Abstract
Mapping DNase I hypersensitive sites (DHSs) within nuclear chromatin is a traditional and powerful method of identifying genetic regulatory elements. DHSs have been mapped by capturing the ends of long DNase I-cut fragments (>100,000 bp), or 100–1200 bp DNase I-double cleavage fragments (also called double-hit fragments). But next generation sequencing requires a DNA library containing DNA fragments of 100–500 bp. Therefore, we used short DNA fragments released by DNase I digestion to generate DNA libraries for next generation sequencing. The short segments are 100–300 bp and can be directly cloned and used for high-throughput sequencing. We identified 83,897 DHSs in 2,343,479 tags across the human genome. Our results indicate that the DHSs identified by this DHS assay are consistent with those identified by longer fragments in previous studies. We also found: (1) the distribution of DHSs in promoter and other gene regions of similarly expressed genes differs among different chromosomes; (2) silenced genes had a more open chromatin structure than previously thought; (3) DHSs in 3′untranslated regions (3′UTRs) are negatively correlated with level of gene expression.
Highlights
In the era of functional genomics, the challenge is to elucidate gene function, regulatory networks and signaling pathways [1]
Among 14,284,385 sequence tags generated by high-throughput sequencing, we identified 10,505,670 unique mapped reads (35 bases in length) in the human genome (Table S2)
Transcriptional regulation is mediated by the interplay between cis-regulatory DNA elements and trans-acting transcription factors, and is perhaps the most important mechanism for controlling gene expression [33]
Summary
In the era of functional genomics, the challenge is to elucidate gene function, regulatory networks and signaling pathways [1]. No method yet has the resolution to precisely identify all regulatory elements or can be readily applied to the entire human genome. The classical method of mapping DNase I hypersensitive sites (DHSs) by Southern blotting has been used to identify many different types of genetic regulatory elements [4], but it can only be applied to one small region of the genome at a time. Boyle et al mapped open chromatin using a DNA library from single DNase I cleavage ends and next-generation sequencing (NGS) [12], while Sabo et al generated a DNase I library of DNA fragments (,,1200 bp) released by two-cleavage ‘hits’ occurring next to each other and identified DNase I hypersensitive sites (DHSs) using microarrays [13,14]
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