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Npgrj Nmeth 890 511..518 ARTICLES Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays s 1,6 1,2,6 1,2 2 2 2 d Peter J Sabo , Michael S Kuehn , Robert Thurman , Brett E Johnson , Ericka M Johnson , Hua Cao , o h 2 2 1 1 1 1 1 t Man Yu , Elizabeth Rosenzweig , Jeff Goldy , Andrew Haydock , Molly Weaver , Anthony Shafer , Kristin Lee , e 1 1 3 3 1 m Fidencio Neri , Richard Humbert , Michael A Singer , Todd A Richmond , Michael O Dorschner , e r u 4 5 3 2 1 t Michael McArthur , Michael Hawrylycz , Roland D Green , Patrick A Navas , William S Noble & a n 1 / John A Stamatoyannopoulos m o c . e r u Localized accessibility of critical DNA sequences to the entire genome and analysis of their relationship to the current t a n regulatory machinery is a key requirement for regulation of annotation of human genes. Comprehensive delineation of the . w human genes. Here we describe a high-resolution, genome-scale accessible chromatin compartment is expected to be of particular w w / approach for quantifying chromatin accessibility by measuring importance for identification of functional human genetic variants / : p t DNase I sensitivity as a continuous function of genome position that mediate individual variation in gene expression and physio- t h using tiling DNA microarrays (DNase-array). We demonstrate this logical phenotypes. On a broader level, human chromosomes p approach across 1% (B30 Mb) of the human genome, wherein have long been thought to be organized into discrete higher- u o r we localized 2,690 classical DNase I hypersensitive sites with order functional domains characterized by ‘open’ (active) G high sensitivity and specificity, and also mapped larger-scale and ‘closed’ (inactive) chromatin8–10. Experimental verification of g n i patterns of chromatin architecture. DNase I hypersensitive sites this concept on a large scale, however, has been difficult using h s exhibit marked aggregation around transcriptional start sites i available technologies. l b (TSSs), though the majority mark nonpromoter functional To address these challenges, we developed a method, DNase- u P elements. We also developed a computational approach for array, capable of measuring chromatin accessibility in vivo at high e r visualizing higher-order features of chromatin structure. This resolution on a chromosomal or even genomic scale in a single u t a revealed that human chromatin organization is dominated by experiment. We demonstrate this method on human B-lympho- N large (100–500 kb) ‘superclusters’ of DNase I hypersensitive blastoid cells, in which we comprehensively map DNase I 6 0 0 sites, which encompass both gene-rich and gene-poor regions. hypersensitive sites across 1% of the human genome with very 2 DNase-array is a powerful and straightforward approach for high sensitivity and specificity. © systematic exposition of the cis-regulatory architecture of complex genomes. RESULTS High-resolution mapping of accessible chromatin The functional landscape of the human genome in vivo is reflected Our approach, named DNase-array (Fig. 1), exploits the well- in the organization and topology of nuclear chromatin1,2.The established phenomenon of chromatin-specific sensitivity to finding, over 25 years ago3–5, that active cis-regulatory sequences DNase I in vivo, and capitalizes on the continuously expanding represent foci of heightened chromatin accessibility has been widely potential of DNA microarrays to effect massively parallel quanti- exploited to provide a window into the cis-regulatory environment fication of genomic DNA samples. of individual mammalian genes and selected loci. Enhanced chro- The hallmark of increased chromatin accessibility is the occur- matin accessibility, classically assayed by hypersensitivity to DNase I rence of multiple cleavage events over a short distance on the same in vivo,isthesine qua non of activated mammalian cis-regulatory linear nuclear chromatin template when intact nuclei are exposed sequences including promoters, enhancers, insulators, boundary to the non-specific endonuclease DNase I. To assay this phenom- elements and locus control regions6,7. enon in human lymphoblastoid cells (GM06990; Coriell), we A major challenge facing modern human genomics is systematic devised an approach for isolating genomic DNA fragments released identification of these transcriptional control elements over the by two cleavage ‘hits’ occurring close to each other (oB1,200 bp) 1Department of Genome Sciences, University of Washington, 1705 NE Pacific St., Box 357730, Seattle, Washington 98195, USA. 2Division of Medical Genetics, Department of Medicine, University of Washington, 1705 NE Pacific St., Box 357730, Seattle, Washington 98195, USA. 3Nimblegen Systems, Inc., 1 Science Court, Madison, Wisconsin 53711, USA. 4Department of Microbiology, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK. 5Allen Institute for Brain Sciences, 551 N. 34th Street, Seattle, Washington 98103, USA. 6These authors contributed equally to this work. Correspondence should be addressed to J.A.S. ([email protected]). RECEIVED 12 APRIL; ACCEPTED 22 MAY; PUBLISHED ONLINE 21 JUNE 2006; DOI:10.1038/NMETH890 NATURE METHODS | VOL.3 NO.7 | JULY 2006 | 511 ARTICLES Nuclear chromatin Naked DNA Figure 1 | Approach for high-resolution mapping of accessible chromatin in human cells using DNase-array. DNase I access to individual chromatin 1 templates is captured with high specificity by isolating DNA fragments created 2 by two DNase I–cutting ‘hits’ occurring in close proximity (o1,200 bp). These Allele . n short fragments are isolated by size fractionation on a sucrose gradient (the Specific Random red box marks lower-density fractions). To control for the possibility of intrinsic sequence preference, equally sized control fragments are isolated from DNase I–treated naked DNA. Chromatin- and nonchromatin–derived Size-fractionate fragments are differentially labeled and hybridized to a genomic tiling DNA microarray. The resulting signal reflects chromatin accessibility (log2 relative DNase I sensitivity) as a function of genomic position. M, marker. to signal ratio 42.1-fold over background), and then s d applying a peak-finding algorithm to delineate the sub-region(s) o h with maximum accessibility (see Methods). Using this approach, t MM e we localized 2,690 DNase I hypersensitive sites within the 30-Mb m e r Isolate <1,200-bp fragments experimental regions, and classified these into 1,191 ‘major’ sites u t a defined by highly significant enrichment (P o 0.001, enrich- n / m ment 46.2-fold over background), and 1,499 less intense o c Label . ‘minor’ DNase I hypersensitive sites (Supplementary Table 1 e (Cy5, Cy3) r online). We observed wide dynamic range in DNase I sensitivity u t a (4200-fold) between the least-intense and most-intense n . w genomic regions, indicative of marked diversity in nuclear chro- w w matin microenvironments. / / Hybridize to genomic : p tiling DNA microarray To assess reproducibility, we compared results from independent t t h DNase-array experiments performed more than two months apart p using chromatin preparations derived from separate cell cultures u o when intact nuclei are treated with DNase I. Consideration of only expanded from frozen cell pellets (Supplementary Fig. 1 online). r G ‘two-hit’ DNase I cutting events in the genome has the potential to Of the 1,191 major DNase I hypersensitive sites detected in the g n impart great specificity, compared with analysis of individual free first sample, we identified 94% (1,113) as hypersensitive sites in the i h DNA ends, as the latter cannot be reliably distinguished a priori second sample (732 at the P 0.001 level and 381 at the P 0.01 s o o i l B b from random shear sites. This approach also maintains a 1:1 level). It is possible that the difference of 6% reflects u P relationship between each DNase I–released fragment and each natural biological variability between different chromatin prepara- e r chromatin template. We labeled these chromatin-specific fragments tions from independent cell expansions, nuclear harvests and u t with Cy5 and labeled similarly sized control fragments (created by DNase I treatments. a N DNase I–treatment of naked genomic DNA) with Cy3. To visualize To determine whether DNase I hypersensitive sites identified by 6 0 the chromatin accessibility pattern as a function of genomic DNase-array correspond with classical DNase I hypersensitive sites, 0 2 position at high resolution, we hybridized the mixture to a high- we performed extensive conventional DNase I hypersensitivity © density oligonucleotide DNA microarray comprising 390,000 mapping in lymphoblast chromatin using the well-established 50-mer oligonucleotides consecutively tiled with a 12-bp overlap Southern end-label strategy3,6,12 (Fig. 2b–g and Supplementary (net resolution of 38 bp) over nonrepetitive regions of 44 genomic Fig. 2 online). Each conventional assay interrogates a restriction loci (the ENCODE regions11), which collectively span B30 Mb, or fragment within which DNase I hypersensitive sites appear as sub- 1% of the human genome. For each genomic position interrogated bands on a Southern blot (for example, Fig. 2b–g and Supple- by the microarray, we quantified chromatin accessibility by com- mentary Fig. 1). To ensure that the conventional assays were puting the ratio of the resulting signals, expressed as the ratio of unbiased, we designed a restriction fragment tiling path to provide chromatin-specific (Cy5) versus nonspecific (Cy3) DNase I activity. end-to-end coverage across two large genomic regions on chromo- This provided a continuous quantitative picture of chromatin somes 5 and 11 (Supplementary Table 2 online). We successfully accessibility as a function of genome position. Exemplary results performed and analyzed 151 conventional DNase I hypersensitiv- from a 1-Mb region of chromosome 5 containing the Th2 cytokine ity assays, collectively interrogating B836 kb of genomic terrain, gene cluster are shown in Figure 2a. and successfully collected data over large (c100 kb) contiguous DNase I hypersensitive sites result from binding of trans-acting blocks of sequence (Supplementary Table 2).
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