Transcriptional Silencing in the Imprinted Igf2-H19 Loci: The Mystique of Epigenetics

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 Igf2-H19 loci located distally on chromosome 7 in mouse and 11p15.5 in human, share common regulatory elements that regulate differential expression. Where the H19 is silenced when paternally inherited, the Igf2 is silenced when maternally inherited. The differentially methylated 5'-flank of H19 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. The H19 ICR exhibits default-silencing functions in promoter-proximal positions. The maximal distance between the H19 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 H19 ICR and H19 promoter. Results suggest that the H19 ICR adopts a chromatin conformation that must be separated by a minimal distance from pivotal cis-regulatory elements to avoid adverse effects on neighbouring promoters. Poly(ADP-ribosy)lation represents a novel post-translational epigenetic mark that segregates with exclusively the maternal derived H19 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 H19 ICR. 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 IGF2 target. Murine hybrid cell lines of human chromosome 11, demonstrated that the ZFP809 overcame the epigenetic marks that repressed maternal IGF2 and paternal H19 allele, respectively. Results suggested that imprinted genes are not normally exposed to strong cis-regulatory elements and that the designed ZFPs can be exploited to develop a therapeutic method for rectifying epigenetic lesions

Transcriptional Silencing in the Imprinted Igf2-H19 Loci: The Mystique of Epigenetics

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 Igf2-H19 loci located distally on chromosome 7 in mouse and 11p15.5 in human, share common regulatory elements that regulate differential expression. Where the H19 is silenced when paternally inherited, the Igf2 is silenced when maternally inherited. The differentially methylated 5'-flank of H19 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. The H19 ICR exhibits default-silencing functions in promoter-proximal positions. The maximal distance between the H19 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 H19 ICR and H19 promoter. Results suggest that the H19 ICR adopts a chromatin conformation that must be separated by a minimal distance from pivotal cis-regulatory elements to avoid adverse effects on neighbouring promoters. Poly(ADP-ribosy)lation represents a novel post-translational epigenetic mark that segregates with exclusively the maternal derived H19 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 H19 ICR. 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 IGF2 target. Murine hybrid cell lines of human chromosome 11, demonstrated that the ZFP809 overcame the epigenetic marks that repressed maternal IGF2 and paternal H19 allele, respectively. Results suggested that imprinted genes are not normally exposed to strong cis-regulatory elements and that the designed ZFPs can be exploited to develop a therapeutic method for rectifying epigenetic lesions

Protein-lysine methyltransferases G9a and GLP1 promote responses to DNA damage

Abstract Upon induction of DNA breaks, ATM activation leads to a cascade of local chromatin modifications that promote efficient recruitment of DNA repair proteins. Errors in this DNA repair pathway lead to genomic instability and cancer predisposition. Here, we show that the protein lysine methyltransferase G9a (also known as EHMT2) and GLP1 (also known as EHMT1) are critical components of the DNA repair pathway. G9a and GLP1 rapidly localizes to DNA breaks, with GLP1 localization being dependent on G9a. ATM phosphorylation of G9a on serine 569 is required for its recruitment to DNA breaks. G9a catalytic activity is required for the early recruitment of DNA repair factors including 53BP and BRCA1 to DNA breaks. Inhibition of G9a catalytic activity disrupts DNA repair pathways and increases sensitivity to ionizing radiation. Thus, G9a is a potential therapeutic target in the DNA repair pathway

Akt-mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer

Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis