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CpG methylation regulates the Igf2/H19 insulator
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Animal Development and Genetics.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Animal Development and Genetics.
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2001 (English)In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 11, no 14, 1128-1130 p.Article in journal (Refereed) Published
Abstract [en]

The differentially methylated 5'-flank of the mouse H19 gene unidirectionally regulates the communication between enhancer elements and gene promoters and presumably represses maternal Igf2 expression in vivo [1-6]. The specific activation of the paternally inherited Igf2 allele has been proposed to involve methylation-mediated inactivation of the H19 insulator function during male germline development [1-4, 6]. Here, we addressed the role of methylation by inserting a methylated fragment of the H19-imprinting control region (ICR) into a nonmethylated episomal H19 minigene construct, followed by the transfection of ligation mixture into Hep3B cells. Individual clones were expanded and analyzed for genotype, methylation status, chromatin conformation, and insulator function. The results show that the methylated status of the H19 ICR could be propagated for several passages without spreading into the episomal vector. Moreover, the nuclease hypersensitive sites, which are typical for the maternally inherited H19 ICR allele [1], were absent on the methylated ICR, underscoring the suggestion that the methylation mark dictates parent of origin-specific chromatin conformations [1] that involve CTCF [2]. Finally, the insulator function was strongly attenuated in stably maintained episomes. Collectively, these results provide the first experimental support that the H19 insulator function is regulated by CpG methylation.

Place, publisher, year, edition, pages
2001. Vol. 11, no 14, 1128-1130 p.
Keyword [en]
Alleles, Animals, Cell Line, CpG Islands, DNA Methylation, Female, Genomic Imprinting, Male, Mice, Plasmids/genetics, RNA; Untranslated/*genetics
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-90356DOI: 10.1016/S0960-9822(01)00314-1PubMedID: 11509237OAI: oai:DiVA.org:uu-90356DiVA: diva2:162681
Available from: 2003-04-24 Created: 2003-04-24 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Epigenetic Regulation of the H19 Chromatin Insulator in Development and Disease
Open this publication in new window or tab >>Epigenetic Regulation of the H19 Chromatin Insulator in Development and Disease
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The coordinated regulation of gene expression must be tightly controlled for normal development to occur. In mammals, this issue is further complicated by the requirement of both the maternal and paternal genomes for normal development, reflecting the fact that a subset of genes are monoallelically expressed depending on parental inheritance, a phenomenon known as genomic imprinting.

The imprinted H19 and Igf2 genes are often considered as paradigms of genomic imprinting, since their monoallelic expression patterns are coordinated via a short stretch of sequence upstream of H19, known as the imprinting control region (ICR). This region is differentially methylated, with specific CpG methylation on the paternal allele. It is shown here that the ICR harbours several maternal-specific hypersensitive sites, located in linker regions between positioned nucleosomes. Furthermore, this region functions as an orientation-dependent insulator, that binds the chromatin insulator factor CTCF. The hypothesis that the methylation status of the ICR dictates the activity of the Igf2 gene 90 kb further upstream was confirmed by the demonstration that the insulator function is lost when the ICR is CpG methylated.

The ICR has previously been shown to act as a silencer when positioned in a promotor-proximal position. The cause of this silencing was shown to be distance-dependent, suggesting that the silencing features of the ICR depend on a chromatin conformation that renders adjacent sequences inaccessible to the RNA polymerase. These data issue a cautionary note with respect to the interpretation of silencer functions.

In several forms of cancer, the normally silent maternal IGF2 gene is expressed, possibly as a result of loss of insulator function at the ICR. The utilisation of CTCF target-sites was analysed in different tumours, and was shown to be highly variable. Methylation analysis showed that potential loss of insulator function and gain of methylation at the maternal ICR did not always correlate with biallelic expression of IGF2. Further investigations uncovered a novel mechanism, in which the activation of the IGF2 promoter was independent of insulator function in some cancers.

This thesis shows that the regulation of the imprinted state of Igf2 depends on the formation of an epigenetically regulated chromatin insulator, and that the loss of IGF2 imprinting in human cancer can be attributed to several mechanisms, including a novel mechanism that neutralises chromatin insulator function.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 57 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 825
Keyword
Developmental biology, Utvecklingsbiologi
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
urn:nbn:se:uu:diva-3405 (URN)91-554-5589-1 (ISBN)
Public defence
2003-05-16, Lindalsalen, EBC, Uppsala, 14:00
Opponent
Supervisors
Available from: 2003-04-24 Created: 2003-04-24 Last updated: 2011-11-10Bibliographically approved
2. 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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