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Binding sites for metabolic disease related transcription factors inferred at base pair resolution by chromatin immunoprecipitation and genomic microarrays
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Systems genomics)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Systems genomics)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
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2005 (English)In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 14, no 22, 3435-3447 p.Article 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.

Place, publisher, year, edition, pages
2005. Vol. 14, no 22, 3435-3447 p.
Keyword [en]
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: urn:nbn:se:uu:diva-80603DOI: 10.1093/hmg/ddi378PubMedID: 16221759OAI: oai:DiVA.org:uu-80603DiVA: diva2:108517
Available from: 2006-05-19 Created: 2006-05-19 Last updated: 2017-02-02Bibliographically approved
In thesis
1. Genome-Wide Studies of Transcriptional Regulation in Mammalian Cells
Open this publication in new window or tab >>Genome-Wide Studies of Transcriptional Regulation in Mammalian Cells
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. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 616
Keyword
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:nbn:se:uu:diva-132882 (URN)978-91-554-7935-0 (ISBN)
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: 2011-01-13Bibliographically approved
2. From Single Gene to Whole Genome Studies of Human Transcription Regulation
Open this publication in new window or tab >>From Single Gene to Whole Genome Studies of Human Transcription Regulation
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transcriptional regulation largely determines which proteins and the protein levels that are found in a cell, and this is crucial in development, differentiation and responses to environmental stimuli. The major effectors of transcriptional regulation are a group of proteins known as transcription factors, which importance is supported by their frequent involvement in mendelian and complex diseases.

In paper I, we attempted to establish the importance of DNA sequence variation in transcriptional control, by analyzing the potential functionality of polymorphic short repetitive elements as cis-regulatory elements. However, the relevance of this study was constrained by the limited number of analyzed sequences and the in vitro nature of the experiments. To overcome these limitations, (paper II) we optimized an in vivo large-scale technology named ChIP-chip, which couples chromatin immunoprecipitation and microarray hybridization. We successfully identified the binding profiles of metabolic-disease associated transcription factors in 1% of the human genome, using a liver cellular model, and inferred the binding sites at base pair resolution.

Another important characteristic of transcriptional regulation is its plasticity, which allows adjusting the cellular transcriptome to cellular and environmental stimuli. In paper III, we investigated such plasticity by treating HepG2 cells with butyrate, a histone deacetylase inhibitor (HDACi) and interrogating the changes in histone H3 and H4 acetylation levels in 1% of the genome. Observation of frequent deacetylation around transcription start sites and hyperacetylation at the nuclear periphery challenges pre-assumed HDACi mechanisms of action.

Finally, in paper IV we extended the DNA binding profiles of the medically relevant transcription factors, USF1 and USF2, and H3 acetylation to the whole non-repetitive fraction of the human genome. Using motif finding tools and chromatin profiling, we uncovered the major determinants of USF-DNA interactions. Furthermore, USFs and H3ac were clearly localized around transcription start sites, frequently in the context of bidirectional promoters.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 222
Keyword
Genetics, transcription, ChIP-chip, genome-wide, transcription factors, in vivo, Genetik
Identifiers
urn:nbn:se:uu:diva-7463 (URN)978-91-554-6790-3 (ISBN)
Public defence
2007-03-02, Rudbeck Hall, Rudbeck Laboratory, Dag Hammarsjöld, 20, Uppsala, 13:15
Opponent
Supervisors
Available from: 2007-02-08 Created: 2007-02-08 Last updated: 2013-09-20Bibliographically approved
3. A Bioinformatics Study of Human Transcriptional Regulation
Open this publication in new window or tab >>A Bioinformatics Study of Human Transcriptional Regulation
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Regulation of transcription is a central mechanism in all living cells that now can be investigated with high-throughput technologies. Data produced from such experiments give new insights to how transcription factors (TFs) coordinate the gene transcription and thereby regulate the amounts of proteins produced. These studies are also important from a medical perspective since TF proteins are often involved in disease. To learn more about transcriptional regulation, we have developed strategies for analysis of data from microarray and massively parallel sequencing (MPS) experiments.

Our computational results consist of methods to handle the steadily increasing amount of data from high-throughput technologies. Microarray data analysis tools have been assembled in the LCB-Data Warehouse (LCB-DWH) (paper I), and other analysis strategies have been developed for MPS data (paper V). We have also developed a de novo motif search algorithm called BCRANK (paper IV).

The analysis has lead to interesting biological findings in human liver cells (papers II-V). The investigated TFs appeared to bind at several thousand sites in the genome, that we have identified at base pair resolution. The investigated histone modifications are mainly found downstream of transcription start sites, and correlated to transcriptional activity. These histone marks are frequently found for pairs of genes in a bidirectional conformation. Our results suggest that a TF can bind in the shared promoter of two genes and regulate both of them.

From a medical perspective, the genes bound by the investigated TFs are candidates to be involved in metabolic disorders. Moreover, we have developed a new strategy to detect single nucleotide polymorphisms (SNPs) that disrupt the binding of a TF (paper IV). We further demonstrated that SNPs can affect transcription in the immediate vicinity. Ultimately, our method may prove helpful to find disease-causing regulatory SNPs.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 566
Keyword
bioinformatics, microarray, ChIP-chip, ChIP-seq, transcription factor, histone modification, motif search
National Category
Bioinformatics (Computational Biology)
Identifiers
urn:nbn:se:uu:diva-9346 (URN)978-91-554-7324-2 (ISBN)
Public defence
2008-11-28, C8:305, BMC, Husargatan 3, Uppsala, 09:00
Opponent
Supervisors
Available from: 2008-11-06 Created: 2008-11-06 Last updated: 2013-09-20Bibliographically approved

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Publisher's full textPubMedhttp://hmg.oxfordjournals.org/content/14/22/3435.long

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Wallerman, OlaEnroth, StefanWester, KennethPontén, FredrikKomorowski, JanWadelius, Claes

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