Histone lysine methylation has been linked to the recruitment of mammalian DNA repair factor 53BP1 and putative fission yeast homolog Crb2 to DNA double-strand breaks (DSBs), but how histone recognition is achieved has not been established. Here we demonstrate that this link occurs through direct binding of 53BP1 and Crb2 to histone H4. Using X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, we show that, despite low amino acid sequence conservation, both 53BP1 and Crb2 contain tandem tudor domains that interact with histone H4 specifically dimethylated at Lys20 (H4-K20me2). The structure of 53BP1/H4-K20me2 complex uncovers a unique five-residue 53BP1 binding cage, remarkably conserved in the structure of Crb2, that best accommodates a dimethyllysine but excludes a trimethyllysine, thus explaining the methylation state-specific recognition of H4-K20. This study reveals an evolutionarily conserved molecular mechanism of targeting DNA repair proteins to DSBs by direct recognition of H4-K20me2.

The pathogenic sequelae of BRCA1 mutation in human and mouse cells are mitigated by concomitant deletion of 53BP1, which binds histone H4 dimethylated at Lys20 (H4K20me2) to promote nonhomologous end-joining, suggesting a balance between BRCA1 and 53BP1 regulates DNA double-strand break (DSB) repair mechanism choice. Here, we document that acetylation is a key determinant of this balance. TIP60 acetyltransferase deficiency reduced BRCA1 at DSB chromatin with commensurate increases in 53BP1, while HDAC inhibition yielded the opposite effect. TIP60 -dependent H4 acetylation diminished 53BP1 binding to H4K20me2 in part through disruption of a salt bridge between H4K16 and Glu1551 in the 53BP1 Tudor domain. Moreover, TIP60 deficiency impaired HR and conferred sensitivity to PARP inhibition in a 53BP1-dependent manner. These findings demonstrate that acetylation in cis to H4K20me2 regulates relative BRCA1 and 53BP1 DSB chromatin occupancy to direct DNA repair mechanism.

DNA methyltransferase 1 (DNMT1) is crucial for maintenance of methylation, gene regulation and chromatin stability1-3. DNA mismatch repair, cell cycle regulation in post-mitotic neurons4,5 and neurogenesis6 are influenced by DNA methylation. Here we show mutations in DNMT1 cause both central and peripheral neurodegeneration in one form of hereditary sensory and autonomic neuropathy (HSAN1) with dementia and hearing loss7,8. Exome sequencing led to the identification of DNMT1 mutation c.A1484G (p.Tyr495Cys) in two American and one Japanese kindreds and a triple nucleotide change c.1470TCC-1472ATA (p.Asp490Glu-Pro491Tyr) in one European kindred. All mutations are within the targeting sequence (TS) domain of DNMT1. These mutations cause premature degradation of mutant proteins, reduced methyltransferase activity and impaired heterochromatin binding during the G2 cell cycle phase, leading to global hypomethylation and site specific hypermethylation. Our study demonstrates DNMT1 mutations cause aberrant methylation implicated in complex pathogenesis. The discovered DNMT1 mutations provide a new framework for the study of neurodegenerative diseases.

The lysine demethylase JMJD2A has the unique property of binding trimethylated peptides from two different histone sequences (H3K4me3 and H4K20me3) through its tudor domains. Here we show using X-ray crystallography and calorimetry that H3K4me3 and H4K20me3, which are recognized with similar affinities by JMJD2A, adopt radically different binding modes, to the extent that we were able to design single point mutations in JMJD2A that inhibited the recognition of H3K4me3 but not H4K20me3 and vice versa.The methylation of histones at lysine residues participates in the regulation of fundamental biological processes, such as gene transcription and cellular response to DNA damage, by attracting effector proteins that bind chromatin only after a target histone has been methylated at a specific lysine 1 . Therefore, there is considerable interest in understanding how specialized domains of these regulatory proteins recognize histone amino acid sequences in a methylation site-specific and sometimes methylation state-specific manner. The previously determined three-dimensional structures of chromo, double-chromo, tandem tudor, hybrid tudor and PHD domains in complex with methylated histone peptides have all revealed a binding cage 2 , made of two to four aromatic amino acid side chains, that accounts for the specific interaction with a methyllysine at the positively charged methylated ammonium group through cation-π interactions 3-9 . In one instance, it was shown that the selectivity for a particular methylation state-a dimethyllysine over a trimethyllysine-is mediated by the carboxylate side chain of an aspartate that forms a hydrogen bond with the dimethylammonium hydrogen 7 . Besides the methylation state, interactions of the effector protein with several histone amino acids adjacent to the methyllysine confer high specificity for a given methylated histone site 3-9 .The tandem hybrid tudor domains of the human histone demethylase JMJD2A (JMJD2A-tudor) can recognize H3K4me3 (residues 1-10) and H4K20me3 (residues 16-25), two histone peptides that do not share any amino acid sequence similarity except the trimethylated lysine 10 . H3K4me3 and H4K20me3 bound with about the same K d to JMJD2A-tudor, as shown by isothermal titration calorimetry (ITC) (Fig. 1a and Table 1). To better understand the histone binding mechanism of JMJD2A and probe possible differences between the recognition of histones H3K4me3 (ref. 6) and H4K20me3 by JMJD2A-tudor, we determined the crystal structure of human JMJD2A-tudor in complex with H4K20me3 at a resolution of 2.8 Å ( Figs. 1b and 2 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript details). In the crystal asymmetric unit, there are two copies of the JMJD2A-tudor-H4K20me3 complex. In one complex, seven residues (residues 17-23) of the peptide were unambiguously modeled in the electron density map ( Fig. 1b and Supplementary Fig. 1 online), whereas in the other complex five residues (residues 17-21) of the peptide were readily modeled. The two H4K20me3 pept...

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