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The ZBED6-IGF2 axis has a major effect on growth of skeletal muscle and internal organs in placental mammals
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Department of Animal Production, Ain Shams University, Cairo, Egypt.
Department of Molecular Medicine and Surgery, Integrative Physiology, Karolinska Institutet.
Department of Molecular Medicine and Surgery, Integrative Physiology, Karolinska Institutet.
Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden.
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2018 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 9, p. E2048-E2057Article in journal (Refereed) Published
Abstract [en]

A single nucleotide substitution in the third intron of insulin-like growth factor 2 (IGF2) is associated with increased muscle mass and reduced subcutaneous fat in domestic pigs. This mutation disrupts the binding of the ZBED6 transcription factor and leads to a threefold up-regulation of IGF2 expression in pig skeletal muscle. Here, we investigated the biological significance of ZBED6-IGF2 interaction in the growth of placental mammals using two mouse models, ZBED6 knock-out (Zbed6(-/-)) and Igf2 knock-in mice that carry the pig IGF2 mutation. These transgenic mice exhibit markedly higher serum IGF2 concentrations, higher growth rate, increased lean mass, and larger heart, kidney, and liver; no significant changes were observed for white adipose tissues. The changes in body and lean mass were most pronounced in female mice. The phenotypic changes were concomitant with a remarkable up-regulation of Igf2 expression in adult tissues. Transcriptome analysis of skeletal muscle identified differential expression of genes belonging to the extracellular region category. Expression analysis using fetal muscles indicated a minor role of ZBED6 in regulating Igf2 expression prenatally. Furthermore, transcriptome analysis of the adult skeletal muscle revealed that this elevated expression of Igf2 was derived from the P1 and P2 promoters. The results revealed very similar phenotypic effects in the Zbed6 knock-out mouse and in the Igf2 knock-in mouse, showing that the effect of ZBED6 on growth of muscle and internal organs is mediated through the binding site in the Igf2 gene. The results explain why this ZBED6 binding site is extremely well conserved among placental mammals.

Place, publisher, year, edition, pages
2018. Vol. 115, no 9, p. E2048-E2057
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-329188DOI: 10.1073/pnas.1719278115ISI: 000426152500018OAI: oai:DiVA.org:uu-329188DiVA, id: diva2:1139886
Funder
Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research Council, 2012-1760Swedish Research Council, 2015-00165Swedish Research Council, 80576801Swedish Research Council, 70374401Available from: 2017-09-10 Created: 2017-09-10 Last updated: 2018-04-26Bibliographically approved
In thesis
1. Functional characterization of the biological significance of the ZBED6/ZC3H11A locus in placental mammals
Open this publication in new window or tab >>Functional characterization of the biological significance of the ZBED6/ZC3H11A locus in placental mammals
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The recent advances in molecular and computational biology have made possible the study of complicated transcriptional regulatory networks that control a wide range of biological processes and phenotypic traits. In this thesis, several approaches were combined including next generation sequencing, gene expression profiling, chromatin and RNA immunoprecipitation, bioinformatics and genome editing methods in order to characterize the biological significance of the ZBED6 and ZC3H11A genes.

A mutation in the binding site of ZBED6, located in an intron of IGF2, disrupts the binding and leads to 3-fold upregulation of IGF2 mRNA in pig muscle tissues. The first part of the thesis presents a detailed functional characterization of ZBED6. Transient silencing of ZBED6 expression in mouse myoblasts led to increased Igf2 expression (~2-fold). ChIP-seq analysis of ZBED6 and histone modifications showed that ZBED6 preferentially binds active promoters and modulates their transcriptional activities (paper I). In the follow-up studies using CRISPR/Cas9 we showed that either the deletion of ZBED6 or its binding site in Igf2 (Igf2ΔGGCT) led to more than 30-fold up-regulation of Igf2 expression in myoblasts. Differentiation of these genetically engineered cells resulted in hypertrophic myotubes. Transcriptome analysis revealed ~30% overlap between the differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with significant enrichment of muscle-specific genes. ZBED6-overexpression in myoblasts led to cell cycle arrest, reduced cell viability, reduced mitochondrial activities and impaired the differentiation of myoblasts (paper II). Further studies on cancer cells showed that ZBED6 influences the growth of colorectal cancer cells with dramatic changes in the transcription of hundreds of cancer-related genes (paper III). The phenotypic characterization of Zbed6-/- and Igf2pA/mG mouse models showed that the ZBED6-Igf2 axis has a major effect on regulating muscle growth and the growth of internal organs. Transcriptome analysis demonstrated a massive up-regulation of Igf2 expression (~30-fold) in adult tissues, but not in fetal tissues, of transgenic mice (paper IV).

In the second part of the thesis we investigated the cellular function of Zc3h11a, the gene harboring ZBED6 in one of its first introns. The function of the ZC3H11A protein is so far poorly characterized. We show that ZC3H11A is a novel stress-induced protein that is required for efficient mRNA export from the nucleus. The inactivation of ZC3H11A inhibited the growth of multiple viruses including HIV, influenza, HSV and adenoviruses (paper V).

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. p. 57
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1372
Keywords
ZBED6, IGF2, ZC3H11A, Muscle development, Transcriptome analysis, CRISPR/Cas9, mRNA export
National Category
Medical Genetics Cell and Molecular Biology
Research subject
Molecular Genetics; Bioinformatics
Identifiers
urn:nbn:se:uu:diva-329190 (URN)978-91-513-0072-6 (ISBN)
Public defence
2017-10-30, B/B42, Biomedicinskt centrum (BMC), Uppsala, 13:15 (English)
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Supervisors
Available from: 2017-10-09 Created: 2017-09-12 Last updated: 2018-01-13

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Younis, ShadySundström, ElisabethGustafson, UllaAndersson, Leif

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