Discovery of functional noncoding elements by digital analysis of chromatin structure

Abstract

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 19͞20-bp genomic DNA tags to localize individual DNase I cutting events in nuclear chromatin and produced Ϸ257,000 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.cis-regulatory elements ͉ DNase I-hypersensitive sites ͉ gene regulation C omprehensive delineation of functional noncoding sequences in complex genomes is a major goal of modern biology. The activation and function of regulatory sequences is linked to focal alterations in chromatin structure (1, 2), which may be detected experimentally through hypersensitivity to DNase I in the context of nuclear chromatin. DNase I-hypersensitive sites (HSs) are the sine qua non of classical cisregulatory elements, including promoters, enhancers, silencers, insulators, and locus control regions (3-9). Systematic mapping of DNase I HSs across the genome should therefore yield a comprehensive library of cis-regulatory elements, but is intractable with conventional approaches.The feasibility of cloning DNase I HSs has recently been demonstrated by using both direct vector-assisted end cloning (10) and a subtractive enrichment approach (11). The former method is limited to quiescent cells and therefore cannot be used in the context of well-studied and widely used cell lines or other proliferating tissues. Moreover, neither method is well suited to efficient recovery of DNase I HSs on a genomewide scale because of the 1:1 mapping between cloning events and sequences; both require large amounts of sequencing and, critically, an independent molecular validation step for each candidate clone.The application of tag-based or ''digital'' methodologies has revolutionized the study of transcriptome biology (12-14), enabling both the generation of genomewide data sets and insight into the tremendous dynamic range of gene expression.Genomewide localization of DNase I HSs and associated cis-regulatory sequences requires development of a highthroughput approach that (i) can efficiently map mi...

Nucleosome-mediated cooperativity between transcription factors

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SoxF factors induce Notch1 expression via direct transcriptional regulation during early arterial development

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The active FMR1 promoter is associated with a large domain of altered chromatin conformation with embedded local histone modifications

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