Chromatin functions
the DNA repair connection
The aim of this master thesis was to examine a possible connection between chromatin functions and the DNA damage repair system, with the imprinted IGF2/H19 locus as a bait. Interactions can occur within and between chromosomes and it has been shown that H19 ICR on both alleles can communicate with alleles on other chromosomes. The methylation-sensitive insulator protein CTCF is bound on the maternal H19 ICR and acts as an insulator by blocking the IGF2 promoter to access its enhancers. This is possible by formation of loop structures by CTCF that enables intra and inter-chromosomal interactions. CTCF is poly(ADP-ribosyl)ated by PARP-1 when it is bound to H19 ICR. It has been shown that poly(ADP-ribosyl)ation by PARP-1 are important in DNA repair, chromatin remodelling and epigenetic regulation of chromatin structures. ATM physically and functionally interacts with poly(ADP-ribosyl)ation and phosphorylation of H2AX to ?H2AX that is important events in DNA repair. Chromatin immunoprecipitation and DNA fluorescent in situ hybridization can help answering the question if the components involved in DNA repair are present at H19 ICR when CTCF is bound and poly(ADP-ribosyl)ated. The conclusion obtained from the results is that PAR, PARP-1, ATM and ?H2AX are present at the H19 ICR only on the maternal allele. The results reliability is low. There was a big variation between replicates in ChIP and there was no time for a 3D analysis of DNA FISH results.