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      Global Mapping of Cell Type–Specific Open Chromatin by FAIRE-seq Reveals the Regulatory Role of the NFI Family in Adipocyte Differentiation

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          Abstract

          Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type–specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation) and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI) transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA–mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our study demonstrates the utility of FAIRE-seq in providing a global view of cell type–specific regulatory elements in the genome and in identifying transcriptional regulators of adipocyte differentiation.

          Author Summary

          Humans consist of a few hundred types of specialized-function cells. Spatial and temporal transcriptional regulation of genes is essential for manifestation of cellular phenotypes. Identification of regulatory regions in the genome is central to understanding the mechanism of cell type–specific gene regulation. Recently developed high-throughput sequencing technology and computational analyses allow genome-wide investigation of the genome's chromatin structure. Using the FAIRE-seq technique, we identified the genome's open chromatin regions, which harbor regulatory elements in adipocytes. Open chromatin regions distal to genes' transcription start sites significantly differ among cell types. Multiple cell type–specific open chromatin regions exist near genes regulated during adipocyte differentiation. Computational motif analysis of adipocyte-specific open chromatin regions revealed enrichment of a binding motif for the NFI transcription factor family. These factors bind to the regulatory elements near adipogenic PPARγ, C/EBPα, and aP2 genes and regulate their expression. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus and knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation. Our study demonstrates the utility of FAIRE-seq in providing a global view of regulatory elements and in identifying transcriptional regulators of cellular functions.

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          Most cited references 86

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          Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.

          DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The procedure first requires uploading a gene list containing any number of common gene identifiers followed by analysis using one or more text and pathway-mining tools such as gene functional classification, functional annotation chart or clustering and functional annotation table. By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.
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            Initial sequencing and analysis of the human genome.

             James Galagan (2001)
            The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
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              Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

              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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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                October 2011
                October 2011
                20 October 2011
                : 7
                : 10
                Affiliations
                [1 ]Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
                [2 ]Functional Regulation of Adipocytes, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
                [3 ]Genome Science Division, Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan
                [4 ]Department of Integrated Molecular Science on Metabolic Diseases, 22nd Century Medical and Research Center, University of Tokyo, Tokyo, Japan
                [5 ]Molecular Medicinal Sciences on Metabolic Regulation, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
                [6 ]Systems Biology and Medicine Division, Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan
                [7 ]Metabolic Medicine Division, Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan
                The University of North Carolina at Chapel Hill, United States of America
                Author notes

                Conceived and designed the experiments: H Waki, M Nakamura, T Yamauchi, K Wakabayashi. Performed the experiments: H Waki, M Nakamura, K Wakabayashi, J Yu, L Hirose-Yotsuya, K Take, W Sun, T Aoyama. Analyzed the data: H Waki, M Nakamura, K Wakabayashi, T Fujita, S Tsutsumi, T Yamauchi, M Iwabu, M Okada-Iwabu. Contributed reagents/materials/analysis tools: H Waki, M Nakamura, K Wakabayashi, T Fujita, S Tsutsumi. Wrote the paper: H Waki, M Nakamura. Supervised the design of the experiments: K Ueki, T Kodama, T Yamauchi, S Tsutsumi, J Sakai, H Aburatani, T Kadowaki.

                Article
                PGENETICS-D-11-00795
                10.1371/journal.pgen.1002311
                3197683
                22028663
                Waki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Counts
                Pages: 16
                Categories
                Research Article
                Biology
                Biochemistry
                Lipids
                Fats
                Fatty Acids
                Lipid Mediators
                Metabolism
                Carbohydrate Metabolism
                Lipid Metabolism
                Metabolic Pathways
                Hormones
                Developmental Biology
                Cell Differentiation
                Cell Fate Determination
                Genetics
                Epigenetics
                Gene Expression
                Gene Function
                Genomics
                Chromosome Biology
                Chromatin
                Molecular Cell Biology
                Gene Expression
                DNA transcription
                Histone Modification
                Cellular Types
                Medicine
                Endocrinology
                Diabetic Endocrinology
                Nutrition
                Obesity

                Genetics

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