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ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2009 (English)In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 7, no 12, e1000256- p.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.

Place, publisher, year, edition, pages
2009. Vol. 7, no 12, e1000256- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-119579DOI: 10.1371/journal.pbio.1000256ISI: 000273060500005PubMedID: 20016685OAI: oai:DiVA.org:uu-119579DiVA: diva2:300548
Available from: 2010-02-26 Created: 2010-02-26 Last updated: 2017-12-12Bibliographically 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. Functional Studies of Genes Associated with Muscle Growth in Pigs and Hair Greying in Horses
Open this publication in new window or tab >>Functional Studies of Genes Associated with Muscle Growth in Pigs and Hair Greying in Horses
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Domestic animals have become very different from their wild ancestors during domestication and animal breeding. This provides a good model to unravel the molecular mechanisms underlying phenotypic variation. In my thesis I have studied genes affecting two important traits, leanness in pigs and hair greying-associated melanoma in horses.

In the first part of the thesis, I focused on an intronic mutation leading to more muscle growth and less fat deposition in domestic pigs to identify a transcription factor (TF) that binds to the regulatory element overlapping with the mutation. The aim has been to further study the function of the previously unknown TF in mouse myoblast cells and in insulin-producing cells (Paper I-III). We discovered a new TF ZBED6 binding to intron 3 of the IGF2 gene, in which a single nucleotide substitution in pigs abrogates the binding and causes increased leanness in domestic pigs. Silencing of ZBED6 expression in mouse myoblasts increased Igf2 expression, cell proliferation and migration, and myotube formation. This result is in line with the increased leanness phenotype in mutant pigs. Chromatin Immunoprecipitation-sequencing (ChIP-seq) using an anti-ZBED6 antibody identified 1200 ZBED6 target genes besides IGF2 and many are TFs controlling fundamental biological processes. In the first follow-up study we found ZBED6 mainly affected the expression of muscle protein genes by directly regulating Igf2 and Twist2 expression, in agreement with our previous observation of faster myotube formation in ZBED6-silenced cells. ChIP-seq with antibodies against six different histone modifications revealed that ZBED6 preferentially binds to active promoters and modulates transcriptional activity by a novel mechanism rather than by recruiting repressive histone modifications. The second follow-up study revealed that ZBED6 affects the morphology and insulin content and release in pancreatic ß cells.

In the second part (Paper IV), we investigate the functional significance of an intronic duplication in the Syntaxin 17 (STX17) gene causing hair greying and melanoma in horses. We found two Microphtalmia-associated transcription factor (MITF) binding sites within the duplication and showed that the duplicated sequence up-regulates reporter gene expression in a melanocyte-specific manner both by reporter assays in mouse melanocytes and in transgenic zebrafish. These results established that the intronic duplication acts as a melanocyte-specific enhancer that becomes much stronger when it is duplicated.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 836
Keyword
QTN, muscle development, IGF2, ZBED6, RNA-seq, ChIP-seq, myoblasts, pancreatic beta cells, Grey horse, melanoma, MITF
National Category
Genetics
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
urn:nbn:se:uu:diva-183715 (URN)978-91-554-8527-6 (ISBN)
Public defence
2012-12-14, room B22, BMC, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2012-11-22 Created: 2012-10-31 Last updated: 2013-02-11Bibliographically approved
3. QTL Analysis in the Pig: From the Identification of Quantitative Trait Loci to the Understanding of Molecular Mechanisms
Open this publication in new window or tab >>QTL Analysis in the Pig: From the Identification of Quantitative Trait Loci to the Understanding of Molecular Mechanisms
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Domestic pigs have become very different form the wild ancestors they originate from. Selection for muscle growth and meat quality has made the pig a good model for genetic studies of muscle development.

The first part of this thesis presents a genome-wide scan for quantitative trait loci (QTL) in a cross between Landrace and Hampshire pigs. Traits such as body composition, fat deposition, body length, meat quality and weight measurements of individual muscles were investigated. In total we identified 15 different QTLs that reached genome-wide significance. The three most significant QTLs were for carcass length on chromosome 17 and two overlapping QTLs on chromosome 1 for body composition and weight of M. biceps femoris, respectively. A strong candidate gene for the body composition QTL is melanocortin 4 receptor (MC4R). We also identified several QTLs for sizes of different muscles, fat deposition and meat quality traits.

In a previous study using a cross between the domestic Large White and wild boar, the mutation underlying a major QTL for muscle growth and fat deposition was identified as a single nucleotide substitution (QTN) in intron 3 of the IGF2 gene. The QTN disrupts the binding of a repressor affecting IGF2 mRNA expression. In the second part of this thesis, the identification of the repressor is presented. The repressor, named ZBED6, is a previously unknown mammalian member of the BED-domain protein family. We could show that Zbed6 specifically binds the wild-type but not the mutated sequence surrounding the QTN. Further studies of silenced Zbed6 in the mouse myoblast cell line C2C12 showed that it represses transcription in a luciferase reporter assay and affects Igf2 mRNA transcription and proliferation. ZBED6 shows very high sequence conservation and has a broad tissue distribution of expression suggesting that ZBED6 also has important biological function outside the muscle cell.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 55 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 420
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-9556 (URN)978-91-554-7412-6 (ISBN)
Public defence
2009-03-13, B41, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2009-02-20 Created: 2009-02-10 Last updated: 2012-11-26Bibliographically approved

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Pontén, FredrikKullander, KlasWadelius, ClaesLindblad-Toh, KerstinAndersson, Leif

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