Published OnlineFirst June 1, 2018; DOI: 10.1158/1541-7786.MCR-17-0688 Minireview Molecular Cancer Research Understanding the Histone DNA Repair Code: H4K20me2 Makes Its Mark Karissa L. Paquin and Niall G. Howlett Abstract Chromatin is a highly compact structure that must be describe the writers, erasers, and readers of this important rapidly rearranged in order for DNA repair proteins to access chromatin mark as well as the combinatorial histone post- sites of damage and facilitate timely and efficient repair. translational modifications that modulate H4K20me recog- Chromatin plasticity is achieved through multiple processes, nition. Finally, we discuss the central role of H4K20me in including the posttranslational modification of histone tails. determining if DNA double-strand breaks (DSB) are repaired In recent years, the impact of histone posttranslational mod- by the error-prone, nonhomologous DNA end joining path- ification on the DNA damage response has become increas- way or the error-free, homologous recombination pathway. ingly well recognized, and chromatin plasticity has been firmly This review article discusses the regulation and function of linked to efficient DNA repair. One particularly important H4K20me2 in DNA DSB repair and outlines the components histone posttranslational modification process is methylation. and modifications that modulate this important chromatin Here, we focus on the regulation and function of H4K20 mark and its fundamental impact on DSB repair pathway methylation (H4K20me) in the DNA damage response and choice. Mol Cancer Res; 16(9); 1335–45. Ó2018 AACR. Introduction recruitment of chromatin reader proteins and/or chromatin remo- deling complexes, which can lead to marked changes in chroma- Chromatin is a highly organized and condensed structure that tin structure and compaction. Single and combinatorial PTMs can allows billions of base pairs of DNA to be tightly packaged into the have distinct signaling and cellular outcomes. Combinatorial nuclei of eukaryotic cells. The basic subunit of chromatin is the marks add to the variability and complexity of chromatin recog- nucleosome, an octamer of histones around which 146 bp of nition and plasticity (3, 4). In this review, we will discuss one DNA is wrapped 1.7 times. Each nucleosome contains two copies aspect of chromatin plasticity, namely histone PTM. Specifically, each of histones H2A, H2B, H3, and H4. Histones are highly we will focus on the dimethylation of histone H4 lysine 20 in conserved among eukaryotes, emphasizing their importance (1). mammalian cell lineages and how this particular PTM has become Chromatin cannot be a rigid and unchanging structure, however. increasingly recognized as a major determinant of DNA repair. For It is highly dynamic in order to facilitate DNA replication, tran- more comprehensive reviews of chromatin plasticity and DNA scription, and repair. Chromatin plasticity is a necessity, as with- repair, please refer to the following excellent reviews (5–7). out it, DNA-interacting proteins would not be able to access this tightly condensed structure. Chromatin plasticity is facilitated by nucleosome repositioning, histone exchange, and the posttrans- DNA DSB Repair lational modification (PTM) of histone tails. Nucleosome repo- DNA damage can arise as a result of endogenous agents, such as sitioning involves the physical sliding of nucleosomes along the reactive oxygen species, a byproduct of normal cellular processes, DNA or their eviction. In histone exchange, histone variants are or by exogenous means, such as exposure to UV light. DNA substituted for the canonical histones H2A, H2B, H3, or H4. For damage must be repaired in an efficient and timely manner in example, H2A can be substituted with the variant H2AX upon the order to continue normal cellular processes like replication and formation of DNA double-strand breaks (DSB; ref. 2). Histone transcription. Although there are many distinct types of DNA PTM is the addition of small molecules, such as acetyl-, methyl-, damage, here we will focus on DNA DSBs. DSBs arise upon and phospho-groups, or small proteins, such as SUMO (small cellular exposure to ionizing radiation and as a consequence of ubiquitin-like modifier) and ubiquitin to the tails of histones, replication fork collapse. DSBs can also arise transiently during which extend from the core nucleosome. These PTMs change DNA repair processes, including nucleotide excision repair and chromatin structure in several ways, for example, by modulating interstrand cross-link repair (8). Upon DSB formation, free ends the strength of histone–DNA interactions, and by facilitating the of broken DNA are recognized by the MRE11–RAD50–NBS1 (MRN) complex, which recruits the ATM (ataxia telangiectasia mutated) kinase (9, 10). ATM phosphorylates a histone variant Department of Cell and Molecular Biology, University of Rhode Island, Kingston, called H2AX on serine 139, forming gH2AX (11, 12). gH2AX was Rhode Island. one of the first recognized histone PTMs and has been extensively Corresponding Author: Niall G. Howlett, University of Rhode Island, 120 Flagg studied in relation to DSB repair (11). Mediator of DNA damage Road, Kingston, RI 02881. Phone: 401-874-4306; Fax: 401-874-2065; E-mail: checkpoint 1 (MDC1) recognizes gH2AX via its BRCA1 C-Termi- [email protected] nus (BRCT) domain (13). MDC1 subsequently recruits additional doi: 10.1158/1541-7786.MCR-17-0688 molecules of ATM via its forkhead-associated (FHA) domain; Ó2018 American Association for Cancer Research. ATM phosphorylates additional H2AX molecules, thereby www.aacrjournals.org 1335 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst June 1, 2018; DOI: 10.1158/1541-7786.MCR-17-0688 Paquin and Howlett amplifying the gH2AX signal up to two megabases proximal to (lysine methyltransferase 5A), a SET-domain (Su(var)3-9, the DSB site (refs. 13–15; Fig. 1A). As one of the first steps in Enhancer-of-zeste and Trithorax) containing methyltransferase DSB repair, H2AX phosphorylation is widely used as a marker for (Table 1). KMT5A, similar to all methyltransferases, is referred DSB formation. to as a writer of chromatin marks. It has recently been shown that DSBs are repaired by one of two ways: homologous recombi- KMT5A prefers the entire nucleosome as its substrate, rather than nation (HR) or nonhomologous DNA end joining (NHEJ). HR is individual H4 histones or peptides, and that it interacts with H2A an error-free repair pathway that uses a homologous DNA and H2B in order to monomethylate H4K20 (41–44). Loss of sequence as a template to repair damaged DNA (16). HR is a Kmt5a in both fly and mouse results in embryonic lethality cell-cycle–dependent pathway, occurring primarily during (45, 46). Studies have shown that with knockout of Kmt5a, S-phase due to the presence of homologous DNA in the sister H4K20 di- and trimethylation are downregulated (45). In HeLa chromatid. Briefly, upon gH2AX phosphorylation, the MRN cells, KMT5A knockdown results in reduced 53BP1 recruitment to complex, CtBP-interacting protein (CtIP), exonuclease 1 (EXO1), DSBs (47, 48). In addition, Kmt5a knockout embryonic stem cells and DNA replication/helicase protein 2 (DNA2) all promote 50-30 and KMT5A-depleted HeLa and U2OS cells display increased DNA end resection, resulting in the generation of 30 single- DSBs and gH2AX formation, even in the absence of exposure to stranded overhangs on each strand (ssDNA; refs. 17–21). The DNA-damaging agents (45, 49, 50). This is likely an accumulation ssDNA overhangs are first coated by replication protein A (RPA) to of spontaneous DNA damage throughout the cell cycle, which protect against nucleolytic degradation. The major DNA strand remains unrepaired due to lack of H4K20 methylation (45). recombinase, RAD51, is subsequently loaded onto ssDNA in a KMT5A-depleted U2OS cells have increased cell-cycle checkpoint process facilitated by functional homologs of the yeast Rad52 activation, decreased cell-cycle progression, and accumulated in epistasis group and the BRCA2 protein (22–24). RAD51 forms a S-phase, also in the absence of DNA damage (50). nucleoprotein filament coating the ssDNA, and a displacement loop (D-loop) is formed upon invasion of the ssDNA into the KMT5B/C. The H4K20me2 mark has been shown to be involved complementary sister chromatid duplex, referred to as the syn- in DNA repair. This histone mark is found throughout the aptic complex (22–26). New DNA is then synthesized using the nucleus; however, it has been reported to be enriched at sites of sister chromatid as a template, and Holliday junctions (branched DNA damage (51). Globally, Kmt5b (Aliases: Suv4-20h1, heteroduplex DNA intermediates comprising newly synthesized SUV420H1) and Kmt5c (Aliases: Suv4-20h2, SUV420H2) are DNA on the invading strand and the template strand) are responsible for H4K20 di- and trimethylation, respectively resolved, resulting in a duplicate of the sister chromatid (gene (52). KMT5B/C has been shown to catalyze dimethylation more conversion), with no loss of genetic information (ref. 16; Fig. 1B). efficiently than trimethylation in vitro (38, 39, 53). This suggests Conversely, NHEJ is typically an error-prone pathway that that additional proteins may be necessary for efficient H4K20 simply ligates the free ends of broken DNA. NHEJ occurs in all trimethylation, or that another HMT catalyzes this reaction phases of the cell cycle and can result in catastrophic events such as (38, 52). Although