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Identification of a melanocyte-specific, microphthalmia-associated transcription factor-dependent regulatory element in the intronic duplication causing hair greying and melanoma in horses
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2012 (English)In: Pigment Cell & Melanoma Research, ISSN 1755-1471, E-ISSN 1755-148X, Vol. 25, no 1, p. 28-36Article 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.

Place, publisher, year, edition, pages
2012. Vol. 25, no 1, p. 28-36
Keyword [en]
melanoma, hair greying, horses, zebrafish, STX17, NR4A3, microphthalmia-associated transcription factor
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-168743DOI: 10.1111/j.1755-148X.2011.00902.xISI: 000298577600007OAI: oai:DiVA.org:uu-168743DiVA: diva2:503690
Available from: 2012-02-16 Created: 2012-02-15 Last updated: 2017-12-07Bibliographically 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. p. 52
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, LinGolovko, AnnaAndersson, Leif

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