<p>Genomic imprinting marks a subset of autosomal loci expressed in parent of origin-dependent monoallelic expression in a non-Mendelian fashion. To restore totipotency and to reset the imprint according to the sex of the individual, the mark must be erased during germline development. The imprinted <i>Igf2-H19</i> loci located distally on chromosome 7 in mouse and 11p15.5 in human, share common regulatory elements that regulate differential expression. Where the <i>H19 </i>is silenced when paternally inherited, the <i>Igf2</i> is silenced when maternally inherited. </p><p>The differentially methylated 5'-flank of <i>H19</i> gene, termed imprinting control region (ICR), shown to display a unique chromatin organisation harbours hypersensitive sites in linker regions flanked by positioned nucleosomes on the maternal allele. This unique chromatin conformation functions as a methylation-sensitive and unidirectional chromatin insulator, which later was found to depend on the chromatin insulator protein CTCF. </p><p>The <i>H19</i> ICR exhibits default-silencing functions in promoter-proximal positions. The maximal distance between the <i>H19</i> ICR and the promoter of the reporter gene required for this effect was 1.2 ± 0.3kb which can be compared to the 1.9 kb distance between the endogenous <i>H19 </i>ICR and <i>H19</i> promoter. Results suggest that the <i>H19</i> ICR adopts a chromatin conformation that must be separated by a minimal distance from pivotal <i>cis</i>-regulatory elements to avoid adverse effects on neighbouring promoters. </p><p>Poly(ADP-ribosy)lation represents a novel post-translational epigenetic mark that segregates with exclusively the maternal derived <i>H19</i> ICR and associated with factors that interact with the CTCF target sites. CTCF is itself poly(ADP-ribosy)lated and the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide relieves the insulator function of the <i>H19</i> ICR. </p><p>Designed zinc finger proteins were applied to examine if epigenetic marks provided an obstacle for targeted activation and silencing. The zinc finger protein ZFP809 with activator/repressor domain able to efficiently activate/silence the <i>IGF2</i> target. Murine hybrid cell lines of human chromosome 11, demonstrated that the ZFP809 overcame the epigenetic marks that repressed maternal <i>IGF2</i> and paternal <i>H19</i> allele, respectively. Results suggested that imprinted genes are not normally exposed to strong <i>cis</i>-regulatory elements and that the designed ZFPs can be exploited to develop a therapeutic method for rectifying epigenetic lesions.</p>
| Identifer | oai:union.ndltd.org:UPSALLA/oai:DiVA.org:uu-2556 |
| Date | January 2002 |
| Creators | Ginjala, Vasudeva |
| Publisher | Uppsala University, Department of Animal Development and Genetics, Uppsala : Acta Universitatis Upsaliensis |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, text |
| Relation | Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1104-232X ; 736 |
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