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DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically 'recognizes' H4K20me3 via its first bromo-adjacent-homology domain (DNMT1BAH1). Engagement of DNMT1BAH1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1's activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

N-alpha-acetyltransferase 40 (NAA40) is an evolutionarily conserved N-terminal acetyltransferase (NAT) linked to oncogenesis and chemoresistance. A recent study reported the generation of a second, shorter NAA40 isoform (NAA40S) through alternative translation, which we proceeded to further characterise. Notably, recombinant NAA40S had a greater in vitro enzymatic activity and affinity towards its histone H2A/H4 substrates compared to full-length NAA40 (NAA40L). Within cells, NAA40S was enzymatically active, based on its ability to suppress the H2A/H4S1Ph antagonistic mark in CRISPR-generated NAA40 knockout cells. Finally, we show that in addition to alternative translation, the NAA40S isoform could be derived from a primate and testis-specific transcript, which may align with the "out-of-testis" origin of recently evolved genes and isoforms. To summarise, our data reveal an even greater functional divergence between the two NAA40 isoforms than had been previously recognised. [ABSTRACT FROM AUTHOR]

Abstract: DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling [...]

Next-generation sequencing technology is used to show that the error-prone polymerase [theta] (Pol[theta]) is needed to promote alternative non-homologous end joining at telomeres, and during chromosomal translocations, while counteracting homologous recombination; inhibition of Pol[theta] represents a potential therapeutic strategy for tumours that have mutations in homology-directed repair genes. Pol[theta] involved in alternative DNA repair The error-prone non-homologous end joining (NHEJ) DNA repair pathway is used as an alternative when the error-free homologous recombination pathway is compromised, or is the pathway of choice in some cellular contexts, such as in the immune system. After broken ends are paired via microhomology, NHEJ depends on DNA synthesis, but the identity of the polymerase involved was unclear. Two studies, from the laboratories of Agnel Sfeir and Alan D'Andrea, now implicate the mammalian POLQ gene, encoding the error-prone polymerase Pol[theta], in this process. Sfeir and colleagues show that upon telomere deprotection, Pol[theta] is needed to prevent alternative end joining at telomeres, and chromosomal translations at non-telomeric sequences. D'Andrea and colleagues focus on the role of Pol[theta] in cancer cells, and show that in a homologous-recombination-deficient background, the absence of Pol[theta] results in a synthetic lethality, suggesting a possible therapeutic approach. The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions.sup.1,2,3. Using next-generation sequencing technology, here we show that repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify polymerase theta (Pol[theta]; encoded by Polq in mice) as a crucial alternative NHEJ factor in mammalian cells. Polq inhibition suppresses alternative NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of Polq in mice results in increased rates of homology-directed repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Pol[theta] has a synergistic effect on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed repair genes., Author(s): Pedro A. Mateos-Gomez [sup.1] , Fade Gong [sup.2] , Nidhi Nair [sup.3] , Kyle M. Miller [sup.2] , Eros Lazzerini-Denchi [sup.3] , Agnel Sfeir [sup.1] Author Affiliations: (1) Department [...]

The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions (1-3). Using next-generation sequencing technology, here we show that repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify polymerase theta (Polθ; encoded by Polq in mice) as a crucial alternative NHEJ factor in mammalian cells. Polq inhibition suppresses alternative NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of Polq in mice results in increased rates of homology-directed repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Polθ has a synergistic effect on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed repair genes., Chromosome end-to-end fusions are inhibited by shelterin; a multi-subunit complex anchored to telomeric DNA by two Myb-containing proteins--TRF1 and TRF2 (ref. 4). Telomere fusions are executed by two independent end-joining [...]

The notion that telomeres are essential for chromosome linearity stems from the existence of two chief dangers: inappropriate DNA damage response (DDR) reactions that mistake natural chromosome ends for double-strand DNA breaks (DSBs), and the progressive loss of DNA from chromosomal termini due to the end replication problem. Telomeres avert the former peril by binding sequence-specific end-protection factors that control the access of DDR activities (1,2). The latter threat is tackled by recruiting telomerase, a reverse transcriptase that uses an integral RNA subunit to template the addition of telomere repeats to chromosome ends3. Here we describe an alternative mode of linear chromosome maintenance in which canonical telomeres are superseded by blocks of heterochromatin. We show that in the absence of telomerase, Schizosaccharomyces pombe cells can survive telomere sequence loss by continually amplifying and rearranging heterochromatic sequences. Because the heterochromatin assembly machinery is required for this survival mode, we have termed it 'HAATI' (heterochromatin amplification-mediated and telomerase-independent). HAATI uses the canonical end-protection protein Potl (ref. 4) and its interacting partner Ccql (ref. 5) to preserve chromosome linearity. The data suggest a model in which Ccql is recruited by the amplified heterochromatin and provides an anchor for Potl, which accomplishes its end-protection function in the absence of its cognate DNA-binding sequence. HAATI resembles the chromosome end-maintenance strategy found in Drosophila melanogaster, which lacks specific telomere sequences but nonetheless assembles terminal heterochromatin structures that recruit end-protection factors. These findings reveal a previously unrecognized mode by which cancer cells might escape the requirement for telomerase activation, and offer a tool for studying genomes that sustain unusually high levels of heterochromatinization., Ten to fifteen percent of cancer cells achieve unlimited proliferative potential without telomerase by using one or more method(s) termed alternative lengthening of telomeres (ALT). The modus operandi of ALT [...]

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair (1,2). Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions (3-6). Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1.We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage (7-11). These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation., Mammalian cells express SUMO1 and the highly-related proteins SUMO2 and SUMO3 (SUMO2/3). These somewhat functionally-redundant proteins (12) are structurally related to ubiquitin and are covalently attached to target proteins by [...]

Author(s): Kyle M. Miller [1, 2, 3]; Ofer Rog [1, 3]; Julia Promisel Cooper (corresponding author) [1] Telomere replication is achieved through the combined action of the conventional DNA replication [...]