Methylation patterns of the mammalian genome are thought to be crucial for development. The precise mechanisms designating specific genomic loci for methylation are not known. Targeted deletion of Lsh results in perinatal lethality with a rather normal development. We report here, however, that Lsh −/− mice show substantial loss of methylation throughout the genome. The hypomethylated loci comprise repetitive elements and single copy genes. This suggests that global genomic methylation is not absolutely required for normal embryogenesis. Based on the similarity of Lsh to other SNF2 chromatin remodeling proteins, it suggests that alteration of chromatin affects global methylation patterns in mice.

Summary Although the BBAP E3 ligase and its binding partner, BAL, are overexpressed in chemotherapy-resistant lymphomas, the role of these proteins in DNA damage responses remains undefined. Since BAL proteins modulate promoter-coupled transcription and contain structural motifs associated with chromatin remodeling and DNA repair, we reasoned that the BBAP E3 ligase might target nucleosomal proteins. Herein, we demonstrate that BBAP selectively monoubiquitylates histone H4 lysine 91 and protects cells exposed to DNA damaging agents. Disruption of BBAP-mediated monoubiquitylation of histone H4K91 is associated with the loss of chromatin-associated H4K20 methylase, mono- and dimethyl H4K20, and a delay in the kinetics of 53BP1 foci formation at sites of DNA damage. Since 53BP1 localizes to DNA damage sites, in part, via an interaction with dimethyl H4K20, these data directly implicate BBAP in the monoubiquitylation and additional post-translational modification of histone H4 and an associated DNA damage response.

The BAL1 macrodomain-containing protein and its partner E3 ligase, BBAP, are overexpressed in chemotherapy-resistant lymphomas. BBAP selectively ubiquitylates histone H4 and indirectly promotes early 53BP1 recruitment to DNA damage sites. However, neither BBAP nor BAL1 has been directly associated with a DNA damage response (DDR), and the function of BAL1 remains undefined. Herein, we describe a direct link between rapid and short-lived poly(ADP-ribose) (PAR) polymerase 1 (PARP1) activation and PARylation at DNA damage sites, PAR-dependent recruitment of the BAL1 macrodomain-containing protein and its partner E3 ligase, local BBAP-mediated ubiquitylation, and subsequent recruitment of the checkpoint mediators 53BP1 and BRCA1. The PARP1-dependent localization of BAL1-BBAP functionally limits both early and delayed DNA damage and enhances cellular viability independent of ATM, MDC1, and RNF8. These data establish that BAL1 and BBAP are bona fide members of a DNA damage response pathway and are directly associated with PARP1 activation, BRCA1 recruitment, and double-strand break repair.

The genome is burdened with repetitive sequences that are generally embedded in silenced chromatin. We have previously demonstrated that Lsh (lymphoid-specific helicase) is crucial for the control of heterochromatin at pericentromeric regions consisting of satellite repeats. In this study, we searched for additional genomic targets of Lsh by examining the effects of Lsh deletion on repeat regions and single copy gene sequences. We found that the absence of Lsh resulted in an increased association of acetylated histones with repeat sequences and transcriptional reactivation of their silenced state. In contrast, selected single copy genes displayed no change in histone acetylation levels, and their transcriptional rate was indistinguishable compared to Lsh-deficient cells and wild-type controls. Microarray analysis of total RNA derived from brain and liver tissues revealed that <0.4% of the 15 247 examined loci were abnormally expressed in Lsh-/-embryos and almost two-thirds of these deregulated sequences contained repeats, mainly retroviral LTR (long terminal repeat) elements. Chromatin immunoprecipitation analysis demonstrated a direct interaction of Lsh with repetitive sites in the genome. These data suggest that the repetitive sites are direct targets of Lsh action and that Lsh plays an important role as 'epigenetic guardian' of the genome to protect against deregulation of parasitic retroviral elements.

contributed equally to this work Methylation of histone tails and CpG methylation are involved in determining heterochromatin structure, but their cause and effect relationship has not been resolved as yet in mammals. Here we report that Lsh, a member of the SNF2 chromatin remodeling family, controls both types of epigenetic modi®cations. Lsh has been shown to be associated with pericentromeric heterochromatin and to be required for normal CpG methylation at pericentromeric sequences. Loss of Lsh, in Lsh-de®cient mice, results in accumulation of di-and tri-methylated histone 3 at lysine 4 (H3-K4me) at pericentromeric DNA and other repetitive sequences. In contrast, di-or tri-methylation of H3-K9 and distribution of HP1 appear unchanged after Lsh deletion, suggesting independent regulatory mechanisms for H3-K4 or K9 methylation. Experimental DNA demethylation with 5¢-azacytidine results in a similar increase of H3-K4me. These results support the model that loss of CpG methylation caused by Lsh de®ciency antecedes elevation of H3-K4me. Thus, Lsh is crucial for the formation of normal heterochromatin, implying a functional role for Lsh in the regulation of transcription and mitosis.

The eukaryotic genome is packaged into distinct domains of transcriptionally active euchromatin and silent heterochromatin. A hallmark of mammalian heterochromatin is CpG methylation. Lsh, a member of the SNF2 family, is a major regulator of DNA methylation in mice and thus crucial for normal heterochromatin formation. In order to define the molecular function of Lsh, we examined its cellular localization and its association with chromatin. Our studies demonstrate that Lsh is an exclusively nuclear protein, and we define a nuclear localization domain within the N-terminal portion of Lsh. Lsh strongly associates with chromatin and requires the internal and C-terminal regions for this interaction. Lsh accumulates at pericentromeric heterochromatin, suggesting a direct role for Lsh in the methylation of centromeric DNA sequences and the formation of heterochromatin. In search of a signal that is responsible for Lsh recruitment to pericentromeric heterochromatin, we found that histone tail modifications were critical. Prolonged treatment with histone deacetylase inhibitors has been reported to disrupt higher-order heterochromatin organization, and this was accompanied by dissociation of Lsh from pericentromeric heterochromatin. These results are consistent with a model in which Lsh is recruited by intact heterochromatin structure and then assists in maintaining heterochromatin organization by establishing CpG methylation patterns.The chromatin of eukaryotic cells is organized into two major types: euchromatin and heterochromatin (6, 39). Euchromatin contains the majority of single-copy genes, replicates during early S phase, and contains acetylated histones. Heterochromatin is composed of long stretches of DNA repeats, usually replicates in late S phase, and contains underacetylated histones. A specific methylation mark in heterochromatin in histone 3 at lysine 9 serves as a recognition signal for binding of the heterochromatin protein 1 (HP1) (26). Heterochromatin regulates many diverse nuclear functions, including gene silencing, normal centromere function and nuclear organization.Another hallmark of mammalian heterochromatin is DNA methylation (5-7, 9, 34, 39). The addition of methyl groups to cytosines within the CpG dinucleotide by DNA methyltransferases is involved in regulating transcription, maintaining genome stability, imprinting, and X chromosome inactivation. In mammalian cells, DNA methylation is catalyzed by two important classes of DNA methyltransferases (5). DNA methyltransferase 1 (Dnmt1) resides at the replication fork and methylates CpG dinucleotides in the newly synthesized daughter strand. The function of Dnmt1 is thought to be essential for maintaining DNA methylation patterns in proliferating cells (30). There are two members of the second class of methyltransferases, Dnmt3a and Dnmt3b, which are required for the initiation of de novo methylation during embryonic development (37).Although DNA methylation and heterochromatin are closely associated, their precise relationship has not been elucid...

The BAL1 macro domain-containing protein and its partner E3 ligase, BBAP, are overexpressed in chemotherapy-resistant lymphomas. BBAP selectively ubiquitylates histone H4 and indirectly promotes early 53BP1 recruitment to DNA damage sites. However, neither BBAP nor BAL1 have been directly associated with a DNA damage response (DDR) and the function of BAL1 remains undefined. DDR proteins assemble in a coordinated, sequential manner at sites of DNA breaks. The initial recruitment phase is rapid, transient and dependent upon PARylation at DNA damage sites. A second phase, which also begins within seconds but lasts for hours, includes the sequential phosphorylation of ATM, MDC1 and the RNF8-dependent ubiquitylation of multiple double-strand break (DSB) repair factors. We have now defined a direct link between the rapid and short-lived PARP1 activation and PARylation at DNA damage sites, PAR-dependent recruitment of the BAL1 macro domain-containing protein and its partner BBAP E3 ligase, local BBAP-mediated ubiquitylation and subsequent recruitment of the checkpoint mediators, 53BP1 and BRCA1. The PARP1-dependent localization of BAL1/BBAP functionally limits both early and delayed DNA damage and enhances cellular viability independent of ATM, MDC1 and RNF8. These data establish BAL1 and BBAP as bona fide DDR pathway members and directly associate PARP1 activation, BRCA1 recruitment and DSB repair. Citation Format: Qingsheng Yan, Rong Xu, Liya Zhu, Xin Cheng, Zhe Wang, John Manis, Margaraet Shipp. BAL1 and its partner E3 Ligase, BBAP, Link PARP activation, Ubiquitylation and double-strand DNA repair independent of ATM, MDC1 and RNF8. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 633. doi:10.1158/1538-7445.AM2013-633

No abstract