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Functional Studies of Genes Associated with Muscle Growth in Pigs and Hair Greying in Horses
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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 [en]
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: urn:nbn:se:uu:diva-183715ISBN: 978-91-554-8527-6 (print)OAI: oai:DiVA.org:uu-183715DiVA: diva2:563920
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
List of papers
1. 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, 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.

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
2. The role of ZBED6 in transcriptional regulation studied by transcriptome  analysis after RNAi in mouse myoblasts
Open this publication in new window or tab >>The role of ZBED6 in transcriptional regulation studied by transcriptome  analysis after RNAi in mouse myoblasts
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(English)Article, review/survey (Other academic) Submitted
Abstract [en]

ZBED6 is a recently discovered transcription factor that has evolved from a domesticated DNA transposon and is unique to placental mammals. Here we further characterize the functional significance of ZBED6 based on transcriptome analysis of mouse myoblasts after Zbed6-silencing. ZBED6 appears as an important transcriptional regulator since differential expression of more than 700 genes was observed after Zbed6-silencing. The most significantly enriched GO term was muscle protein and contractile fiber, which is consistent with increased myotube formation. Twenty small nucleolar RNAs showed differential expression and all increased in expression after Zbed6-silencing. This is particularly interesting because ZBED6 localization is strongly enriched in the nucleolus. There was an overrepresentation of genes with ZBED6 binding sites among the differentially expressed genes after silencing, suggesting that ZBED6 acts as a transcriptional regulator at many loci. Many genes showed significant down-regulation after Zbed6-silencing, which begs the question of whether ZBED6 acts as an activator at some of these loci or if the decreased mRNA levels of these genes all represent secondary effects. The co-localization of histone marks and ZBED6 binding sites and the effect of ZBED6-silencing on distribution of histone marks was evaluated by ChIP-seq. There was a strong association between ZBED6 binding sites and the H3K4me3, H3K4me2 and H3K27ac modifications, which are usually found at active promoters, but no association with the repressive marks H3K27me3. We propose that ZBED6 preferentially binds to active promoters and modulates transcriptional activity by a novel mechanism rather than by recruiting repressive histone modifications.  

Keyword
Transcriptome analysis, RNAi
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
urn:nbn:se:uu:diva-183721 (URN)
Available from: 2012-10-31 Created: 2012-10-31 Last updated: 2012-11-23Bibliographically approved
3. Stable silencing of ZBED6 affects morphology, gene expression and  insulin release in insulin-producing islet cells
Open this publication in new window or tab >>Stable silencing of ZBED6 affects morphology, gene expression and  insulin release in insulin-producing islet cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Zbed6 has evolved from a domesticated DNA transposon and encodes a novel transcription factor unique to placental mammals. Here we have investigated the function of ZBED6 in insulin-producing beta cells based on whole transcriptome analysis of MIN6 cells with lentiviral shRNA-mediated stable silencing of either Zbed6 (shZbed6) or mock mRNA (shMock). Zbed6-silencing was associated with altered cell morphology as the shZbed6 cells showed increased neuron-like protrusions compared with shMock cells. ZBED6 appeared as an important transcriptional regulator in islet cells since more than 700 genes showed differential expression in shZbed6 cells when compared with control cells. The most significantly enriched GO categories among differentially expressed genes were neuronal differentiation and cell adhesion, which is consistent with the changes in morphology in the silenced cells. A ChIP-seq analysis identified more than 4,000 putative binding sites in the genome of MIN6 cells and there was a significant overrepresentation of genes with ZBED6 sites among the differentially expressed genes after silencing. This suggests that ZBED6 acts as a transcriptional regulator for many genes in MIN6 cells. The genes showing differential expression included Pdx1, Mafa and Nkx6-1, three crucial transcription factors in beta-cell maturation, which were all up-regulated after Zbed6-silencing. Finally, in shZbed6 MIN6 cells the content and release of insulin was increased. We conclude that ZBED6 is expressed in insulin-producing islet cells and has a significant role for the modulation of cellular functions in this cell type.   

Keyword
Transcriptome analysis, insulin, beta cells
National Category
Basic Medicine
Identifiers
urn:nbn:se:uu:diva-183722 (URN)
Available from: 2012-10-31 Created: 2012-10-31 Last updated: 2012-11-23
4. Identification of a melanocyte-specific, microphthalmia-associated transcription factor-dependent regulatory element in the intronic duplication causing hair greying and melanoma in horses
Open this publication in new window or tab >>Identification of a melanocyte-specific, microphthalmia-associated transcription factor-dependent regulatory element in the intronic duplication causing hair greying and melanoma in horses
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2012 (English)In: Pigment Cell & Melanoma Research, ISSN 1755-1471, E-ISSN 1755-148X, Vol. 25, no 1, 28-36 p.Article in journal (Refereed) Published
Abstract [en]

Greying with age in horses is an autosomal dominant trait, characterized by hair greying, high incidence of melanoma and vitiligo-like depigmentation. Previous studies have revealed that the causative mutation for this phenotype is a 4.6-kb intronic duplication in STX17 (Syntaxin 17). By using reporter constructs in transgenic zebrafish, we show that a construct containing two copies of the duplicated sequence acts as a strong enhancer in neural crest cells and has subsequent melanophore-specific activity during zebrafish embryonic development whereas a single copy of the duplicated sequence acts as a weak enhancer, consistent with the phenotypic manifestation of the mutation in horses. We further used luciferase assays to investigate regulatory regions in the duplication, to reveal tissue-specific activities of these elements. One region upregulated the reporter gene expression in a melanocyte-specific manner and contained two microphthalmia-associated transcription factor (MITF) binding sites, essential for the activity. Microphthalmia-associated transcription factor regulates melanocyte development, and these binding sites are outstanding candidates for mediating the melanocyte-specific activity of the element. These results provide strong support for the causative nature of the duplication and constitute an explanation for the melanocyte-specific effects of the Grey allele.

Keyword
melanoma, hair greying, horses, zebrafish, STX17, NR4A3, microphthalmia-associated transcription factor
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-168743 (URN)10.1111/j.1755-148X.2011.00902.x (DOI)000298577600007 ()
Available from: 2012-02-16 Created: 2012-02-15 Last updated: 2017-12-07Bibliographically approved

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