Publications by authors named "Peter J Sabo"

30 Publications

  • Page 1 of 1

Mapping and Dynamics of Regulatory DNA in Maturing Siliques.

Front Plant Sci 2019 14;10:1434. Epub 2019 Nov 14.

Department of Genome Sciences, University of Washington, Seattle, WA, United States.

The genome is reprogrammed during development to produce diverse cell types, largely through altered expression and activity of key transcription factors. The accessibility and critical functions of epidermal cells have made them a model for connecting transcriptional events to development in a range of model systems. In and many other plants, fertilization triggers differentiation of specialized epidermal seed coat cells that have a unique morphology caused by large extracellular deposits of polysaccharides. Here, we used DNase I-seq to generate regulatory landscapes of seeds at two critical time points in seed coat maturation (4 and 7 DPA), enriching for seed coat cells with the INTACT method. We found over 3,000 developmentally dynamic regulatory DNA elements and explored their relationship with nearby gene expression. The dynamic regulatory elements were enriched for motifs for several transcription factors families; most notably the TCP family at the earlier time point and the MYB family at the later one. To assess the extent to which the observed regulatory sites in seeds added to previously known regulatory sites in we compared our data to 11 other data sets generated with 7-day-old seedlings for diverse tissues and conditions. Surprisingly, over a quarter of the regulatory, i.e. accessible, bases observed in seeds were novel. Notably, plant regulatory landscapes from different tissues, cell types, or developmental stages were more dynamic than those generated from bulk tissue in response to environmental perturbations, highlighting the importance of extending studies of regulatory DNA to single tissues and cell types during development.
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http://dx.doi.org/10.3389/fpls.2019.01434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868056PMC
November 2019

Integrative analysis of 111 reference human epigenomes.

Nature 2015 Feb;518(7539):317-30

1] Department of Cellular and Molecular Medicine, Institute of Genomic Medicine, Moores Cancer Center, Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA. [2] Ludwig Institute for Cancer Research, 9500 Gilman Drive, La Jolla, California 92093, USA.

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.
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http://dx.doi.org/10.1038/nature14248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4530010PMC
February 2015

Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution.

Science 2014 Nov;346(6212):1007-12

Howard Hughes Medical Institute. Division of Hematology/Oncology, Children's Hospital Boston and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA.

To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I-hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes.
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http://dx.doi.org/10.1126/science.1246426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337786PMC
November 2014

Conservation of trans-acting circuitry during mammalian regulatory evolution.

Nature 2014 Nov;515(7527):365-70

1] Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA [2] Department of Medicine, University of Washington, Seattle, Washington 98195, USA.

The basic body plan and major physiological axes have been highly conserved during mammalian evolution, yet only a small fraction of the human genome sequence appears to be subject to evolutionary constraint. To quantify cis- versus trans-acting contributions to mammalian regulatory evolution, we performed genomic DNase I footprinting of the mouse genome across 25 cell and tissue types, collectively defining ∼8.6 million transcription factor (TF) occupancy sites at nucleotide resolution. Here we show that mouse TF footprints conjointly encode a regulatory lexicon that is ∼95% similar with that derived from human TF footprints. However, only ∼20% of mouse TF footprints have human orthologues. Despite substantial turnover of the cis-regulatory landscape, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through evolutionary innovation of TF recognition sequences. Furthermore, the higher-level organization of mouse TF-to-TF connections into cellular network architectures is nearly identical with human. Our results indicate that evolutionary selection on mammalian gene regulation is targeted chiefly at the level of trans-regulatory circuitry, enabling and potentiating cis-regulatory plasticity.
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http://dx.doi.org/10.1038/nature13972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405208PMC
November 2014

A comparative encyclopedia of DNA elements in the mouse genome.

Nature 2014 Nov;515(7527):355-64

Bioinformatics and Genomics, Centre for Genomic Regulation (CRG) and UPF, Doctor Aiguader, 88, 08003 Barcelona, Catalonia, Spain.

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.
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http://dx.doi.org/10.1038/nature13992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4266106PMC
November 2014

Mapping and dynamics of regulatory DNA and transcription factor networks in A. thaliana.

Cell Rep 2014 Sep 15;8(6):2015-2030. Epub 2014 Sep 15.

Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA. Electronic address:

Our understanding of gene regulation in plants is constrained by our limited knowledge of plant cis-regulatory DNA and its dynamics. We mapped DNase I hypersensitive sites (DHSs) in A. thaliana seedlings and used genomic footprinting to delineate ∼ 700,000 sites of in vivo transcription factor (TF) occupancy at nucleotide resolution. We show that variation associated with 72 diverse quantitative phenotypes localizes within DHSs. TF footprints encode an extensive cis-regulatory lexicon subject to recent evolutionary pressures, and widespread TF binding within exons may have shaped codon usage patterns. The architecture of A. thaliana TF regulatory networks is strikingly similar to that of animals in spite of diverged regulatory repertoires. We analyzed regulatory landscape dynamics during heat shock and photomorphogenesis, disclosing thousands of environmentally sensitive elements and enabling mapping of key TF regulatory circuits underlying these fundamental responses. Our results provide an extensive resource for the study of A. thaliana gene regulation and functional biology.
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http://dx.doi.org/10.1016/j.celrep.2014.08.019DOI Listing
September 2014

An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level.

Science 2013 Oct;342(6155):253-7

Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA.

Genome-wide association studies (GWASs) have ascertained numerous trait-associated common genetic variants, frequently localized to regulatory DNA. We found that common genetic variation at BCL11A associated with fetal hemoglobin (HbF) level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping uncovers a motif-disrupting common variant associated with reduced transcription factor (TF) binding, modestly diminished BCL11A expression, and elevated HbF. The surrounding sequences function in vivo as a developmental stage-specific, lineage-restricted enhancer. Genome engineering reveals the enhancer is required in erythroid but not B-lymphoid cells for BCL11A expression. These findings illustrate how GWASs may expose functional variants of modest impact within causal elements essential for appropriate gene expression. We propose the GWAS-marked BCL11A enhancer represents an attractive target for therapeutic genome engineering for the β-hemoglobinopathies.
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http://dx.doi.org/10.1126/science.1242088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4018826PMC
October 2013

Comprehensive characterization of erythroid-specific enhancers in the genomic regions of human Krüppel-like factors.

BMC Genomics 2013 Aug 28;14:587. Epub 2013 Aug 28.

CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, P,R, China.

Background: Mapping of DNase I hypersensitive sites (DHSs) is a powerful tool to experimentally identify cis-regulatory elements (CREs). Among CREs, enhancers are abundant and predominantly act in driving cell-specific gene expression. Krüppel-like factors (KLFs) are a family of eukaryotic transcription factors. Several KLFs have been demonstrated to play important roles in hematopoiesis. However, transcriptional regulation of KLFs via CREs, particularly enhancers, in erythroid cells has been poorly understood.

Results: In this study, 23 erythroid-specific or putative erythroid-specific DHSs were identified by DNase-seq in the genomic regions of 17 human KLFs, and their enhancer activities were evaluated using dual-luciferase reporter (DLR) assay. Of the 23 erythroid-specific DHSs, the enhancer activities of 15 DHSs were comparable to that of the classical enhancer HS2 in driving minimal promoter (minP). Fifteen DHSs, some overlapping those that increased minP activities, acted as enhancers when driving the corresponding KLF promoters (KLF-Ps) in erythroid cells; of these, 10 DHSs were finally characterized as erythroid-specific KLF enhancers. These 10 erythroid-specific KLF enhancers were further confirmed using chromatin immunoprecipitation coupled to sequencing (ChIP-seq) data-based bioinformatic and biochemical analyses.

Conclusion: Our present findings provide a feasible strategy to extensively identify gene- and cell-specific enhancers from DHSs obtained by high-throughput sequencing, which will help reveal the transcriptional regulation and biological functions of genes in some specific cells.
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http://dx.doi.org/10.1186/1471-2164-14-587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3846580PMC
August 2013

Genome-scale mapping of DNase I hypersensitivity.

Curr Protoc Mol Biol 2013 Jul;Chapter 27:Unit 21.27

Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

DNase I-seq is a global and high-resolution method that uses the nonspecific endonuclease DNase I to map chromatin accessibility. These accessible regions, designated as DNase I hypersensitive sites (DHSs), define the regulatory features, (e.g., promoters, enhancers, insulators, and locus control regions) of complex genomes. In this unit, methods are described for nuclei isolation, digestion of nuclei with limiting concentrations of DNase I, and the biochemical fractionation of DNase I hypersensitive sites in preparation for high-throughput sequencing. DNase I-seq is an unbiased and robust method that is not predicated on an a priori understanding of regulatory patterns or chromatin features.
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http://dx.doi.org/10.1002/0471142727.mb2127s103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405172PMC
July 2013

Probing DNA shape and methylation state on a genomic scale with DNase I.

Proc Natl Acad Sci U S A 2013 Apr 1;110(16):6376-81. Epub 2013 Apr 1.

Department of Electrical Engineering and Biological Sciences, Columbia University, New York, NY 10027, USA.

DNA binding proteins find their cognate sequences within genomic DNA through recognition of specific chemical and structural features. Here we demonstrate that high-resolution DNase I cleavage profiles can provide detailed information about the shape and chemical modification status of genomic DNA. Analyzing millions of DNA backbone hydrolysis events on naked genomic DNA, we show that the intrinsic rate of cleavage by DNase I closely tracks the width of the minor groove. Integration of these DNase I cleavage data with bisulfite sequencing data for the same cell type's genome reveals that cleavage directly adjacent to cytosine-phosphate-guanine (CpG) dinucleotides is enhanced at least eightfold by cytosine methylation. This phenomenon we show to be attributable to methylation-induced narrowing of the minor groove. Furthermore, we demonstrate that it enables simultaneous mapping of DNase I hypersensitivity and regional DNA methylation levels using dense in vivo cleavage data. Taken together, our results suggest a general mechanism by which CpG methylation can modulate protein-DNA interaction strength via the remodeling of DNA shape.
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http://dx.doi.org/10.1073/pnas.1216822110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3631675PMC
April 2013

Systematic localization of common disease-associated variation in regulatory DNA.

Science 2012 Sep 5;337(6099):1190-5. Epub 2012 Sep 5.

Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.

Genome-wide association studies have identified many noncoding variants associated with common diseases and traits. We show that these variants are concentrated in regulatory DNA marked by deoxyribonuclease I (DNase I) hypersensitive sites (DHSs). Eighty-eight percent of such DHSs are active during fetal development and are enriched in variants associated with gestational exposure-related phenotypes. We identified distant gene targets for hundreds of variant-containing DHSs that may explain phenotype associations. Disease-associated variants systematically perturb transcription factor recognition sequences, frequently alter allelic chromatin states, and form regulatory networks. We also demonstrated tissue-selective enrichment of more weakly disease-associated variants within DHSs and the de novo identification of pathogenic cell types for Crohn's disease, multiple sclerosis, and an electrocardiogram trait, without prior knowledge of physiological mechanisms. Our results suggest pervasive involvement of regulatory DNA variation in common human disease and provide pathogenic insights into diverse disorders.
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http://dx.doi.org/10.1126/science.1222794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771521PMC
September 2012

The accessible chromatin landscape of the human genome.

Nature 2012 Sep;489(7414):75-82

Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA.

DNase I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ∼2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ∼580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation.
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http://dx.doi.org/10.1038/nature11232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3721348PMC
September 2012

An encyclopedia of mouse DNA elements (Mouse ENCODE).

Genome Biol 2012 Aug 13;13(8):418. Epub 2012 Aug 13.

To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.
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http://dx.doi.org/10.1186/gb-2012-13-8-418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3491367PMC
August 2012

Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR.

Dev Biol 2012 Jun 19;366(2):185-94. Epub 2012 Apr 19.

Stem Cell Program and Division of Hematology/Oncology, Children's Hospital and Dana Farber Cancer Institute, and Harvard Stem Cell Institute, Harvard Medical School, 1 Blackfan Cir., Karp 7, Boston, MA 02115, USA.

Globin gene switching is a complex, highly regulated process allowing expression of distinct globin genes at specific developmental stages. Here, for the first time, we have characterized all of the zebrafish globins based on the completed genomic sequence. Two distinct chromosomal loci, termed major (chromosome 3) and minor (chromosome 12), harbor the globin genes containing α/β pairs in a 5'-3' to 3'-5' orientation. Both these loci share synteny with the mammalian α-globin locus. Zebrafish globin expression was assayed during development and demonstrated two globin switches, similar to human development. A conserved regulatory element, the locus control region (LCR), was revealed by analyzing DNase I hypersensitive sites, H3K4 trimethylation marks and GATA1 binding sites. Surprisingly, the position of these sites with relation to the globin genes is evolutionarily conserved, despite a lack of overall sequence conservation. Motifs within the zebrafish LCR include CACCC, GATA, and NFE2 sites, suggesting functional interactions with known transcription factors but not the same LCR architecture. Functional homology to the mammalian α-LCR MCS-R2 region was confirmed by robust and specific reporter expression in erythrocytes of transgenic zebrafish. Our studies provide a comprehensive characterization of the zebrafish globin loci and clarify the regulation of globin switching.
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http://dx.doi.org/10.1016/j.ydbio.2012.03.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378398PMC
June 2012

Dynamic reprogramming of chromatin accessibility during Drosophila embryo development.

Genome Biol 2011 11;12(5):R43. Epub 2011 May 11.

Department of Genome Sciences, University of Washington, Foege S310A, 1705 NE Pacific Street, Box 355065, Seattle, WA 98195, USA.

Background: The development of complex organisms is believed to involve progressive restrictions in cellular fate. Understanding the scope and features of chromatin dynamics during embryogenesis, and identifying regulatory elements important for directing developmental processes remain key goals of developmental biology.

Results: We used in vivo DNaseI sensitivity to map the locations of regulatory elements, and explore the changing chromatin landscape during the first 11 hours of Drosophila embryonic development. We identified thousands of conserved, developmentally dynamic, distal DNaseI hypersensitive sites associated with spatial and temporal expression patterning of linked genes and with large regions of chromatin plasticity. We observed a nearly uniform balance between developmentally up- and down-regulated DNaseI hypersensitive sites. Analysis of promoter chromatin architecture revealed a novel role for classical core promoter sequence elements in directing temporally regulated chromatin remodeling. Another unexpected feature of the chromatin landscape was the presence of localized accessibility over many protein-coding regions, subsets of which were developmentally regulated or associated with the transcription of genes with prominent maternal RNA contributions in the blastoderm.

Conclusions: Our results provide a global view of the rich and dynamic chromatin landscape of early animal development, as well as novel insights into the organization of developmentally regulated chromatin features.
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http://dx.doi.org/10.1186/gb-2011-12-5-r43DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3219966PMC
February 2012

The role of chromatin accessibility in directing the widespread, overlapping patterns of Drosophila transcription factor binding.

Genome Biol 2011 7;12(4):R34. Epub 2011 Apr 7.

Genomics Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road MS 84-171, Berkeley, CA 94720, USA.

Background: In Drosophila embryos, many biochemically and functionally unrelated transcription factors bind quantitatively to highly overlapping sets of genomic regions, with much of the lowest levels of binding being incidental, non-functional interactions on DNA. The primary biochemical mechanisms that drive these genome-wide occupancy patterns have yet to be established.

Results: Here we use data resulting from the DNaseI digestion of isolated embryo nuclei to provide a biophysical measure of the degree to which proteins can access different regions of the genome. We show that the in vivo binding patterns of 21 developmental regulators are quantitatively correlated with DNA accessibility in chromatin. Furthermore, we find that levels of factor occupancy in vivo correlate much more with the degree of chromatin accessibility than with occupancy predicted from in vitro affinity measurements using purified protein and naked DNA. Within accessible regions, however, the intrinsic affinity of the factor for DNA does play a role in determining net occupancy, with even weak affinity recognition sites contributing. Finally, we show that programmed changes in chromatin accessibility between different developmental stages correlate with quantitative alterations in factor binding.

Conclusions: Based on these and other results, we propose a general mechanism to explain the widespread, overlapping DNA binding by animal transcription factors. In this view, transcription factors are expressed at sufficiently high concentrations in cells such that they can occupy their recognition sequences in highly accessible chromatin without the aid of physical cooperative interactions with other proteins, leading to highly overlapping, graded binding of unrelated factors.
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http://dx.doi.org/10.1186/gb-2011-12-4-r34DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3218860PMC
February 2012

Diverse gene reprogramming events occur in the same spatial clusters of distal regulatory elements.

Genome Res 2011 May 6;21(5):697-706. Epub 2011 Apr 6.

Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-5055, USA.

The spatial organization of genes in the interphase nucleus plays an important role in establishment and regulation of gene expression. Contradicting results have been reported to date, with little consensus about the dynamics of nuclear organization and the features of the contact loci. In this study, we investigated the properties and dynamics of genomic loci that are in contact with glucocorticoid receptor (GR)-responsive loci. We took a systematic approach, combining genome-wide interaction profiling by the chromosome conformation capture on chip (4C) technology with expression, protein occupancy, and chromatin accessibility profiles. This approach allowed a comprehensive analysis of how distinct features of the linear genome are organized in the three-dimensional nuclear space in the context of rapid gene regulation. We found that the transcriptional response to GR occurs without dramatic nuclear reorganization. Moreover, contrary to the view of transcription-driven organization, even genes with opposite transcriptional responses colocalize. Regions contacting GR-regulated genes are not particularly enriched for GR-regulated loci or for any functional group of genes, suggesting that these subnuclear environments are not organized to respond to a specific factor. The contact regions are, however, highly enriched for DNase I-hypersensitive sites that comprehensively mark cell-type-specific regulatory sites. These findings indicate that the nucleus is pre-organized in a conformation allowing rapid transcriptional reprogramming, and this organization is significantly correlated with cell-type-specific chromatin sites accessible to regulatory factors. Numerous open chromatin loci may be arranged in nuclear domains that are poised to respond to diverse signals in general and to permit efficient gene regulation.
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http://dx.doi.org/10.1101/gr.111153.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083086PMC
May 2011

Quantitative models of the mechanisms that control genome-wide patterns of transcription factor binding during early Drosophila development.

PLoS Genet 2011 Feb 3;7(2):e1001290. Epub 2011 Feb 3.

Department of Molecular and Cell Biology, California Institute of Quantitative Biosciences, University of California Berkeley, Berkeley, California, United States of America.

Transcription factors that drive complex patterns of gene expression during animal development bind to thousands of genomic regions, with quantitative differences in binding across bound regions mediating their activity. While we now have tools to characterize the DNA affinities of these proteins and to precisely measure their genome-wide distribution in vivo, our understanding of the forces that determine where, when, and to what extent they bind remains primitive. Here we use a thermodynamic model of transcription factor binding to evaluate the contribution of different biophysical forces to the binding of five regulators of early embryonic anterior-posterior patterning in Drosophila melanogaster. Predictions based on DNA sequence and in vitro protein-DNA affinities alone achieve a correlation of ∼0.4 with experimental measurements of in vivo binding. Incorporating cooperativity and competition among the five factors, and accounting for spatial patterning by modeling binding in every nucleus independently, had little effect on prediction accuracy. A major source of error was the prediction of binding events that do not occur in vivo, which we hypothesized reflected reduced accessibility of chromatin. To test this, we incorporated experimental measurements of genome-wide DNA accessibility into our model, effectively restricting predicted binding to regions of open chromatin. This dramatically improved our predictions to a correlation of 0.6-0.9 for various factors across known target genes. Finally, we used our model to quantify the roles of DNA sequence, accessibility, and binding competition and cooperativity. Our results show that, in regions of open chromatin, binding can be predicted almost exclusively by the sequence specificity of individual factors, with a minimal role for protein interactions. We suggest that a combination of experimentally determined chromatin accessibility data and simple computational models of transcription factor binding may be used to predict the binding landscape of any animal transcription factor with significant precision.
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http://dx.doi.org/10.1371/journal.pgen.1001290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033374PMC
February 2011

Chromatin accessibility pre-determines glucocorticoid receptor binding patterns.

Nat Genet 2011 Mar 23;43(3):264-8. Epub 2011 Jan 23.

Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

Development, differentiation and response to environmental stimuli are characterized by sequential changes in cellular state initiated by the de novo binding of regulated transcriptional factors to their cognate genomic sites. The mechanism whereby a given regulatory factor selects a limited number of in vivo targets from a myriad of potential genomic binding sites is undetermined. Here we show that up to 95% of de novo genomic binding by the glucocorticoid receptor, a paradigmatic ligand-activated transcription factor, is targeted to preexisting foci of accessible chromatin. Factor binding invariably potentiates chromatin accessibility. Cell-selective glucocorticoid receptor occupancy patterns appear to be comprehensively predetermined by cell-specific differences in baseline chromatin accessibility patterns, with secondary contributions from local sequence features. The results define a framework for understanding regulatory factor-genome interactions and provide a molecular basis for the tissue selectivity of steroid pharmaceuticals and other agents that intersect the living genome.
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http://dx.doi.org/10.1038/ng.759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386452PMC
March 2011

Comprehensive analysis of the chromatin landscape in Drosophila melanogaster.

Nature 2011 Mar 22;471(7339):480-5. Epub 2010 Dec 22.

Center for Biomedical Informatics, Harvard Medical School, Boston, Massachusetts 02115, USA.

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.
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http://dx.doi.org/10.1038/nature09725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109908PMC
March 2011

CCCTC-binding factor and the transcription factor T-bet orchestrate T helper 1 cell-specific structure and function at the interferon-gamma locus.

Immunity 2009 Oct 8;31(4):551-64. Epub 2009 Oct 8.

Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195, USA.

How cell type-specific differences in chromatin conformation are achieved and their contribution to gene expression are incompletely understood. Here we identify a cryptic upstream orchestrator of interferon-gamma (IFNG) transcription, which is embedded within the human IL26 gene, compromised of a single CCCTC-binding factor (CTCF) binding site and retained in all mammals, even surviving near-complete evolutionary deletion of the equivalent gene encoding IL-26 in rodents. CTCF and cohesins occupy this element in vivo in a cell type-nonspecific manner. This element is juxtaposed to two other sites located within the first intron and downstream of Ifng, where CTCF, cohesins, and the transcription factor T-bet bind in a T helper 1 (Th1) cell-specific manner. These interactions, close proximity of other elements within the locus to each other and to the gene encoding interferon-gamma, and robust murine Ifng expression are dependent on CTCF and T-bet. The results demonstrate that cooperation between architectural (CTCF) and transcriptional enhancing (T-bet) factors and the elements to which they bind is required for proper Th1 cell-specific expression of Ifng.
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http://dx.doi.org/10.1016/j.immuni.2009.08.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2810421PMC
October 2009

Comprehensive mapping of long-range interactions reveals folding principles of the human genome.

Science 2009 Oct;326(5950):289-93

Broad Institute of Harvard and Massachusetts Institute of Technology (MIT), MA 02139, USA.

We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1 megabase. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes. We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free, polymer conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.
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http://dx.doi.org/10.1126/science.1181369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2858594PMC
October 2009

Global mapping of protein-DNA interactions in vivo by digital genomic footprinting.

Nat Methods 2009 Apr 22;6(4):283-9. Epub 2009 Mar 22.

Department of Genome Sciences, University of Washington, Seattle, USA.

The orchestrated binding of transcriptional activators and repressors to specific DNA sequences in the context of chromatin defines the regulatory program of eukaryotic genomes. We developed a digital approach to assay regulatory protein occupancy on genomic DNA in vivo by dense mapping of individual DNase I cleavages from intact nuclei using massively parallel DNA sequencing. Analysis of >23 million cleavages across the Saccharomyces cerevisiae genome revealed thousands of protected regulatory protein footprints, enabling de novo derivation of factor binding motifs and the identification of hundreds of new binding sites for major regulators. We observed striking correspondence between single-nucleotide resolution DNase I cleavage patterns and protein-DNA interactions determined by crystallography. The data also yielded a detailed view of larger chromatin features including positioned nucleosomes flanking factor binding regions. Digital genomic footprinting should be a powerful approach to delineate the cis-regulatory framework of any organism with an available genome sequence.
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http://dx.doi.org/10.1038/nmeth.1313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2668528PMC
April 2009

Assaying the regulatory potential of mammalian conserved non-coding sequences in human cells.

Genome Biol 2008 2;9(12):R168. Epub 2008 Dec 2.

Department of Genetic Medicine and Development, University of Geneva Medical School, 1 rue Michel Servet, 1211, Geneva 4, Switzerland.

Background: Conserved non-coding sequences in the human genome are approximately tenfold more abundant than known genes, and have been hypothesized to mark the locations of cis-regulatory elements. However, the global contribution of conserved non-coding sequences to the transcriptional regulation of human genes is currently unknown. Deeply conserved elements shared between humans and teleost fish predominantly flank genes active during morphogenesis and are enriched for positive transcriptional regulatory elements. However, such deeply conserved elements account for <1% of the conserved non-coding sequences in the human genome, which are predominantly mammalian.

Results: We explored the regulatory potential of a large sample of these 'common' conserved non-coding sequences using a variety of classic assays, including chromatin remodeling, and enhancer/repressor and promoter activity. When tested across diverse human model cell types, we find that the fraction of experimentally active conserved non-coding sequences within any given cell type is low (approximately 5%), and that this proportion increases only modestly when considered collectively across cell types.

Conclusions: The results suggest that classic assays of cis-regulatory potential are unlikely to expose the functional potential of the substantial majority of mammalian conserved non-coding sequences in the human genome.
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http://dx.doi.org/10.1186/gb-2008-9-12-r168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2646272PMC
March 2009

Interaction of the glucocorticoid receptor with the chromatin landscape.

Mol Cell 2008 Mar;29(5):611-24

Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-5055, USA.

The generality and spectrum of chromatin-remodeling requirements for nuclear receptor function are unknown. We have characterized glucocorticoid receptor (GR) binding events and chromatin structural transitions across GR-induced or -repressed genes. This analysis reveals that GR binding invariably occurs at nuclease-accessible sites (DHS). A remarkable diversity of mechanisms, however, render these sites available for GR binding. Accessibility of the GR binding sites is either constitutive or hormone inducible. Within each category, some DHS sites require the Brg1-containing Swi/Snf complex, but others are Brg1 independent, implicating a different remodeling complex. The H2A.Z histone variant is highly enriched at both inducible and constitutive DHS sites and is subject to exchange during hormone activation. The DHS profile is highly cell specific, implicating cell-selective organization of the chromatin landscape as a critical determinant of tissue-selective receptor function. Furthermore, the widespread requirement for chromatin remodeling supports the recent hypothesis that the rapid exchange of receptor proteins occurs during nucleosome reorganization.
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http://dx.doi.org/10.1016/j.molcel.2008.02.010DOI Listing
March 2008

Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

Nature 2007 Jun;447(7146):799-816

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2212820PMC
http://dx.doi.org/10.1038/nature05874DOI Listing
June 2007

Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays.

Nat Methods 2006 Jul;3(7):511-8

Department of Genome Sciences, University of Washington, 1705 NE Pacific St., Box 357730, Seattle, Washington 98195, USA.

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.
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http://dx.doi.org/10.1038/nmeth890DOI Listing
July 2006

High-throughput localization of functional elements by quantitative chromatin profiling.

Nat Methods 2004 Dec 18;1(3):219-25. Epub 2004 Nov 18.

Department of Molecular Biology, Regulome, 2211 Elliott Avenue, Suite 600, Seattle, Washington 98121, USA.

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 approximately 300 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, high-throughput 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.
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http://dx.doi.org/10.1038/nmeth721DOI Listing
December 2004

Discovery of functional noncoding elements by digital analysis of chromatin structure.

Proc Natl Acad Sci U S A 2004 Nov 18;101(48):16837-42. Epub 2004 Nov 18.

Department of Molecular Biology, Regulome, 2211 Elliott Avenue, Suite 600, Seattle, WA 98121, USA.

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 19/20-bp genomic DNA tags to localize individual DNase I cutting events in nuclear chromatin and produced approximately 257,000 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.
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http://dx.doi.org/10.1073/pnas.0407387101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC534745PMC
November 2004

Genome-wide identification of DNaseI hypersensitive sites using active chromatin sequence libraries.

Proc Natl Acad Sci U S A 2004 Mar 19;101(13):4537-42. Epub 2004 Mar 19.

Department of Molecular Biology, Regulome, Canal View Building, 551 North 34th Street, Seattle, WA 98103, USA.

Comprehensive identification of sequences that regulate transcription is one of the major goals of genome biology. Focal alteration in chromatin structure in vivo, detectable through hypersensitivity to DNaseI and other nucleases, is the sine qua non of a diverse cast of transcriptional regulatory elements including enhancers, promoters, insulators, and locus control regions. We developed an approach for genome-scale identification of DNaseI hypersensitive sites (HSs) via isolation and cloning of in vivo DNaseI cleavage sites to create libraries of active chromatin sequences (ACSs). Here, we describe analysis of >61,000 ACSs derived from erythroid cells. We observed peaks in the density of ACSs at the transcriptional start sites of known genes at non-gene-associated CpG islands, and, to a lesser degree, at evolutionarily conserved noncoding sequences. Peaks in ACS density paralleled the distribution of DNaseI HSs. ACSs and DNaseI HSs were distributed between both expressed and nonexpressed genes, suggesting that a large proportion of genes reside within open chromatin domains. The results permit a quantitative approximation of the distribution of HSs and classical cis-regulatory sequences in the human genome.
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http://dx.doi.org/10.1073/pnas.0400678101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC384782PMC
March 2004
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