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Stable silencing of ZBED6 affects morphology, gene expression and  insulin release in insulin-producing islet cells
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 Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (ola.wallerman@imbim.uu.se)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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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 [en]
Transcriptome analysis, insulin, beta cells
National Category
Basic Medicine
Identifiers
URN: urn:nbn:se:uu:diva-183722OAI: oai:DiVA.org:uu-183722DiVA: diva2:563926
Available from: 2012-10-31 Created: 2012-10-31 Last updated: 2012-11-23
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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