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The Binding Sites for the Chromatin Insulator Protein CTCF Map to DNA Methylation-Free Domains Genome-Wide
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology.
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2004 (English)In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 14, no 8, 1594-1602 p.Article in journal (Refereed) Published
Abstract [en]

All known vertebrate chromatin insulators interact with the highly conserved, multivalent 11-zinc finger nuclear factor CTCF to demarcate expression domains by blocking enhancer or silencer signals in a position-dependent manner. Recent observations document that the properties of CTCF include reading and propagating the epigenetic state of the differentially methylated H19 imprinting control region. To assess whether these findings may reflect a universal role for CTCF targets, we identified more than 200 new CTCF target sites by generating DNA microarrays of clones derived from chromatin-immunopurified (ChIP) DNA followed by ChIP-on-chip hybridization analysis. Target sites include not only known loci involved in multiple cellular functions, such as metabolism, neurogenesis, growth, apoptosis, and signalling, but potentially also heterochromatic sequences. Using a novel insulator trapping assay, we also show that the majority of these targets manifest insulator functions with a continuous distribution of stringency. As these targets are generally DNA methylation-free as determined by antibodies against 5-methylcytidine and a methyl-binding protein (MBD2), a CTCF-based network correlates with genome-wide epigenetic states.

Place, publisher, year, edition, pages
2004. Vol. 14, no 8, 1594-1602 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-91998DOI: 10.1101/gr.2408304PubMedID: 15256511OAI: oai:DiVA.org:uu-91998DiVA: diva2:164932
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Genomic Imprinting in Development and Evolution
Open this publication in new window or tab >>Genomic Imprinting in Development and Evolution
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Genetic information is encoded by the linear sequence of the DNA double helix, while epigenetic information is overlayed as the packaging of DNA and associated proteins into the chromatin structure. Variations in chromatin structure play a vital role in establishing and maintaining patterns of gene expression during differentiation and development of higher eukaryotes, and disruption of this epigenetic gene regulation can lead to cancer. Mammals display an epigenetic phenomenon known as genomic imprinting, which provides an ideal model system for the study of epigenetics. Genes subject to genomic imprinting are differentially expressed within a single cell depending on the parental origin of the chromosome. Imprinting of the maternally expressed H19 gene and the adjacent paternally expressed Igf2 gene is regulated by the chromatin insulator protein CTCF. The studies presented in this thesis aim to investigate the functional mechanisms of CTCF-dependent gene regulation at the H19/Igf2 locus and at numerous other target sites throughout the genome. We have investigated the role of CTCF and a related protein BORIS in establishing the maternal to paternal imprint transition in chromatin structure at the H19/Igf2 locus in the male germline. We have developed novel microarray based methods to identify and characterize numerous new CTCF target sites throughout the mouse genome. We have shown that CTCF acts as part of the RNA polymerase II complex. We have identified the post-translational modification by addition of ADP-ribose polymers to CTCF, and demonstrated that this modification regulates its insulating ability. The results of these studies of CTCF-dependent epigenetic gene regulation are discussed in light of the evolution of genomic imprinting and chromatin insulators, and a novel role for poly ADP-ribosylation of CTCF in the progression of cancer is proposed.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 74 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 1000
Keyword
Developmental biology, Utvecklingsbiologi
National Category
Developmental Biology
Identifiers
urn:nbn:se:uu:diva-4491 (URN)91-554-6015-1 (ISBN)
Public defence
2004-09-22, Lindahlsalen, Evolutionary Biology Centre, Norbyvägen 18A, Uppsala, 10:00 (English)
Opponent
Supervisors
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2013-05-16Bibliographically approved
2. The Control of the Epigenome
Open this publication in new window or tab >>The Control of the Epigenome
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The genetic information required for the existence of a living cell of any kind is encoded in the sequence information scripted in the double helix DNA. A modern trend in biology struggles to come to grip with the amazing fact that there are so many different cell types in our body and that they are directed from the same genomic blueprint. It is clear, that the key to this feature is provided by epigenetic information that dictates how, where and when genes should be expressed. Epigenetic states “dress up” the genome by packaging it in chromatin conformations that differentially regulate accessibility for key nuclear factors and in coordination with differential localizations within the nucleus will dictate the ultimate task, expression.

In the imprinted Igf2/H19 domain, this feature is determined by the interaction between the chromatin insulator protein CTCF and the unmethylated H19 imprinting control region. Here I show that CTCF interacts with many sites genome-wide and that these sites are generally protected from DNA methylation, suggesting that CTCF function has been recruited to manifest novel imprinted states during mammalian development.

This thesis also describes the discovery of an epigenetically regulated network of intra and interchromosomal complexes, identified by the invented 4C method. Importantly, the disruption of CTCF binding sites at the H19 imprinting control region not only disconnects this network, but also leads to significant changes in expression patterns in the interacting partners.

Interestingly, CTCF plays an important role in the regulation of the replication timing not only of the Igf2 gene, but also of all other sequences binding this factor potentially by a cell cycle-specific relocation of CTCF-DNA complexes to subnuclear compartments.

Finally, I show that epigenetic marks signifying active or inactive states can be gained and lost, respectively, upon exposure to stress. As many genes belonging to the apoptotic pathway are upregulated we propose that stress-induced epigenetic lesions represent a surveillance system marking the affected cells for death to the benefit of the individual. This important observation opens our minds to the view of new intrinsic mechanisms that the cell has in order to maintain proper gene expression, and in the case of misleads there are several check points that direct the cell to towards important survival decisions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 230
Keyword
Biology, Epigenetics, chromosome interactions, replication timing, histone modifications, epigenetic surveillance, Biologi
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-7190 (URN)91-554-6680-X (ISBN)
Public defence
2006-10-31, Lindahlsalen, EBC, Nörbyvagen 18a, uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2006-10-10 Created: 2006-10-10 Last updated: 2011-05-17Bibliographically approved
3. Chromatin Insulators and CTCF: Architects of Epigenetic States during Development.
Open this publication in new window or tab >>Chromatin Insulators and CTCF: Architects of Epigenetic States during Development.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A controlled and efficient coordination of gene expression is the key for normal development of an organism. In mammals, a subset of autosomal genes is expressed monoallelically depending on the sex of the transmitting parent, a phenomenon known as genomic imprinting.

The imprinted state of the H19 and Igf2 genes is controlled by a short stretch of sequences upstream of H19 known as the imprinting control region (ICR). This region is differentially methylated and is responsible for the repression of the maternally inherited Igf2 allele. It harbors hypersensitive sites on the unmethylated maternal allele and functions as an insulator that binds a chromatin insulator protein CTCF. Hence the H19 ICR, which plays an important role in maintaining the imprinting status of H19 and Igf2, was shown to lose the insulator property upon CpG methylation.

Another ICR in the Kcnq1 locus regulates long-range repression of p57Kip2 and Kcnq1 on the paternal allele, and is located on the neighboring subdomain of the imprinted gene cluster containing H19 and Igf2, on the distal end of mouse chromosome 7. Similarly to the H19 ICR, the Kcnq1 ICR appears to possess a unidirectional and methylation-sensitive chromatin insulator property in two different somatic cell types. Hence, methylation dependent insulator activity emerges as a common feature of imprinting control regions.

The protein CTCF is required for the interpretation and propagation of the differentially methylated status of the H19 ICR. Work in this thesis shows that this feature applies genomewide. The mapping of CTCF target sites demonstrated not only a strong link between CTCF, formation of insulator complexes and maintaining methylation-free domains, but also a network of target sites that are involved in pivotal functions. The pattern of CTCF in vivo occupancy varies in a lineage-specific manner, although a small group of target sites show constitutive binding.

In conclusion, the work of this thesis shows that epigenetic marks play an important role in regulating the insulator property. The studies also confirm the importance of CTCF in maintaining methylation-free domains and its role in insulator function. Our study unravels a new range of target sites for CTCF involved in divergent functions and their developmental control.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 39 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 972
Keyword
Developmental biology, DNA methylation, CTCF, Chromatin insulators, Microarray, Utvecklingsbiologi
National Category
Developmental Biology
Identifiers
urn:nbn:se:uu:diva-4241 (URN)91-554-5952-8 (ISBN)
Public defence
2004-05-18, Lindahlsalen, EBC, Norbyvägen 18A, Uppsala, Sweden, Uppsala, 13:30
Opponent
Supervisors
Available from: 2004-04-20 Created: 2004-04-20 Last updated: 2014-01-22Bibliographically approved

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