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The role of ZBED6 in transcriptional regulation studied by transcriptome  analysis after RNAi in mouse myoblasts
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.
Virginia Polytechnic Institute and State University, Department of Animal and Poultry Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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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 [en]
Transcriptome analysis, RNAi
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-183721OAI: oai:DiVA.org:uu-183721DiVA: diva2:563923
Available from: 2012-10-31 Created: 2012-10-31 Last updated: 2012-11-23Bibliographically approved
In thesis
1. 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

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Jiang, LinRubin, Carl-JohanSundström, ElisabethWallerman, OlaAndersson, Leif

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