Localized accessibility of critical DNA sequences to the regulatory machinery is a key requirement for regulation of human genes. Here we describe a high-resolution, genome-scale approach for quantifying chromatin accessibility by measuring DNase I sensitivity as a continuous function of genome position using tiling DNA microarrays (DNase-array). We demonstrate this approach across 1% ( approximately 30 Mb) of the human genome, wherein we localized 2,690 classical DNase I hypersensitive sites with high sensitivity and specificity, and also mapped larger-scale patterns of chromatin architecture. DNase I hypersensitive sites exhibit marked aggregation around transcriptional start sites (TSSs), though the majority mark nonpromoter functional elements. We also developed a computational approach for visualizing higher-order features of chromatin structure. This revealed that human chromatin organization is dominated by large (100-500 kb) 'superclusters' of DNase I hypersensitive sites, which encompass both gene-rich and gene-poor regions. DNase-array is a powerful and straightforward approach for systematic exposition of the cis-regulatory architecture of complex genomes.

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...

Identification of functional, noncoding elements that regulate transcription in the context of complex genomes is a major goal of modern biology. Localization of functionality to specific sequences is a requirement for genetic and computational studies. Here, we describe a generic approach, quantitative chromatin profiling, that uses quantitative analysis of in vivo chromatin structure over entire gene loci to rapidly and precisely localize cis-regulatory sequences and other functional modalities encoded by DNase I hypersensitive sites. To demonstrate the accuracy of this approach, we analyzed B300 kilobases of human genome sequence from diverse gene loci and cleanly delineated functional elements corresponding to a spectrum of classical cis-regulatory activities including enhancers, promoters, locus control regions and insulators as well as novel elements. Systematic, highthroughput identification of functional elements coinciding with DNase I hypersensitive sites will substantially expand our knowledge of transcriptional regulation and should simplify the search for noncoding genetic variation with phenotypic consequences.Understanding the human genome will require comprehensive delineation of functional elements within the 98% of genomic terrain that does not encode protein. In vivo, cis-regulatory modalities colocalize with focal alterations in chromatin structure [1][2][3][4] , and this governs the accessibility of genomic sequences to critical regulatory factors. Exploitation of the close connection between functional elements and chromatin structure should offer a powerful and generic approach for de novo identification of cis-regulatory sequences in the context of complex gene domains.Active regulatory elements within complex genomes are distinguished by pronounced sensitivity to the nonspecific endonuclease DNase I 3-5 when exposed in the context of intact nuclei. DNase I hypersensitive sites are the sine qua non of a diverse spectrum of classical transcriptional and chromosomal regulatory activities including enhancers, promoters, silencers, insulators, boundary elements and locus control regions 1,3,6 . Indeed, in the human genome, many functional elements were first identified as major DNase I hypersensitive sites and only later were found to have specific regulatory roles. Analysis of chromatin structure may enable generic delineation of functional elements across the genome, provided it exhibits direct sequence specificity, quantitative data output that permits automated analysis, and adaptability to a high-throughput format.DNase I hypersensitive sites in native genomic domains have traditionally been localized by an approach relying on Southern transfer followed by indirect end-labeling 5 . Although widely applied, this technique is not quantitative and has numerous technical and resource-related limitations that prevent its application on a genome-wide scale. The major limitations of conventional hypersensitivity assays are the low throughput and the lack of sequence specificity. Conventional So...

We employ real-time PCR to allow us to quantify the sensitivity of chromatin to digestion by DNaseI. This approach has three clear advantages over the more conventional use of the Southern hybridization assay: the accuracy of quantification is improved; the resolution of the assay is enhanced, by designing primers to amplify small amplicons it is possible to analyze sequences both co-incident and proximal to sites of DNaseI-hypersensitivity; less material is needed, as little as 5 ng of treated genomic DNA. We applied this method in an analysis of the chromatin structure of the previously described mouse β-globin locus control region (LCR) using fetal liver cells. The four hypersensitive sites of the canonical mouse LCR, HS1 to HS4, are shown to have kinetics of digestion consistent with these sequences being nucleosome-free in vivo. A different pattern was seen for HS6, a recently described "weak" hypersensitive site. The site was also rapidly lost but more of the sites proved resistant, we interpreted this to show that this hypersensitive was only forming in a portion of the erythroid cells. This finding implies that in vivo the LCR is structurally heterogeneous. Sequences proximal to the hypersensitive sites show a third pattern of intermediate sensitivity, consistent with the chromatin being unfolded but the sites still bound by a continual nucleosomal array. Our results demonstrate that this method has the potential to achieve accurate and detailed mapping of chromatin structure from small amounts of tissue samples.