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Genome-Wide Studies of Transcriptional Regulation in Mammalian Cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The key to the complexity of higher organisms lies not in the number of protein coding genes they carry, but rather in the intrinsic complexity of the gene regulatory networks. The major effectors of transcriptional regulation are proteins called transcription factors, and in this thesis four papers describing genome-wide studies of seven such factors are presented, together with studies on components of the chromatin and transcriptome.

In Paper I, we optimized a large-scale in vivo method, ChIP-chip, to study protein – DNA interactions using microarrays. The metabolic-disease related transcription factors USF1, HNF4a and FOXA2 were studied in 1 % of the genome, and a surprising number of binding sites were found, mostly far from annotated genes.

In Paper II, a novel sequencing based method, ChIP-seq, was applied to FOXA2, HNF4a and GABPa, allowing a true genome-wide view of binding sites. A large overlap between the datasets were seen, and molecular interactions were verified in vivo. Using a ChIP-seq specific motif discovery method, we identified both the expected motifs and several for co-localized transcription factors.

In Paper III, we identified and studied a novel transcription factor, ZBED6, using the ChIP-seq method. Here, we went from one known binding site to several hundred sites throughout the mouse genome. Finally, in Paper IV, we studied the chromatin landscape by deep sequencing of nucleosomal DNA, and further used RNA-sequencing to quantify expression levels, and extended the knowledge about the binding profiles for the transcription factors NFY and TCF7L2.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2010. , p. 61
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 616
Keywords [en]
ChIP, ChIP-chip, ChIP-seq, transcription factors, motif discovery, nucleosome positioning, HepG2, genome-wide, RNA-seq
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-132882ISBN: 978-91-554-7935-0 (print)OAI: oai:DiVA.org:uu-132882DiVA, id: diva2:359637
Public defence
2010-12-10, Rudbeck Hall, Rudbeck Laboratory, Dag Hammarskjölds v 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-11-19 Created: 2010-10-28 Last updated: 2018-01-12Bibliographically approved
List of papers
1. Binding sites for metabolic disease related transcription factors inferred at base pair resolution by chromatin immunoprecipitation and genomic microarrays
Open this publication in new window or tab >>Binding sites for metabolic disease related transcription factors inferred at base pair resolution by chromatin immunoprecipitation and genomic microarrays
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2005 (English)In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 14, no 22, p. 3435-3447Article in journal (Refereed) Published
Abstract [en]

We present a detailed in vivo characterization of hepatocyte transcriptional regulation in HepG2 cells, using chromatin immunoprecipitation and detection on PCR fragment-based genomic tiling path arrays covering the encyclopedia of DNA element (ENCODE) regions. Our data suggest that HNF-4α and HNF-3β, which were commonly bound to distal regulatory elements, may cooperate in the regulation of a large fraction of the liver transcriptome and that both HNF-4α and USF1 may promote H3 acetylation to many of their targets. Importantly, bioinformatic analysis of the sequences bound by each transcription factor (TF) shows an over-representation of motifs highly similar to the in vitro established consensus sequences. On the basis of these data, we have inferred tentative binding sites at base pair resolution. Some of these sites have been previously found by in vitro analysis and some were verified in vitro in this study. Our data suggests that a similar approach could be used for the in vivo characterization of all predicted/uncharacterized TF and that the analysis could be scaled to the whole genome.

Keywords
Base Pairing/*genetics, Binding Sites/genetics, Cell Line; Tumor, Chromatin/*metabolism, Chromatin Immunoprecipitation/methods, Consensus Sequence, Genome; Human, Hepatocyte Nuclear Factor 3-beta/physiology, Hepatocyte Nuclear Factor 4/physiology, Hepatocytes/metabolism, Histones/metabolism, Humans, Metabolic Diseases/*metabolism, Oligonucleotide Array Sequence Analysis/methods, Promoter Regions (Genetics), Research Support; N.I.H.; Extramural, Research Support; Non-U.S. Gov't, Sequence Analysis; DNA, Transcription Factors/genetics/*metabolism, Upstream Stimulatory Factors/metabolism
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-80603 (URN)10.1093/hmg/ddi378 (DOI)16221759 (PubMedID)
Available from: 2006-05-19 Created: 2006-05-19 Last updated: 2017-12-14Bibliographically approved
2. Molecular interactions between HNF4a, FOXA2 and GABP identified at regulatory DNA elements through ChIP-sequencing
Open this publication in new window or tab >>Molecular interactions between HNF4a, FOXA2 and GABP identified at regulatory DNA elements through ChIP-sequencing
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2009 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 22, p. 7498-7508Article in journal (Refereed) Published
Abstract [en]

Gene expression is regulated by combinations of transcription factors, which can be mapped to regulatory elements on a genome-wide scale using ChIP experiments. In a previous ChIP-chip study of USF1 and USF2 we found evidence also of binding of GABP, FOXA2 and HNF4a within the enriched regions. Here, we have applied ChIP-seq for these transcription factors and identified 3064 peaks of enrichment for GABP, 7266 for FOXA2 and 18783 for HNF4a. Distal elements with USF2 signal was frequently bound also by HNF4a and FOXA2. GABP peaks were found at transcription start sites, whereas 94% of FOXA2 and 90% of HNF4a peaks were located at other positions. We developed a method to accurately define TFBS within peaks, and found the predicted sites to have an elevated conservation level compared to peak centers; however the majority of bindings were not evolutionary conserved. An interaction between HNF4a and GABP was seen at TSS, with one-third of the HNF4a positive promoters being bound also by GABP, and this interaction was verified by co-immunoprecipitations.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-121011 (URN)10.1093/nar/gkp823 (DOI)000272935000022 ()19822575 (PubMedID)
Available from: 2010-03-18 Created: 2010-03-18 Last updated: 2017-12-12Bibliographically approved
3. ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth
Open this publication in new window or tab >>ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth
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2009 (English)In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 7, no 12, p. e1000256-Article in journal (Refereed) Published
Abstract [en]

A single nucleotide substitution in intron 3 of IGF2 in pigs abrogates a binding site for a repressor and leads to a 3-fold up-regulation of IGF2 in skeletal muscle. The mutation has major effects on muscle growth, size of the heart, and fat deposition. Here, we have identified the repressor and find that the protein, named ZBED6, is previously unknown, specific for placental mammals, and derived from an exapted DNA transposon. Silencing of Zbed6 in mouse C2C12 myoblasts affected Igf2 expression, cell proliferation, wound healing, and myotube formation. Chromatin immunoprecipitation (ChIP) sequencing using C2C12 cells identified about 2,500 ZBED6 binding sites in the genome, and the deduced consensus motif gave a perfect match with the established binding site in Igf2. Genes associated with ZBED6 binding sites showed a highly significant enrichment for certain Gene Ontology classifications, including development and transcriptional regulation. The phenotypic effects in mutant pigs and ZBED6-silenced C2C12 myoblasts, the extreme sequence conservation, its nucleolar localization, the broad tissue distribution, and the many target genes with essential biological functions suggest that ZBED6 is an important transcription factor in placental mammals, affecting development, cell proliferation, and growth.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-119579 (URN)10.1371/journal.pbio.1000256 (DOI)000273060500005 ()20016685 (PubMedID)
Available from: 2010-02-26 Created: 2010-02-26 Last updated: 2017-12-12Bibliographically approved
4. Nucleosome landscape in HepG2 cells in relation to NFY, HNF4a and FOXA2 binding sites
Open this publication in new window or tab >>Nucleosome landscape in HepG2 cells in relation to NFY, HNF4a and FOXA2 binding sites
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Nucleosomes are the building blocks that compact the DNA in the nucleus, thereby regulating its accessibility. Here we report a genome-wide nucleosome positioning analysis on the HepG2 cell line, based on deep sequencing of mononucleosomal DNA. In concordance with other studies, we found nucleosomes to be well positioned at the TSS, with a nucleosome free region in the proximal promoter. Focusing on the importance of nucleosomal positioning at distal elements we found that TFBS sites often are flanked by well-positioning nucleosomes at a distance that indicate that the TF complexes replaces the nucleosome, and we also find that some transposable elements have distinct nucleosomal signatures. To build on previous regulatory data for HepG2 cells we also produced ChIP-seq reads for the transcription factors NF-Y and TCF7L2 and correlate this to the HepG2 transcriptome as defined by RNA-seq and Pol-II ChIP-seq. NF-Y was found primarily at promoters, contrary to what has been suggested from previous studies, and binds genes involved in cell cycle and chromatin organization.

Keywords
ChIP-seq, NFY, Pol-II, TCF7L2, nucleosome positioning
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-132879 (URN)
Available from: 2010-10-28 Created: 2010-10-28 Last updated: 2018-01-12Bibliographically approved

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