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Transcriptional modulator ZBED6 affects cell cycle and growth of human colorectal cancer cells
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
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. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
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2015 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 25, 7743-7748 p.Article in journal (Refereed) Published
Abstract [en]

The transcription factor ZBED6 (zinc finger, BED-type containing 6) is a repressor of IGF2 whose action impacts development, cell proliferation, and growth in placental mammals. In human colorectal cancers, IGF2 overexpression is mutually exclusive with somatic mutations in PI3K signaling components, providing genetic evidence for a role in the PI3K pathway. To understand the role of ZBED6 in tumorigenesis, we engineered and validated somatic cell ZBED6 knock-outs in the human colorectal cancer cell lines RKO and HCT116. Ablation of ZBED6 affected the cell cycle and led to increased growth rate in RKO cells but reduced growth in HCT116 cells. This striking difference was reflected in the transcriptome analyses, which revealed enrichment of cell-cycle-related processes among differentially expressed genes in both cell lines, but the direction of change often differed between the cell lines. ChIP sequencing analyses displayed enrichment of ZBED6 binding at genes up-regulated in ZBED6-knockout clones, consistent with the view that ZBED6 modulates gene expression primarily by repressing transcription. Ten differentially expressed genes were identified as putative direct gene targets, and their down-regulation by ZBED6 was validated experimentally. Eight of these genes were linked to the Wnt, Hippo, TGF-beta, EGF receptor, or PI3K pathways, all involved in colorectal cancer development. The results of this study show that the effect of ZBED6 on tumor development depends on the genetic background and the transcriptional state of its target genes.

Place, publisher, year, edition, pages
2015. Vol. 112, no 25, 7743-7748 p.
Keyword [en]
ZBED6, colorectal cancer, IGF2, PI3K pathway
National Category
Genetics
Identifiers
URN: urn:nbn:se:uu:diva-258330DOI: 10.1073/pnas.1509193112ISI: 000356731300067OAI: oai:DiVA.org:uu-258330DiVA: diva2:841846
Funder
Swedish Research Council
Available from: 2015-07-15 Created: 2015-07-13 Last updated: 2017-09-12
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. 57 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1372
Keyword
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)
Opponent
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Available from: 2017-10-09 Created: 2017-09-12 Last updated: 2017-10-18

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Younis, ShadyGupta, RajeshAndersson, LeifSjoblöm, Tobias
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Science for Life Laboratory, SciLifeLabDepartment of Immunology, Genetics and PathologyDepartment of Medical Biochemistry and MicrobiologyVascular BiologyExperimental and Clinical Oncology
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