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Epigenetic Regulation of Replication Timing and Signal Transduction
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Cell Biology.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Upon fertilization the paternal and maternal genomes unite, giving rise to the embryo, with its unique genetic code. All cells in the human body are derived from the fertilized ovum: hence they all contain (with a few exceptions) the same genetic composition. However, by selective processes, genes are turned on and off in an adaptable, and cell type-specific, manner. The aim of this thesis is to investigate how signals coming from outside the cell and epigenetic factors residing in the cell nucleus, cooperate to control gene expression.

The transforming growth factor-β (TGF-β) superfamily consists of around 30 cytokines, which are essential for accurate gene regulation during embryonic development and adult life. Among these are the ligands TGF-β1 and bone morphogenetic (BMP) -7, which interact with diverse plasma membrane receptors, but signal via partly the same Smad proteins. Smad4 is essential to achieve TGF-β-dependent responses. We observed that by regulating transcription factors such as Id2 and Id3 in a specific manner, TGF-β1 and BMP-7 achieve distinct physiological responses.

Moreover, we demonstrate that CTCF, an insulator protein regulating higher order chromatin conformation, is able to direct transcription by recruiting RNA polymerase II to its target sites. This is the first mechanistic explanation of how an insulator protein can direct transcription, and reveals a link between epigenetic modifications and classical regulators of transcription. We also detected that DNA loci occupied by CTCF replicate late. The timing of replication is a crucial determinant of gene activity. Genes replicating early tend to be active, whereas genes replicating late often are silenced. Thus, CTCF can regulate transcription at several levels. Finally, we detected a substantial cross-talk between CTCF and TGF-β signaling. This is the first time that a direct interplay between a signal transduction pathway and the chromatin insulator CTCF is demonstrated.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2008. , p. 72
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 302
Keywords [en]
Cell and molecular biology, Chromatin, CTCF, Epigenetics, Genomic Imprinting, H19, Id, Igf2, Insulator, Replication, Signal Transduction, Smad, TGF-β, Transcription
Keywords [sv]
Cell- och molekylärbiologi
Identifiers
URN: urn:nbn:se:uu:diva-8413ISBN: 978-91-554-7069-2 (print)OAI: oai:DiVA.org:uu-8413DiVA, id: diva2:171299
Public defence
2008-02-15, B41, Uppsala Biomedicinska Centrum, BMC, Husarg. 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2008-01-25 Created: 2008-01-25 Last updated: 2009-03-26Bibliographically approved
List of papers
1. Id2 and Id3 Define the Potency of Cell Proliferation and Differentiation Responses to Transforming Growth Factor β and Bone Morphogeenetic Protein
Open this publication in new window or tab >>Id2 and Id3 Define the Potency of Cell Proliferation and Differentiation Responses to Transforming Growth Factor β and Bone Morphogeenetic Protein
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2004 (English)In: Molecular Cell Biology, Vol. 24, no 10, p. 4241-54Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-96649 (URN)
Available from: 2008-01-25 Created: 2008-01-25 Last updated: 2009-03-26Bibliographically approved
2. CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide
Open this publication in new window or tab >>CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide
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2007 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 27, no 5, p. 1631-1648Article in journal (Refereed) Published
Abstract [en]

CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. "Serial" chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-96650 (URN)10.1128/MCB.01993-06 (DOI)000244305500008 ()17210645 (PubMedID)
Available from: 2008-01-25 Created: 2008-01-25 Last updated: 2017-12-14Bibliographically approved
3. CTCF Regulates Asynchronous Replication of the Imprinted H19/Igf2 Domain
Open this publication in new window or tab >>CTCF Regulates Asynchronous Replication of the Imprinted H19/Igf2 Domain
2007 (English)In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 6, no 4, p. 450-454Article in journal (Refereed) Published
Abstract [en]

Asynchronous replication during S phase is a universal characteristic of genomically imprinted genes. Replication timing in imprinted domains is determined epigenetically, as it is parent of origin specific, and is seen in the absence of sequence divergence between the two alleles. At the imprinted H19/lgf2 domain, the methylated paternal allele replicates early while the CTCF-bound maternal allele replicates late during S phase. CTCF regulates the allele-specific epigenetic characteristics of this domain, including methylation, transcription and chromosome conformation. Here we show that maternal, but not paternal inheritance of a mutated H19 imprinting control region, lacking functional CTCF binding sites, underlies a late to early switch in replication timing of the maternal H19/ lgf2 domain.

Keywords
replication timing, CTCF, H19/Igf2, genomic imprinting
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-96651 (URN)000245495600013 ()17329968 (PubMedID)
Available from: 2008-01-25 Created: 2008-01-25 Last updated: 2017-12-14Bibliographically approved
4. CTCF and Smad Proteins of the TGF-β Pathway interact during regulation of gene expression from the H19 imprinted control region
Open this publication in new window or tab >>CTCF and Smad Proteins of the TGF-β Pathway interact during regulation of gene expression from the H19 imprinted control region
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(English)Manuscript (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:uu:diva-96652 (URN)
Available from: 2008-01-25 Created: 2008-01-25 Last updated: 2010-01-14Bibliographically approved

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