Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Functional characterization of the biological significance of the ZBED6/ZC3H11A locus in placental mammals
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.
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
Keyword [en]
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: urn:nbn:se:uu:diva-329190ISBN: 978-91-513-0072-6 (print)OAI: oai:DiVA.org:uu-329190DiVA, id: diva2:1140332
Public defence
2017-10-30, B/B42, Biomedicinskt centrum (BMC), Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2017-10-09 Created: 2017-09-12 Last updated: 2018-01-13
List of papers
1. ZBED6 Modulates the Transcription of Myogenic Genes in Mouse Myoblast Cells
Open this publication in new window or tab >>ZBED6 Modulates the Transcription of Myogenic Genes in Mouse Myoblast Cells
Show others...
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 4, p. e94187-Article in journal (Refereed) Published
Abstract [en]

ZBED6 is a recently discovered transcription factor, unique to placental mammals, that has evolved from a domesticated DNA transposon. It acts as a repressor at the IGF2 locus. Here we show that ZBED6 acts as a transcriptional modulator in mouse myoblast cells, where more than 700 genes were differentially expressed after Zbed6-silencing. The most significantly enriched GO term was muscle protein and contractile fiber, which was consistent with increased myotube formation. Twenty small nucleolar RNAs all showed increased expression after Zbed6-silencing. 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 analysis. 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 mark H3K27me3. Zbed6-silencing led to increased enrichment of active marks at myogenic genes, in agreement with the RNA-seq findings. We propose that ZBED6 preferentially binds to active promoters and modulates transcriptional activity without recruiting repressive histone modifications.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-225025 (URN)10.1371/journal.pone.0094187 (DOI)000334160900101 ()
Available from: 2014-05-26 Created: 2014-05-26 Last updated: 2017-12-05Bibliographically approved
2. Changes in myotube development and mitochondrial activity in mouse C2C12 cells after ZBED6 silencing is largely due to increased IGF2 expression
Open this publication in new window or tab >>Changes in myotube development and mitochondrial activity in mouse C2C12 cells after ZBED6 silencing is largely due to increased IGF2 expression
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-329186 (URN)
Available from: 2017-09-10 Created: 2017-09-10 Last updated: 2018-01-13
3. Transcriptional modulator ZBED6 affects cell cycle and growth of human colorectal cancer cells
Open this publication in new window or tab >>Transcriptional modulator ZBED6 affects cell cycle and growth of human colorectal cancer cells
Show others...
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, p. 7743-7748Article 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.

Keyword
ZBED6, colorectal cancer, IGF2, PI3K pathway
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-258330 (URN)10.1073/pnas.1509193112 (DOI)000356731300067 ()
Funder
Swedish Research Council
Available from: 2015-07-15 Created: 2015-07-13 Last updated: 2017-09-12
4. The ZBED6-IGF2 axis has a major effect on growth of skeletal muscle and internal organs in placental mammals
Open this publication in new window or tab >>The ZBED6-IGF2 axis has a major effect on growth of skeletal muscle and internal organs in placental mammals
Show others...
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.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-329188 (URN)10.1073/pnas.1719278115 (DOI)000426152500018 ()
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, 70374401
Available from: 2017-09-10 Created: 2017-09-10 Last updated: 2018-04-26Bibliographically approved
5. Multiple viruses rely on the stress-induced protein ZC3H11A for efficient replication
Open this publication in new window or tab >>Multiple viruses rely on the stress-induced protein ZC3H11A for efficient replication
Show others...
2017 (English)Manuscript (preprint) (Other academic)
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-329189 (URN)
Available from: 2017-09-10 Created: 2017-09-10 Last updated: 2018-01-13

Open Access in DiVA

fulltext(1071 kB)122 downloads
File information
File name FULLTEXT01.pdfFile size 1071 kBChecksum SHA-512
196a491ad5283ecfff825fbb1500888d0dbec00937d07290baccc64413afb24f55ae8ed065134e36f546b2a6faaba8d6631907e78586102f903086b91ce2c367
Type fulltextMimetype application/pdf
Buy this publication >>

Search in DiVA

By author/editor
Younis, Shady
By organisation
Department of Medical Biochemistry and MicrobiologyScience for Life Laboratory, SciLifeLab
Medical GeneticsCell and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 122 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1024 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf