79me2, H3K9me1, H3K9me3, H4K20me1, H3K9ac, and H3K27ac) and one histone variant (H2A.Z) were generated by the Broad/MGH ENCODE group using ChIP-Seq , and are available from the Gene Expression Omnibus (GEO; accession number GSE29611). DNase I hypersensitivity was measured genome-wide using the Digital DNaseI methodology , and can be accessed via GEO accession number GSE32970. Uniformly processed genome-wide signal tracks for these signals were downloaded in bigwig format from the ENCODE project website . "
[Show abstract][Hide abstract] ABSTRACT: Background
Previous work has demonstrated that chromatin feature levels correlate with gene expression. The ENCODE project enables us to further explore this relationship using an unprecedented volume of data. Expression levels from more than 100,000 promoters were measured using a variety of high-throughput techniques applied to RNA extracted by different protocols from different cellular compartments of several human cell lines. ENCODE also generated the genome-wide mapping of eleven histone marks, one histone variant, and DNase I hypersensitivity sites in seven cell lines.
We built a novel quantitative model to study the relationship between chromatin features and expression levels. Our study not only confirms that the general relationships found in previous studies hold across various cell lines, but also makes new suggestions about the relationship between chromatin features and gene expression levels. We found that expression status and expression levels can be predicted by different groups of chromatin features, both with high accuracy. We also found that expression levels measured by CAGE are better predicted than by RNA-PET or RNA-Seq, and different categories of chromatin features are the most predictive of expression for different RNA measurement methods. Additionally, PolyA+ RNA is overall more predictable than PolyA- RNA among different cell compartments, and PolyA+ cytosolic RNA measured with RNA-Seq is more predictable than PolyA+ nuclear RNA, while the opposite is true for PolyA- RNA.
Our study provides new insights into transcriptional regulation by analyzing chromatin features in different cellular contexts.