DNA damage activates signaling pathways that lead to modification of local chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins having ubiquitin ligase activity are recruited to sites of DNA damage, where they ubiquitinate histones and other substrates. This DNA damage-induced histone ubiquitination is thought to play a critical role in mediating the DNA damage response. We now report that the polycomb protein BMI1 is rapidly recruited to sites of DNA damage, where it persists for more than 8 h. The sustained localization of BMI1 to damage sites is dependent on intact ATM and ATR and requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads to impaired repair of DNA double-strand breaks by homologous recombination and the accumulation of cells in G 2 /M. These data support a crucial role for BMI1 in the cellular response to DNA damage.The induction of a DNA break leads to activation of multiple signaling pathways that lead to local modification of chromatin structure and recruitment of DNA repair complexes (18,22,55). Histone H2AX is rapidly phosphorylated near sites of DNA breaks by ATM, ATR, and DNA-PK (39, 54) and can spread to encompass a region of chromatin covering several megabases (40, 41).H2AX phosphorylation facilitates the recruitment of other proteins, including MDC1 (52) and the E3 ubiquitin ligases RNF8 and RNF168, which in turn participate locally in the K63-linked polyubiquitination of histones H2A and H2AX (23,32,50,51). Polyubiquitinated K63-linked histones provide a recognition element that recruits RAP80 through its ubiquitin interaction motifs (28,49,56). RAP80 can then promote the recruitment of other DNA repair factors such as BRCA1 and Abraxas, which are essential for efficient repair. RNF8 and RNF168 function are also required for proper localization of 53BP1, although the exact mechanism is unclear (12,23,32,51). 53BP1 recruitment to regions of DNA damage is dependent upon its Tudor domains, which have been found to specifically interact with methylated histone residues (6, 24, 42). A model has been proposed in which RNF8-and RNF168-mediated ubiquitination of histones confers local changes in chromatin structure, leading to exposure of methylated lysine residues in core histones, allowing the subsequent recruitment of 53BP1 (50). Enzymes involved in deubiquitination, such as BRCC36, USP3, and USP28, are also critical for efficient DNA repair, demonstrating that a dynamic regulation of ubiquitin conjugation and hydrolysis is necessary for optimal DNA repair (37,46,47,61).Polycomb group proteins BMI1 and RING1B/RNF2 form an active heterodimer E3 ligase that catalyzes the monoubiquitination of histone H2A at Lysine 119. (7,8,44,53,57). This activity is important for BMI1-mediated transcriptional silencing during organism development and cellular differentiation (27,48,58). Ubiquitination of H2A at lysine 119 is also induced locally at sites of DNA damage, both at s...

Chromatin insulators demarcate expression domains by blocking the cis effects of enhancers or silencers in a positiondependent manner 1,2 . We show that the chromatin insulator protein CTCF carries a post-translational modification: poly(ADP-ribosyl)ation. Chromatin immunoprecipitation analysis showed that a poly(ADP-ribosyl)ation mark, which exclusively segregates with the maternal allele of the insulator domain in the H19 imprinting control region, requires the bases that are essential for interaction with CTCF 3 . Chromatin immunoprecipitation-on-chip analysis documented that the link between CTCF and poly(ADP-ribosyl)ation extended to more than 140 mouse CTCF target sites. An insulator trap assay showed that the insulator function of most of these CTCF target sites is sensitive to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase activity. We suggest that poly(ADP-ribosyl)ation imparts chromatin insulator properties to CTCF at both imprinted and nonimprinted loci, which has implications for the regulation of expression domains and their demise in pathological lesions.Poly(ADP-ribosyl)ation is traditionally associated with DNA repair and apoptosis 4 , but this view may be too limited 5,6 . For example, one of the poly(ADP-ribose) (PAR) polymerases, PARP-1, is associated both with formation of heterochromatin and with regions of high transcriptional activity in fruit flies 7 . To explore a potential correlation between poly(ADP-ribosyl)ation and expression domains in the mouse, we analyzed the allelic distribution of poly(ADP-ribosyl)ated protein complexes on the chromatin insulator at the H19 imprinting control region (ICR), which partitions expression domains in a parent of origin-specific manner 8 . We analyzed chromatin-immunoprecipitated DNA of fetal liver of M. musculus domesticus  M. musculus musculus intraspecific hybrid crosses by a PCR assay, which exploited a polymorphic BsmAI restriction site at the second CTCF target site 9 .Only the maternally inherited allele was specifically captured using a specific antibody that detects polymers containing ten or more ADP-ribose units (Fig. 1a).As the chromatin insulator protein CTCF is the only factor known to interact preferentially with the maternal H19 ICR allele in vivo 3 , we examined the interaction between poly(ADP-ribosyl)ated proteins and the H19 ICR with point-mutated CTCF target sites 10 . We carried out chromatin immunoprecipitation (ChIP) analysis of primary mouse fibroblast cultures, with the mutation inherited maternally or paternally, followed by PCR of the H19 ICR. The H19 ICR was associated with a poly(ADP-ribosyl)ation mark only if the wild-type allele was inherited maternally (Fig. 1b). Although this result suggested that the poly(ADP-ribosyl)ation mark of the maternal H19 ICR allele requires functional CTCF target sites, we could not rule out indirect effects from de novo methylation 3 . We therefore mixed equimolar amounts of plasmids containing the wild-type H19 ICR and plasmids containing the H19 ICR with mutations of CTC...

All known vertebrate chromatin insulators interact with the highly conserved, multivalent 11-zinc finger nuclear factor CTCF to demarcate expression domains by blocking enhancer or silencer signals in a position-dependent manner. Recent observations document that the properties of CTCF include reading and propagating the epigenetic state of the differentially methylated H19 imprinting control region. To assess whether these findings may reflect a universal role for CTCF targets, we identified more than 200 new CTCF target sites by generating DNA microarrays of clones derived from chromatin-immunopurified (ChIP) DNA followed by ChIP-on-chip hybridization analysis. Target sites include not only known loci involved in multiple cellular functions, such as metabolism, neurogenesis, growth, apoptosis, and signalling, but potentially also heterochromatic sequences. Using a novel insulator trapping assay, we also show that the majority of these targets manifest insulator functions with a continuous distribution of stringency. As these targets are generally DNA methylation-free as determined by antibodies against 5-methylcytidine and a methyl-binding protein (MBD2), a CTCF-based network correlates with genome-wide epigenetic states.

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.

The unmethylated 5' flank of the H19 gene adopts an unusual and maternal-specific chromatin conformation in somatic cells and regulates enhancer-promoter communications, thereby providing an explanation for its role in manifesting the repressed state of the maternally inherited Igf2 allele.