On DNA Methylation Carcinogenesis 2006 Dec;27(12):2409-23. Epub 2006 Sep 4 2006-12-01 2006-09-04 http://dx.doi.org/10.1093/carcin/bgl161 http://carcin.oxfordjournals.org/cgi/reprint/27/12/2409 http://carcin.oxfordjournals.org/cgi/content/full/bgl161/DC1 Using a unique microarray platform for cytosine methylation profiling, the DNA methylation landscape of the human genome was monitored at more than 21,000 sites, including 79% of the annotated transcriptional start sites (TSS). Analysis of an oligodendroglioma derived cell line LN-18 revealed more than 4,000 methylated TSS. The gene-centric analysis indicated a complex pattern of DNA methylation exists along each autosome, with a trend of increasing density approaching the telomeres. Remarkably, 2% of CpG islands (CGI) were densely methylated, and 17% had significant levels of 5mC, whether or not they corresponded to a TSS. Substantial independent verification, obtained from 95 loci, suggested that this approach is capable of large scale detection of cytosine methylation with an accuracy approaching 90%. In addition, we detected large genomic domains that are also susceptible to DNA methylation reinforced inactivation, such as the HOX cluster on chromosome 7 (CH7). Extrapolation from the data suggests that more than 2000 genomic loci may be susceptible to methylation and associated inactivation, and most have yet to be identified. Finally, we report six new targets of epigenetic inactivation (IRX3, WNT10A, WNT6, RAR{alpha}, BMP7, and ZGPAT). These targets displayed cell line and tumor specific differential methylation when compared with normal brain samples, suggesting they may have utility as biomarkers. Uniquely, hypermethylation of the CGI within an IRX3 exon was correlated with over-expression of IRX3 in tumor tissues and cell lines relative to normal brain samples. 1 Ordway JM J. M. Ordway 1 2 Bedell JA J. A. Bedell 1 3 Citek RW R. W. Citek 1 4 Nunberg A A. Nunberg 1 5 Garrido A A. Garrido 1 6 Kendall R R. Kendall 2 7 Stevens JR J. R. Stevens 2 8 Cao D D. Cao 2 9 Doerge RW R. W. Doerge 2 10 Korshunova Y Y. Korshunova 1 11 Holemon H H. Holemon 1 12 McPherson JD J. D. McPherson 3 13 Lakey N N. Lakey 1 14 Leon J J. Leon 1 15 Martienssen RA R. A. Martienssen 4 16 Jeddeloh JA J. A. Jeddeloh 1 Orion Genomics, St. Louis, MO, USA Department of Agronomy, Purdue University, W. Lafayette, IN, USA & Department of Statistics, Purdue University, W. Lafayette, IN, USA Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX Cold Spring Harbor Laboratory, Cold Spring Harbor, NY On DNA Methylation Genome Res. 2006 Aug;16(8):1046-55. Epub 2006 Jun 29. 2006-08-01 2006-06-29 http://dx.doi.org/10.1101/gr.5273806 http://www.genome.org/cgi/reprint/16/8/1046 http://www.genome.org/cgi/content/full/gr.5273806/DC1 The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome. 1 Khulan B Batbayar Khulan 1 2 Thompson RF Reid F. Thompson 1 3 Ye K Kenny Ye 2 4 Fazzari MJ Melissa J. Fazzari 2 5 Suzuki M Masako Suzuki 3 6 Stasiek E Edyta Stasiek 3 7 Figueroa ME Maria E. Figueroa 4 8 Glass JL Jacob L. Glass 1 9 Chen Q Quan Chen 5 10 Montagna C Cristina Montagna 1,5 11 Hatchwell E Eli Hatchwell 6 12 Selzer RR Rebecca R. Selzer 7 13 Richmond TA Todd A. Richmond 7 14 Green RD Roland D. Green 7 15 Melnick A Ari Melnick 4 16 Greally JM John M. Greally 1,3 Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York Epidemiology and Population Health Albert Einstein College of Medicine, Bronx, New York Medicine (Hematology) Albert Einstein College of Medicine, Bronx, New York Developmental and Molecular Biology Albert Einstein College of Medicine, Bronx, New York Pathology, Albert Einstein College of Medicine, Bronx, New York Cold Spring Harbor Laboratories, Cold Spring Harbor, New York NimbleGen Systems Inc., Madison, Wisconsin On DNase Hypersensitivity Nature Methods 2006 Jul;3(7):511-8. 2006-07-01 2006-06-21 http://dx.doi.org/10.1038/nmeth890 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% (approx30 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. 1 Sabo PJ Peter J Sabo 1,6 2 Kuehn MS Michael S Kuehn 1,2,6 3 Thurman R Robert Thurman 1,2 4 Johnson BE Brett E Johnson 2 5 Johnson EM Ericka M Johnson 2 6 Cao H Hua Cao 2 7 Yu M Man Yu 2 8 Rosenzweig E Elizabeth Rosenzweig 2 9 Goldy J Jeff Goldy 1 10 Haydock A Andrew Haydock 1 11 Weaver M Molly Weaver 1 12 Shafer A Anthony Shafer 1 13 Lee K Kristin Lee 1 14 Neri F Fidencio Neri 1 15 Humbert R Richard Humbert 1 16 Singer MA Michael A Singer 3 17 Richmond TA Todd A Richmond 3 18 Dorschner MO Michael O Dorschner 1 19 McArthur M Michael McArthur 4 20 Hawrylycz M Michael Hawrylycz 5 21 Green RD Roland D Green 3 22 Navas PA Patrick A Navas 2 23 Noble WS William S Noble 1 24 Stamatoyannopoulos JA John A Stamatoyannopoulos 1 Department of Genome Sciences, University of Washington, 1705 NE Pacific St., Box 357730, Seattle, Washington 98195, USA Division of Medical Genetics, Department of Medicine, University of Washington, 1705 NE Pacific St., Box 357730, Seattle, Washington 98195, USA Nimblegen Systems, Inc., 1 Science Court, Madison, Wisconsin 53711, USA Department of Microbiology, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK Allen Institute for Brain Sciences, 551 N. 34th Street, Seattle, Washington 98103, USA These authors contributed equally to this work. On DNase Hypersensitivity Nature Methods 2006 Jul;3(7):501-2. 2006-07-01 2006-06-21 http://dx.doi.org/10.1038/nmeth0706-501 DNase-chip and DNase-array: similar names for two different new approaches that give a genomic perspective to the conventional DNase I hypersensitivity assay used to measure chromatin accessibility. 1 Giresi PG Paul G Giresi 1 2 Lieb JD Jason D Lieb 1 Department of Biology and Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina On DNase Hypersensitivity Nature Methods 2006 Jul;3(7):503-9. 2006-08-01 2006-06-21 http://dx.doi.org/10.1038/nmeth888 Mapping DNase I hypersensitive sites is an accurate method of identifying the location of gene regulatory elements, including promoters, enhancers, silencers and locus control regions. Although Southern blots are the traditional method of identifying DNase I hypersensitive sites, the conventional manual method is not readily scalable to studying large chromosomal regions, much less the entire genome. Here we describe DNase-chip, an approach that can rapidly identify DNase I hypersensitive sites for any region of interest, or potentially for the entire genome, by using tiled microarrays. We used DNase-chip to identify DNase I hypersensitive sites accurately from a representative 1% of the human genome in both primary and immortalized cell types. We found that although most DNase I hypersensitive sites were present in both cell types studied, some of them were cell-type specific. This method can be applied globally or in a targeted fashion to any tissue from any species with a sequenced genome. 1 Crawford GE Crawford GE 1,3 2 Davis S Davis S 1 3 Scacheri PC Scacheri PC 1 4 Renaud G Renaud G 1 5 Halawi MJ Halawi MJ 1 6 Erdos MR Erdos MR 1 7 Green R Green R 2 8 Meltzer PS Meltzer PS 1 9 Wolfsberg TG Wolfsberg TG 1 10 Collins FS Collins FS 1 National Human Genome Research Institute, National Institutes of Health, Building 31, Room 4B09, Bethesda, Maryland 20892, USA. NimbleGen Systems, Incorporated, 1 Science Court, Madison, Wisconsin 53711, USA. Present address: Institute for Genome Sciences & Policy, and Department of Pediatrics, 101 Science Drive, CIEMAS Building, Duke University, Box 3382, Durham, North Carolina 27708, USA. On ChIP-chip Genome Res. 2006 Dec;16(12):1585-95. Epub 2006 Oct 19. 2006-12-01 2006-10-19 http://dx.doi.org/10.1101/gr.5520206 http://www.genome.org/cgi/content/full/gr.5520206/DC1 Advances in high-throughput technologies, such as ChIP-chip, and the completion of human and mouse genomic sequences now allow analysis of the mechanisms of gene regulation on a systems level. In this study, we have developed a computational genomics approach (termed ChIPModules), which begins with experimentally determined binding sites and integrates positional weight matrices constructed from transcription factor binding sites, a comparative genomics approach, and statistical learning methods to identify transcriptional regulatory modules. We began with E2F1 binding site information obtained from ChIP-chip analyses of ENCODE regions, from both HeLa and MCF7 cells. Our approach not only distinguished targets from nontargets with a high specificity, but it also identified five regulatory modules for E2F1. One of the identified modules predicted a colocalization of E2F1 and AP-2alpha on a set of target promoters with an intersite distance of <270 bp. We tested this prediction using ChIP-chip assays with arrays containing approximately 14,000 human promoters. We found that both E2F1 and AP-2alpha bind within the predicted distance to a large number of human promoters, demonstrating the strength of our sequence-based, unbiased, and universal protocol. Finally, we have used our ChIPModules approach to develop a database that includes thousands of computationally identified and/or experimentally verified E2F1 target promoters. 1 Jin VX Jin VX 1 2 Rabinovich A Rabinovich A 1 3 Squazzo SL Squazzo SL 1 4 Green R Green R 1 5 Farnham PJ Farnham PJ 1 Department of Pharmacology and the Genome Center, University of California-Davis, Davis, California 95616, USA On ChIP-chip Mol. Cell. Biol. 2006 Oct;26(19):7056-67 2006-10-01 2006-10-01 http://dx.doi.org/10.1128/MCB.01033-06 The reciprocal expression of GATA-1 and GATA-2 during hematopoiesis is an important determinant of red blood cell development. Whereas Gata2 is preferentially transcribed early in hematopoiesis, elevated GATA-1 levels result in GATA-1 occupancy at sites upstream of the Gata2 locus and transcriptional repression. GATA-2 occupies these sites in the transcriptionally active locus, suggesting that a "GATA switch" abrogates GATA-2-mediated positive autoregulation. Chromatin immunoprecipitation (ChIP) coupled with genomic microarray analysis and quantitative ChIP analysis with GATA-1-null cells expressing an estrogen receptor ligand binding domain fusion to GATA-1 revealed additional GATA switches 77 kb upstream of Gata2 and within intron 4 at +9.5 kb. Despite indistinguishable GATA-1 occupancy at -77 kb and +9.5 kb versus other GATA switch sites, GATA-1 functioned uniquely at the different regions. GATA-1 induced histone deacetylation at and near Gata2 but not at the -77 kb region. The -77 kb region, which was DNase I hypersensitive in both active and inactive states, conferred equivalent enhancer activities in GATA-1- and GATA-2-expressing cells. By contrast, the +9.5 kb region exhibited considerably stronger enhancer activity in GATA-2- than in GATA-1-expressing cells, and other GATA switch sites were active only in GATA-1- or GATA-2-expressing cells. Chromosome conformation capture analysis demonstrated higher-order interactions between the -77 kb region and Gata2 in the active and repressed states. These results indicate that dispersed GATA factor complexes function via long-range chromatin interactions and qualitatively distinct activities to regulate Gata2 transcription. 1 Grass JA Grass JA 1 2 Jing H Jing H 1 3 Kim SI Kim SI 1 4 Martowicz ML Martowicz ML 1 5 Pal S Pal S 1 6 Blobel GA Blobel GA 1 7 Bresnick EH Bresnick EH 1 University of Wisconsin Medical School, Department of Pharmacology, 1300 University Avenue, Madison, WI On ChIP-chip Genes Dev. 2006 May 1;20(9):1123-36. Epub 2006 Apr 17. 2006-05-01 2006-04-17 http://dx.doi.org/10.1101/gad.381706 http://www.genesdev.org/cgi/reprint/20/9/1123 http://www.genesdev.org/cgi/content/full/gad.381706/DC1 The Polycomb group (PcG) proteins form chromatin-modifying complexes that are essential for embryonic development and stem cell renewal and are commonly deregulated in cancer. Here, we identify their target genes using genome-wide location analysis in human embryonic fibroblasts. We find that Polycomb-Repressive Complex 1 (PRC1), PRC2, and tri-methylated histone H3K27 co-occupy >1000 silenced genes with a strong functional bias for embryonic development and cell fate decisions. We functionally identify 40 genes derepressed in human embryonic fibroblasts depleted of the PRC2 components (EZH2, EED, SUZ12) and the PRC1 component, BMI-1. Interestingly, several markers of osteogenesis, adipogenesis, and chrondrogenesis are among these genes, consistent with the mesenchymal origin of fibroblasts. Using a neuronal model of differentiation, we delineate two different mechanisms for regulating PcG target genes. For genes activated during differentiation, PcGs are displaced. However, for genes repressed during differentiation, we paradoxically find that they are already bound by the PcGs in nondifferentiated cells despite being actively transcribed. Our results are consistent with the hypothesis that PcGs are part of a preprogrammed memory system established during embryogenesis marking certain key genes for repressive signals during subsequent developmental and differentiation processes. 1 Bracken AP Adrian P. Bracken 1 2 Dietrich N Nikolaj Dietrich 1 3 Pasini D Diego Pasini 1 4 Hansen KH Klaus H. Hansen 1 5 Helin K Kristian Helin 1,2 Biotech Research and Innovation Centre (BRIC), 2100 Copenhagen Ø, Denmark; Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark On ChIP-chip Plant J. 2006 Jul;47(1):152-62. 2006-07-01 2006-07-01 http://dx.doi.org/10.1111/j.1365-313X.2006.02770.x We have developed two long-oligonucleotide microarrays for the analysis of genome features in Arabidopsis thaliana, in particular for the high-throughput identification of transcription factor-binding sites. The first platform contains 190 000 probes representing the 2-kb regions upstream of all annotated genes at a density of seven probes per promoter. The second platform is divided into three chips, each of over 390 000 features, and represents the entire Arabidopsis genome at a density of one probe per 90 bases. Protein–DNA complexes resulting from the formaldehyde fixation of leaves of plants 2 h after exposure to 1 mm salicylic acid (SA) were immunoprecipitated using antibodies against the TGA2 transcription factor. After reversal of the cross-links and amplification, the resulting ChIP sample was hybridized to both platforms. High signal ratios of the ChIP sample versus raw chromatin for clusters of neighboring probes provided evidence for 51 putative binding sites for TGA2, including the only previously confirmed site in the promoter of PR-1 (At2g14610). Enrichment of several regions was confirmed by quantitative real-time PCR. Motif search revealed that the palindromic octamer TGACGTCA was found in 55% of the enriched regions. Interestingly, 15 of the putative binding sites for TGA2 lie outside the presumptive promoter regions. The effect of the 2-h SA treatment on gene expression was measured using Affymetrix ATH1 arrays, and SA-induced genes were found to be significantly over-represented among genes neighboring putative TGA2-binding sites. 1 Thibaud-Nissen F Françoise Thibaud-Nissen 1 2 Wu H Hank Wu 1 3 Richmond T Todd Richmond 2 4 Redman JC Julia C. Redman 1 5 Johnson C Christopher Johnson 3 6 Green R Roland Green 2 7 Arias J Jonathan Arias 3 8 Town CD Christopher D. Town 1 The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA NimbleGen Systems Inc., Madison, WI 53711, USA University of Maryland, Baltimore County, Baltimore, MD 21250, USA On ChIP-chip Genome Res. 2006 Jul;16(7):890-900. Epub 2006 Jun 2. 2006-07-01 2006-06-02 http://dx.doi.org/10.1101/gr.5306606 http://www.genome.org/cgi/content/full/gr.5306606/DC1 Suz12 is a component of the Polycomb group complexes 2, 3, and 4 (PRC 2/3/4). These complexes are critical for proper embryonic development, but very few target genes have been identified in either mouse or human cells. Using a variety of ChIP-chip approaches, we have identified a large set of Suz12 target genes in five different human and mouse cell lines. Interestingly, we found that Suz12 target promoters are cell type specific, with transcription factors and homeobox proteins predominating in embryonal cells and glycoproteins and immunoglobulin-related proteins predominating in adult tumors. We have also characterized the localization of other components of the PRC complex with Suz12 and investigated the overall relationship between Suz12 binding and markers of active versus inactive chromatin, using both promoter arrays and custom tiling arrays. Surprisingly, we find that the PRC complexes can be localized to discrete binding sites or spread through large regions of the mouse and human genomes. Finally, we have shown that some Suz12 target genes are bound by OCT4 in embryonal cells and suggest that OCT4 maintains stem cell self-renewal, in part, by recruiting PRC complexes to certain genes that promote differentiation. 1 Squazzo SL Sharon L. Squazzo 1 2 O’Geen H Henriette O’Geen 1 3 Komashko VM Vitalina M. Komashko 1 4 Krig SR Sheryl R. Krig 1 5 Jin VX Victor X. Jin 1 6 Jang SW Sung Jang 4 7 Margueron R Raphael Margueron 3 8 Reinberg D Danny Reinberg 3 9 Green R Roland Green 2 10 Farnham PJ Peggy J. Farnham 1 Department of Pharmacology and the Genome Center, University of California-Davis, Davis, California 95616, USA NimbleGen Systems Inc., Madison, Wisconsin 53711, USA Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA Graduate Program in Cellular and Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, USA On ChIP-chip J. Biol. Chem. Vol. 281, Issue 17, 11992-12000, April 28, 2006 2006-04-28 2006-02-10 http://dx.doi.org/10.1074/jbc.M513613200 Recently developed target gene identification strategies based upon the chromatin immunoprecipitation assay provide a powerful method to determine the localization of transcription factor binding within mammalian genomes. However, in many cases, it is unclear if the binding capacity of a transcription factor correlates with an obligate role in gene regulation in diverse contexts. It is therefore important to carefully examine the relationship between transcription factor binding and its ability to functionally regulate gene expression. T-bet is a T-box transcription factor expressed in several hematopoietic cell types. By utilizing a chromatin immunoprecipitation assay coupled to genomic microarray technology approach, we identified numerous promoters, including CXCR3, IL2Rbeta, and CCL3, that are bound by T-bet in B cells. Most surprisingly, the ability of T-bet to associate with the target promoters is not dependent upon the cell type background. Several of the promoters appear to be functionally regulated by T-bet. However, we could not detect a functional consequence for T-bet association with many of the identified promoters in overexpression studies or an examination of wild type and T-bet-/- primary B, CD4+, and CD8+ T cells. Thus, there is a high variability in the functional consequences, if any, that result from the association of T-bet with individual target promoters. 1 Beima KM Beima KM 1 2 Miazgowicz MM Miazgowicz MM 1 3 Lewis MD Lewis MD 1 4 Yan PS Yan PS 1 5 Huang TH Huang TH 1 6 Weinmann AS Weinmann AS 1 Immunology Dept., University of Washington, Seattle, WA On ChIP-chip Genome Res. 2006 May;16(5):595-605. Epub 2006 Apr 10. 2006-05-01 2006-04-10 http://dx.doi.org/10.1101/gr.4887606 http://www.genome.org/cgi/reprint/16/5/595 http://www.genome.org/cgi/content/full/gr.4887606/DC1 The E2F family of transcription factors regulates basic cellular processes. Here, we take an unbiased approach towards identifying E2F1 target genes by examining localization of E2F1-binding sites using high-density oligonucleotide tiling arrays. To begin, we developed a statistically-based methodology for analysis of ChIP-chip data obtained from arrays that represent 30 Mb of the human genome. Using this methodology, we identified regions bound by E2F1, MYC, and RNA Polymerase II (POLR2A). We found a large number of binding sites for all three factors; extrapolation suggests there may be approximately 20,000-30,000 E2F1- and MYC-binding sites and approximately 12,000-17,000 active promoters in HeLa cells. In contrast to our results for MYC, we find that the majority of E2F1-binding sites (>80%) are located in core promoters and that 50% of the sites overlap transcription starts. Only a small fraction of E2F1 sites possess the canonical binding motif. Surprisingly, we found that approximately 30% of genes in the 30-Mb region possessed an E2F1 binding site in a core promoter and E2F1 was bound near to 83% of POLR2A-bound sites. To determine if these results were representative of the entire human genome, we performed ChIP-chip analyses of approximately 24,000 promoters and confirmed that greater than 20% of the promoters were bound by E2F1. Our results suggest that E2F1 is recruited to promoters via a method distinct from recognition of the known consensus site and point toward a new understanding of E2F1 as a factor that contributes to the regulation of a large fraction of human genes. 1 Bieda M Mark Bieda 1 2 Xu X Xiaoqin Xu 1 3 Singer M Mike Singer 2 4 Green R Roland Green 2 5 Farnham PJ Peggy J. Farnham1 3 Department of Pharmacology and the Genome Center, University of California-Davis, Davis, California NimbleGen Systems Inc., Madison, Wisconsin On ChIP-chip Genes Dev. 2006 Apr 1;20(7):848-57. Epub 2006 Mar 17. 2006-04-01 2006-03-17 http://dx.doi.org/10.1101/gad.1400206 http://www.genesdev.org/cgi/reprint/20/7/848 http://www.genesdev.org/cgi/content/full/gad.1400206/DC1 X-chromosome dosage compensation in Drosophila requires the male-specific lethal (MSL) complex, which up-regulates gene expression from the single male X chromosome. Here, we define X-chromosome-specific MSL binding at high resolution in two male cell lines and in late-stage embryos. We find that the MSL complex is highly enriched over most expressed genes, with binding biased toward the 3' end of transcription units. The binding patterns are largely similar in the distinct cell types, with ~600 genes clearly bound in all three cases. Genes identified as clearly bound in one cell type and not in another indicate that attraction of MSL complex correlates with expression state. Thus, sequence alone is not sufficient to explain MSL targeting. We propose that the MSL complex recognizes most X-linked genes, but only in the context of chromatin factors or modifications indicative of active transcription. Distinguishing expressed genes from the bulk of the genome is likely to be an important function common to many chromatin organizing and modifying activities. 1 Alekseyenko AA Artyom A. Alekseyenko 1,2,3 2 Larschan E Erica Larschan 2,3 3 Lai WR Weil R. Lai 2 4 Park PJ Peter J. Park 2,4 5 Kuroda MI Mitzi I. Kuroda1 2,3 Howard Hughes Medical Institute Harvard-Partners Center for Genetics and Genomics, Brigham and Women's Hospital, Boston, Massachusetts Department of Genetics, Harvard Medical School, Boston, Massachusetts Children's Hospital Informatics Program, Boston, Massachusetts On ChIP-chip Genes Dev. 2006 Apr 1;20(7):858-70. Epub 2006 Mar 17 2006-04-01 2006-03-17 http://dx.doi.org/10.1101/gad.1399406 http://www.genesdev.org/cgi/reprint/20/7/858 http://www.genesdev.org/cgi/content/full/gad.1399406/DC1 The dosage compensation complex (DCC) of Drosophila melanogaster is capable of distinguishing the single male X from the other chromosomes in the nucleus. It selectively interacts in a discontinuous pattern with much of the X chromosome. How the DCC identifies and binds the X, including binding to the many genes that require dosage compensation, is currently unknown. To identify bound genes and attempt to isolate the targeting cues, we visualized male-specific lethal 1 (MSL1) protein binding along the X chromosome by combining chromatin immunoprecipitation with high-resolution microarrays. More than 700 binding regions for the DCC were observed, encompassing more than half the genes found on the X chromosome. In addition, several rare autosomal binding sites were identified. Essential genes are preferred targets, and genes binding high levels of DCC appear to experience the most compensation (i.e., greatest increase in expression). DCC binding clearly favors genes over intergenic regions, and binds most strongly to the 3' end of transcription units. Within the targeted genes, the DCC exhibits a strong preference for exons and coding sequences. Our results demonstrate gene-specific binding of the DCC, and identify several sequence elements that may partly direct its targeting. 1 Gilfillan GD Gregor D. Gilfillan 1 2 Straub T Tobias Straub 1 3 de Wit E Elzo de Wit 2 4 Greil F Frauke Greil 2 5 Lamm R Rosemarie Lamm 1 6 van Steensel B Bas van Steense 1,2 7 Becker PB Peter B. Becker 1 Adolf-Butenandt-Institut, Molekularbiologie, Ludwig-Maximilians-Universität München, Germany Netherlands Cancer Institute, Amsterdam, The Netherlands On ChIP-chip PLoS Genet. 2006 Apr;2(4):e51. Epub 2006 Apr 7. 2006-04-07 2006-04-07 http://dx.doi.org/10.1371/journal.pgen.0020051 Multiple Endocrine Neoplasia, type I (MEN1) is a familial cancer syndrome characterized primarily by tumors of multiple endocrine glands. The gene for MEN1 encodes a ubiquitously expressed tumor suppressor protein called menin. Menin was recently shown to interact with several components of a trithorax family histone methyltransferase complex including ASH2, Rbbp5, WDR5, and the leukemia proto-oncoprotein MLL. To elucidate menin's role as a tumor suppressor and gain insights to the endocrine-specific tumor phenotype in MEN1, we mapped the genomic binding sites of menin, MLL1, and Rbbp5, to ~20,000 promoters in Hela S3, HepG2 and pancreatic islet cells using the strategy of chromatin-immunoprecipitation coupled with microarray analysis (ChIP-chip). We found that menin, MLL1, and Rbbp5 localize to the promoters of thousands of human genes, but do not always bind together. These data suggest that menin functions as a general regulator of transcription. We also found that factor occupancy generally correlates with high gene expression, but the loss of menin does not result in significant changes in most transcript levels. One exception is the developmentally programmed transcription factor, HLXB9, which is overexpressed in islets in the absence of menin. Our findings expand the realm of menin-targeted genes several hundred-fold beyond that previously described, and provide potential insights to the endocrine tumor bias observed in MEN1 patients. 1 Scacheri PC Peter C Scacheri 1 2 Davis S Sean Davis 2 3 Odom DT Duncan T Odom 3 4 Crawford GE Gregory E Crawford 2 5 Perkins S Stacie Perkins 2 6 Halawi MJ Mohamad J Halawi 2 7 Agarwal SK Sunita K Agarwal 4 8 Marx SJ Stephen J Marx 4 9 Spiegel AM Allen M Spiegel 5 10 Meltzer PS Paul S Meltzer 6 11 Collins FS Francis S Collins 7,8 NIH, NHGRI, Bethesda, MD, United States of America NIH, Bethesda, MD, United States of America Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, United States of America NIH, NIDDK, Bethesda, MD, United States of America NIH, NIDCD, Bethesda, MD, United States of America Cancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD, National Human Genome Research Institute, Bethesda, MD, USA, National Institutes of Health, National Human Genome Research Institute, Bethesda, Maryland, United States of America To whom correspondence should be addressed. E-mail: fc23a@nih.gov On ChIP-chip Nat. Genet. 2005 Oct;37(10):1090-7. Epub 2005 Sep 11 2005-10-01 2005-09-11 http://dx.doi.org/10.1038/ng1637 Histones of multicellular organisms are assembled into chromatin primarily during DNA replication. When chromatin assembly occurs at other times, the histone H3.3 variant replaces canonical H3. Here we introduce a new strategy for profiling epigenetic patterns on the basis of H3.3 replacement, using microarrays covering roughly one-third of the Drosophila melanogaster genome at 100-bp resolution. We identified patterns of H3.3 replacement over active genes and transposons. H3.3 replacement occurred prominently at sites of abundant RNA polymerase II and methylated H3 Lys4 throughout the genome and was enhanced on the dosage-compensated male X chromosome. Active genes were depleted of histones at promoters and were enriched in H3.3 from upstream to downstream of transcription units. We propose that deposition and inheritance of actively modified H3.3 in regulatory regions maintains transcriptionally active chromatin. 1 Mito Y Yoshiko Mito 1 2 Henikoff JG Jorja G. Henikoff 1 3 Henikoff S Steven Henikoff 1,2 Fred Hutchinson Cancer Research Center, Seattle Washington Howard Hughes Medical Institute On ChIP-chip J. Bacteriol. 2005 Sep;187(17):6166-74. 2005-09-01 2005-09-01 http://dx.doi.org/10.1128/JB.187.17.6166-6174.2005 http://jb.asm.org/cgi/reprint/187/17/6166 http://jb.asm.org/cgi/content/full/187/17/6166/DC1 The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the beta' subunit. The DNA was amplified and hybridized to "tiled" oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known sigma70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors in E. coli and an important evaluation of the chIP-chip technique. 1 Herring CD Christopher D. Herring 1,2,4 2 Raffaelle M Marni Raffaelle 2 3 Allen TE Timothy E. Allen 1 4 Kanin EI Elenita I. Kanin 2 5 Landick R Robert Landick 4 6 Ansari AZ Aseem Z. Ansari 2,3 7 Palsson BO Bernhard Ø. Palsson 1 Department of Bioengineering, University of California San Diego, San Diego, California Department of Biochemistry, University of Wisconsin Madison, Madison, Wisconsin Genome Center of Wisconsin, Madison, Wisconsin Department of Bacteriology, University of Wisconsin Madison, Madison, Wisconsin On ChIP-chip Nature 2005 Aug 11;436(7052):876-80. Epub 2005 Jun 29. 2005-08-11 2005-06-29 http://dx.doi.org/10.1038/nature03877 In eukaryotic cells, transcription of every protein-coding gene begins with the assembly of an RNA polymerase II preinitiation complex (PIC) on the promoter. The promoters, in conjunction with enhancers, silencers and insulators, define the combinatorial codes that specify gene expression patterns. Our ability to analyse the control logic encoded in the human genome is currently limited by a lack of accurate information regarding the promoters for most genes. Here we describe a genome-wide map of active promoters in human fibroblast cells, determined by experimentally locating the sites of PIC binding throughout the human genome. This map defines 10,567 active promoters corresponding to 6,763 known genes and at least 1,196 un-annotated transcriptional units. Features of the map suggest extensive use of multiple promoters by the human genes and widespread clustering of active promoters in the genome. In addition, examination of the genome-wide expression profile reveals four general classes of promoters that define the transcriptome of the cell. These results provide a global view of the functional relationships among transcriptional machinery, chromatin structure and gene expression in human cells. 1 Kim TH Tae Hoon Kim 1,5 2 Barrera LO Leah O. Barrera 1,5 3 Zheng M Ming Zheng 3 4 Qu C Chunxu Qu 1 5 Singer MA Michael A. Singer 4 6 Richmond TA Todd A. Richmond 4 7 Wu Y Yingnian Wu 3 8 Green RD Roland D. Green 4 9 Ren B Bing Ren 1,2 Ludwig Institute for Cancer Research Department of Cellular and Molecular Medicine and Moores Cancer Center, UCSD School of Medicine Department of Statistics, University of California, Los Angeles NimbleGen Systems, Inc. These authors contributed equally to this work On ChIP-chip Genes Dev. 2004 Jul 1;18(13):1592-605 2004-07-01 2004-07-01 http://dx.doi.org/10.1101/gad.1200204 http://www.genesdev.org/cgi/reprint/18/13/1592 http://www.genesdev.org/cgi/content/full/18/13/1592/DC1 Polycomb group (PcG) complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. Here we report the use of RNA expression arrays and CpG-island DNA arrays to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, we first identified a cohort of genes whose expression changes upon siRNA-mediated removal of Suz12, a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, we combined a high-resolution chromatin immunoprecipitation (ChIP) analysis with custom oligonucleotide promoter arrays. We next identified additional putative Suz12 target genes by using ChIP coupled to CpG-island microarrays. We showed that HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27. 1 Kirmizis A Antonis Kirmizis 1 2 Bartley SM Stephanie M. Bartley 1 3 Kuzmichev A Andrei Kuzmichev 2 4 Margueron R Raphael Margueron 2 5 Reinberg D Danny Reinberg 2 6 Green R Roland Green 3 7 Farnham PJ Peggy J. Farnham 1,4 McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA NimbleGen Systems Inc., Madison, Wisconsin 53711, USA Corresponding author. E-MAIL farnham@oncology.wisc.edu; FAX (608) 262-2824. On CGH Nat. Rev. Genet. 2006 Feb;7(2):85-97. 2006-02-01 2006-02-01 http://dx.doi.org/10.1038/nrg1767 The first wave of information from the analysis of the human genome revealed SNPs to be the main source of genetic and phenotypic human variation. However, the advent of genome-scanning technologies has now uncovered an unexpectedly large extent of what we term 'structural variation' in the human genome. This comprises microscopic and, more commonly, submicroscopic variants, which include deletions, duplications and large-scale copy-number variants — collectively termed copy-number variants or copy-number polymorphisms — as well as insertions, inversions and translocations. Rapidly accumulating evidence indicates that structural variants can comprise millions of nucleotides of heterogeneity within every genome, and are likely to make an important contribution to human diversity and disease susceptibility. 1 Feuk L Feuk L 1 2 Carson AR Carson AR 1 3 Scherer SW Scherer SW 1 The Centre for Applied Genomics and Program in Genetics and Genomic Biology, The Hospital for Sick Children and Department of Molecular and Medical Genetics, University of Toronto, MaRS Centre East Tower, 101 College Street, Room 14-701, Ontario M5G 1L7, Canada On CGH J. Med. Genet. 2007 Jan;44(1):51-8. Epub 2006 Sep 13. 2007-01-01 2006-09-13 http://dx.doi.org/10.1136/jmg.2006.044909 Objective: The authors describe a significant advance of the method of array painting which allows the rapid, ultra-high resolution mapping of translocation breakpoints such that rearrangement junction fragments can be amplified directly and sequenced. Method: Ultra-high resolution array painting involves the hybridisation of probes generated by PCR of small numbers of flow sorted derivative chromosomes to oligonucelotide arrays designed to tile breakpoint regions at extremely high resolution. Results and Discussion: The authors demonstrate how ultra-high resolution array painting of four balanced translocation cases rapidly and efficiently maps breakpoints to a point where junction fragments can be amplified easily and sequenced. With this new development, breakpoints can be mapped using just two array experiments, the first utilising whole genome array painting to tiling resolution large insert clone arrays, the second utilising ultra-high reolution oligonucleotide arrays targeted to the breakpoint regions. In this way breakpoints can be mapped and then sequenced in a matter of a few weeks. 1 Gribble SM Gribble SM 1 2 Kalaitzopoulos D Kalaitzopoulos D 1 3 Burford DC Burford DC 1 4 Prigmore E Prigmore E 1 5 Selzer RR Selzer RR 1 6 Ng BL Ng BL 1 7 Matthews NS Matthews NS 1 8 Porter KM Porter KM 1 9 Curley R Curley R 1 10 Lindasy SJ Lindasy SJ 1 11 Baptista J Baptista J 1 12 Richmond TA Richmond TA 1 13 Carter NP Carter NP 1 The Wellcome Trust Sanger Institute, United Kingdom On CGH Nat. Genet. 2006 Sep;38(9):1038-42. Epub 2006 Aug 13. 2006-09-01 2006-08-13 http://dx.doi.org/10.1038/ng1862 Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic disorders1. We tested 290 individuals with mental retardation by BAC array comparative genomic hybridization and identified 16 pathogenic rearrangements, including de novo microdeletions of 17q21.31 found in four individuals. Using oligonucleotide arrays, we refined the breakpoints of this microdeletion, defining a 478-kb critical region containing six genes that were deleted in all four individuals. We mapped the breakpoints of this deletion and of four other pathogenic rearrangements in 1q21.1, 15q13, 15q24 and 17q12 to flanking segmental duplications, suggesting that these are also sites of recurrent rearrangement. In common with the 17q21.31 deletion, these breakpoint regions are sites of copy number polymorphism in controls, indicating that these may be inherently unstable genomic regions. 1 Sharp AJ Andrew J Sharp 1 2 Hansen S Sierra Hansen 1 3 Selzer RR Rebecca R Selzer 2 4 Cheng Z Ze Cheng 1 5 Regan R Regina Regan 3 6 Hurst JA Jane A Hurst 4 7 Stewart H Helen Stewart 4 8 Price SM Sue M Price 4 9 Blair E Edward Blair 4 10 Hennekam RC Raoul C Hennekam 5,6 11 Fitzpatrick CA Carrie A Fitzpatrick 7 12 Segraves R Rick Segraves 8 13 Richmond TA Todd A Richmond 2 14 Guiver C Cheryl Guiver 3 15 Albertson DG Donna G Albertson 8,9 16 Pinkel Daniel Pinkel 8 17 Eis PS Peggy S Eis 2 18 Schwartz S Stuart Schwartz 7 19 Knight SJ Samantha J L Knight 3 20 Eichler EE Evan E Eichler 1 Department of Genome Sciences and The Howard Hughes Medical Institute, University of Washington School of Medicine, 1705 NE Pacific St., Seattle, Washington 98195, USA. NimbleGen Systems, Inc., Madison, Wisconsin 53711, USA. Oxford Genetics Knowledge Park, The Wellcome Trust Centre for Human Genetics, Churchill Hospital, Oxford OX3 7BN, UK. Department of Clinical Genetics, Oxford Radcliffe Hospitals National Health Service (NHS) Trust, Churchill Hospital, Oxford OX3 7LJ, UK. Clinical and Molecular Genetics Unit, Institute of Child Health, University College London, London, UK. Department of Pediatrics, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. Department of Human Genetics, University of Chicago, Chicago, Illinois 60637, USA. Comprehensive Cancer Center, University of California San Francisco (UCSF), San Francisco, California 94143, USA. Cancer Research Institute, UCSF, San Francisco, California 94143, USA On CGH Am. J. Med. Genet. 2006 Aug 15;140(16):1711-8 2006-08-15 2006-07-11 http://dx.doi.org/10.1002/ajmg.a.31335 Anophthalmia and pituitary gland hypoplasia are both debilitating conditions where the underlying genetic defect is unknown in the majority of cases. We identified a patient with bilateral anophthalmia and absence of the optic nerves, chiasm and tracts, as well as pituitary gland hypoplasia and ear anomalies with a de novo apparently balanced chromosomal translocation, 46,XY,t(3;14)(q28;q23.2). Translocation breakpoint analysis using FISH and high-resolution microarray comparative genomic hybridization (CGH) has identified a 9.66 Mb deleted region on the long arm of chromosome 14 which includes the genes BMP4, OTX2, RTN1, SIX6, SIX1, and SIX4. Three other patients with interstitial deletions involving 14q22-23 have been described, all with bilateral anophthalmia, pituitary abnormalities, ear anomalies, and a facial phenotype similar to our patient. OTX2 is involved in ocular developmental defects, and the severity of the ocular phenotype in our patient and the other 14q22-23 deletion patients, suggests this genomic region harbors other gene/s involved in ocular development. BMP4 haploinsufficiency is predicted to contribute to the ocular phenotype on the basis of its expression pattern and observed murine mutant phenotypes. In addition, deletion of BMP4 and SIX6 is likely to contribute to the abnormal pituitary development, and SIX1 deletion may contribute to the ear and other craniofacial features. This indicates that contiguous gene deletion may contribute to the phenotypic features in the 14q22-23 deletion patients. 1 Nolen LD Leisha D. Nolen 1,7 2 Amor D David Amor 2 3 Haywood A Ashley Haywood 1,3,4 4 St Heaps L Luke St. Heaps 3 5 Willcock C Chris Willcock 1 6 Mihelec M Marija Mihelec 1 7 Tam P Patrick Tam 5 8 Billson F Frank Billson 6 9 Grigg J John Grigg 6 10 Peters G Greg Peters 3 11 Jamieson RV Robyn V. Jamieson 1,8 Eye Genetics Research Group, Children's Medical Research Institute, The Children's Hospital at Westmead and Save Sight Institute, Sydney, New South Wales, Australia Department of Genetic Health Services Victoria, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia Department of Cytogenetics, Western Sydney Genetics Program, The Children's Hospital at Westmead, New South Wales, Australia Department of Biological Sciences, Macquarie University, Sydney, New South Wales, Australia Embryology Unit, Children's Medical Research Institute, Westmead, New South Wales, Australia Discipline of Ophthalmology and Save Sight Institute, Faculty of Medicine, University of Sydney, New South Wales, Australia School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, New South Wales, Australia On CGH Am. J. Med. Genet. 2006 Jul 11;140(15):1637-1643 2006-07-11 2006-07-11 http://dx.doi.org/10.1002/ajmg.a.31349 We describe two boys with cytogenetically identical interstitial deletions in the q42.11-q42.13 region of the long arm of chromosome 1 detected by high-resolution G-banding analysis. These children share some phenotypic features but also exhibit distinct morphologic differences. We further characterized the deletions using a new technical strategy - microdissection-based high-resolution genomic array (MHGA) analysis - to define the breakpoints, genomic sizes, and gene contents of the deletions. This showed that the patients had distinguishable deletions that were adjacent but did not overlap, thus explaining the observed phenotypic differences. These results were surprising because we expected at least some degree of overlap to explain the features that were shared. MHGA can quickly give precise and detailed information about any rearrangement in the genome using as little material as a single cell. This novel strategy provides unique advantages for both clinical diagnosis and genomic research. 1 Rice GM G.M. Rice 1,6 2 Qi Z Z. Qi 2,5 3 Selzer R R. Selzer 3 4 Richmond T T. Richmond 3 5 Thompson K K. Thompson 2 6 Pauli RM R.M. Pauli 1 7 Yu J J. Yu 2,4,5 Departments of Pediatrics and Medical Genetics, University of Wisconsin - Madison, Madison, Wisconsin Wisconsin State Laboratory of Hygiene, University of Wisconsin - Madison, Madison, Wisconsin NimbleGen Systems, Inc., Madison, Wisconsin Department of Pathology and Laboratory Medicine, University of Wisconsin - Madison, Madison, Wisconsin Department of Laboratory Medicine, University of California San Francisco, San Francisco, California Waisman Center, University of Wisconsin - Madison, Madison, Wisconsin On CGH Cell 2006 Jun 30;125(7):1269-81. 2006-06-30 2006-06-30 http://dx.doi.org/10.1016/j.cell.2006.06.008 http://www.cell.com/cgi/content/full/125/7/1269/DC1/ Genomes of human cancer cells are characterized by numerous chromosomal aberrations of uncertain pathogenetic significance. Here, in an inducible mouse model of melanoma, we characterized metastatic variants with an acquired focal chromosomal amplification that corresponds to a much larger amplification in human metastatic melanomas. Further analyses identified Nedd9, an adaptor protein related to p130CAS, as the only gene within the minimal common region that exhibited amplification-associated overexpression. A series of functional, biochemical, and clinical studies established NEDD9 as a bona fide melanoma metastasis gene. NEDD9 enhanced invasion in vitro and metastasis in vivo of both normal and transformed melanocytes, functionally interacted with focal adhesion kinase and modulated focal contact formation, and exhibited frequent robust overexpression in human metastatic melanoma relative to primary melanoma. Thus, comparative oncogenomics has enabled the identification and facilitated the validation of a highly relevant cancer gene governing metastatic potential in human melanoma. 1 Kim M Minjung Kim 1 2 Gans JD Joseph D. Gans 1 3 Nogueira C Cristina Nogueira 1,2 4 Wang A Audrey Wang 1 5 Paik JH Ji-Hye Paik 1 6 Feng B Bin Feng 3 7 Brennan C Cameron Brennan 4 8 Hahn WC William C. Hahn 1,5 9 Cordon-Cardo C Carlos Cordon-Cardo 6 10 Wagner SN Stephan N. Wagner 7 11 Flotte TJ Thomas J. Flotte 8 12 Duncan LM Lyn M. Duncan 8 13 Granter SR Scott R. Granter 9 14 Chin L Lynda Chin 1,3,10 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Medical Faculty, University of Porto, Porto, Portugal Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, MA 02115, USA Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA DIAID, Department of Dermatology, Medical University of Vienna and Center of Molecular Medicine, Austrian Academy of Sciences, Wahringer Gurtel 18-20, A-1090 Vienna, Austria Dermatopathology Unit, Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115, USA. Department of Dermatology, Harvard Medical School, Boston, MA 02115, USA On CGH Genome Res. 2006 Aug;16(8):949-61. Epub 2006 Jun 29. 2006-08-01 2006-06-29 http://dx.doi.org/10.1101/gr.3677206 DNA copy number variation has long been associated with specific chromosomal rearrangements and genomic disorders, but its ubiquity in mammalian genomes was not fully realized until recently. Although our understanding of the extent of this variation is still developing, it seems likely that, at least in humans, copy number variants (CNVs) account for a substantial amount of genetic variation. Since many CNVs include genes that result in differential levels of gene expression, CNVs may account for a significant proportion of normal phenotypic variation. Current efforts are directed toward a more comprehensive cataloging and characterization of CNVs that will provide the basis for determining how genomic diversity impacts biological function, evolution, and common human diseases. 1 Freeman JL Jennifer L. Freeman 1,2 2 Perry GH George H. Perry 1,3 3 Feuk L Lars Feuk 4 4 Redon R Richard Redon 5 5 McCarroll SA Steven A. McCarroll 6 6 Altshuler DM David M. Altshuler 6 7 Aburatani H Hiroyuki Aburatani 7 8 Jones KW Keith W. Jones 8 9 Tyler-Smith C Chris Tyler-Smith 5 10 Hurles ME Matthew E. Hurles 5 11 Carter NP Nigel P. Carter 5 12 Scherer SW Stephen W. Scherer 4 13 Lee C Charles Lee 1,2,9 Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA Harvard Medical School, Boston, Massachusetts 02115, USA School of Human Evolution and Social Change, Arizona State University, Tempe, Arizona 85287, USA Department of Genetics and Genomic Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom Program in Medical and Population Genetics, Broad Institute of Harvard University and Massachusetts Institute of Technology, Cambridge, Massachusetts 02141, USA Genome Science Division, University of Tokyo, Tokyo, 153-8904 Japan Molecular Genetics Division, Affymetrix, Inc., Santa Clara, California 95051, USA Corresponding author. On CGH Am. J. Hum. Genet. 2006 Aug;79(2):275-90. Epub 2006 Jun 15 2006-08-01 2006-06-15 http://dx.doi.org/10.1086/505653 http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16826518#figures-tables-sec Studies of copy number variation and linkage disequilibrium have typically excluded complex regions of the genome that are rich in duplications which are prone to rearrangement. In an attempt to assess the heritability and linkage disequilibrium of copy number polymorphisms in duplication-rich regions of the genome, we profiled copy number variation in 130 putative rearrangement hotspot regions among 269 individuals of European, Yoruba, Chinese, and Japanese ancestry analyzed by the International HapMap Consortium. Eighty four hotspot regions, corresponding to 257 BAC probes, showed evidence of copy number differences. Despite a predisposing genetic architecture, polymorphism was never observed in the remaining 46 rearrangement hotspots, and we suggest these represent excellent candidates sites for pathogenic rearrangements. We used a combination of BAC-based and high-density customized oligonucleotide arrays to resolve the molecular basis of structural rearrangements. For common variants (>10% frequency), we observed a distinct bias against copy number losses, suggesting that deletions are subject to purifying selection. Heritability estimates did not differ significantly from 1.0 among the majority (30/34) of loci analyzed, consistent with normal Mendelian inheritance. Some of the CNPs in duplication-rich regions showed strong linkage disequilibrium with nearby SNPs and were observed to segregate on ancestral SNP haplotypes. However, linkage disequilibrium with the best available SNP markers is weaker than has been reported for deletion polymorphisms in less-complex regions of the genome. These observations may be accounted for by a low density of SNP data in duplicated regions, challenges in mapping and typing the CNPs, and the possibility that CNPs in these regions have rearranged on multiple haplotype backgrounds. Our results underscore the need for complete maps of genetic variation in duplication-rich regions of the genome. 1 Locke DP Devin P. Locke 1 2 Sharp AJ Andrew J. Sharp 1 3 McCarroll SA Steven A. McCarroll 2, 3 4 McGrath SD Sean D. McGrath 1 5 Newman TL Tera L. Newman 1 6 Cheng Z Ze Cheng 1,4 7 Schwartz S Stuart Schwartz 5 8 Albertson DG Donna G. Albertson 6 9 Pinkel D Daniel Pinkel 6 10 Altshuler DM David M. Altshuler 2, 3, 7, 8 11 Eichler EE Evan E. Eichler 1,4 Department of Genome Sciences, University of Washington School of Medicine. Department of Molecular Biology and Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA Program in Medical and Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA Howard Hughes Medical Institute 1705 NE Pacific St. Seattle, WA Department of Genetics, University of Chicago, Chicago, IL Comprehensive Cancer Center, UCSF, San Francisco, CA Harvard Medical School, Boston, MA Department of Medicine, Massachusetts General Hospital, Boston, MA On CGH PNAS 2006 May 23;103(21):8167-72. Epub 2006 May 15. 2006-03-21 2006-03-14 http://dx.doi.org/10.1073/pnas.0602360103 http://www.pnas.org/cgi/reprint/103/12/4534 http://www.pnas.org/cgi/content/full/0511340103/DC1 Deletions and amplifications of the human genomic sequence (copy number polymorphisms) are the cause of numerous diseases and a potential cause of phenotypic variation in the normal population. Comparative genomic hybridization (CGH) has been developed as a useful tool for detecting alterations in DNA copy number that involve blocks of DNA several kilobases or larger in size. We have developed high-resolution CGH (HR-CGH) to detect accurately and with relatively little bias the presence and extent of chromosomal aberrations in human DNA. Maskless array synthesis was used to construct arrays containing 385,000 oligonucleotides with isothermal probes of 45-85 bp in length; arrays tiling the beta-globin locus and chromosome 22q were prepared. Arrays with a 9-bp tiling path were used to map a 622-bp heterozygous deletion in the beta-globin locus. Arrays with an 85-bp tiling path were used to analyze DNA from patients with copy number changes in the pericentromeric region of chromosome 22q. Heterozygous deletions and duplications as well as partial triploidies and partial tetraploidies of portions of chromosome 22q were mapped with high resolution (typically up to 200 bp) in each patient, and the precise breakpoints of two deletions were confirmed by DNA sequencing. Additional peaks potentially corresponding to known and novel additional CNPs were also observed. Our results demonstrate that HR-CGH allows the detection of copy number changes in the human genome at an unprecedented level of resolution. 1 Strefford JC Jon C. Strefford 1 2 van Delft FW Frederik W. van Delft 2,3 3 Robinson HM Hazel M. Robinson 1 4 Worley H Helen Worley 1 5 Yiannikouris O Olga Yiannikouris 2,3 6 Selzer R Rebecca Selzer 4 7 Richmond T Todd Richmond 4 8 Hann I Ian Hann