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Epigenetic Regulation of the H19 Chromatin Insulator in Development and Disease
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Animal Development and Genetics.
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. , p. 57
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 825
Keywords [en]
Developmental biology
Keywords [sv]
Utvecklingsbiologi
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
URN: urn:nbn:se:uu:diva-3405ISBN: 91-554-5589-1 (print)OAI: oai:DiVA.org:uu-3405DiVA, id: diva2:162684
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
List of papers
1. The 5' flank of mouse H19 in an unusual chromatin conformation unidirectionally blocks enhancer-promoter communication
Open this publication in new window or tab >>The 5' flank of mouse H19 in an unusual chromatin conformation unidirectionally blocks enhancer-promoter communication
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2000 (English)In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 10, no 8, p. 449-457Article in journal (Refereed) Published
Keywords
Alleles, Animals, Blotting; Southern, Cell Line, Chromatin/*chemistry, Enhancer Elements (Genetics), Female, Fetus, Humans, Insulin-Like Growth Factor II/*genetics, Male, Mice, Muscle Proteins/*genetics/metabolism, Plasmids, Polymerase Chain Reaction, Promoter Regions (Genetics), Protein Conformation, RNA; Untranslated, Terminal Repeat Sequences, Transformation; Genetic
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-24765 (URN)10.1016/S0960-9822(00)00442-5 (DOI)10801414 (PubMedID)
Available from: 2007-02-08 Created: 2007-02-08 Last updated: 2017-12-07Bibliographically approved
2. CpG methylation regulates the Igf2/H19 insulator
Open this publication in new window or tab >>CpG methylation regulates the Igf2/H19 insulator
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2001 (English)In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 11, no 14, p. 1128-1130Article 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.

Keywords
Alleles, Animals, Cell Line, CpG Islands, DNA Methylation, Female, Genomic Imprinting, Male, Mice, Plasmids/genetics, RNA; Untranslated/*genetics
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-90356 (URN)10.1016/S0960-9822(01)00314-1 (DOI)11509237 (PubMedID)
Available from: 2003-04-24 Created: 2003-04-24 Last updated: 2017-12-14Bibliographically approved
3. Multiple cis elements within the Igf2/H19 insulator domain organize a distance-dependent silencer: A cautionary note
Open this publication in new window or tab >>Multiple cis elements within the Igf2/H19 insulator domain organize a distance-dependent silencer: A cautionary note
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2002 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 8, p. 5707-5710Article in journal (Refereed) Published
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-90357 (URN)10.1074/jbc.C100552200 (DOI)11777900 (PubMedID)
Available from: 2003-04-24 Created: 2003-04-24 Last updated: 2017-12-14Bibliographically approved
4. The direct bypass of the chromatin insulator function of the H19 imprinting control region in human cancer cells: A novel mechanism of loss of IGF2 imprinting.
Open this publication in new window or tab >>The direct bypass of the chromatin insulator function of the H19 imprinting control region in human cancer cells: A novel mechanism of loss of IGF2 imprinting.
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Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-90358 (URN)
Available from: 2003-04-24 Created: 2003-04-24 Last updated: 2010-01-13Bibliographically approved

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