Epigenetic Regulation and Reprogramming of the H19 Imprinting Control Region

• Main Author: Mariano, Piero
• Format: Doctoral Thesis
• Language: English
• Published: Uppsala universitet, Zoologisk utvecklingsbiologi 2006
• Subjects: Developmental biology; Imprinting; chromatin; H19; Igf2; CTCF; Insulator; methylation; Boris; Utvecklingsbiologi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6299; http://nbn-resolving.de/urn:isbn:91-554-6446-7

The development of a new individual from the fertilized oocyte can ultimately be seen as the consequence of the establishment and maintenance of specific patterns of gene expression. Although regulation of gene activity occurs at different levels, cellular specialization and differentiation are the results of developmental cues that essentially take place at the transcriptional level. The involvement of epigenetics in this process has become increasingly clear during the last decade. Imprinted genes constitute an excellent example as monoallelic expression seems to reflect differential epigenetic marks on the two alleles. This is the case of the imprinted H19 and Igf2 genes were the monoallelic expression is coordinated through a differentially methylated region (hypermethylated on the paternal allele), known as ICR (imprinted control region). In the mouse the ICR harbours four binding sites for the methylation sensitive insulator protein CTCF. Previous studies with episomal constructs had shown that this region behaved as an insulator and that CTCF is required for the insulator activity of the H19 ICR This thesis establish a clear link between the insulator function and the chromatin structure at the H19 ICR and indicates that the precise allocation of the CTCF target sites in the linker regions can play a critical role in this process. The importance of the CTCF interaction at the ICR was also confirmed in vivo using a mouse model that showed how intact CTCF target sites are needed to manifest insulator activity and methylation protection. 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. This thesis also describe the development of a new technique for the localization of chromatin associated factors and modifications with higher sensitivity and resolution compared to existing approaches.