Characterization of a H4K20ME2 Methyl-Binding Domain in the Fanconi Anemia Protein FANCD2

Fanconi anemia (FA) is a rare genetic disease that results on early onset bone marrow failure, congenital defects, and increased cancer susceptibility. It is caused by mutation in any one of twenty two genes, whose protein products work in conjunction as part of the FA-BRCA pathway, a DNA repair pathway that specifically repairs DNA interstrand crosslinks (ICLs). One of the key steps in pathway activation is the monoubiquitination of the FANCD2 protein. Upon pathway activation, FANCD2 is recruited to sites of DNA damage, where is interacts with downstream repair protiens to promote DNA repair via homologous recombination (HR). How FANCD2 recognizes DNA damage in condensed compact chromatin, however, has remained unknown. Here, we uncover a FANCD2 methyl-binding domain, which specifically binds for H4K20me2 in order to recruit FANCD2 to sites on DNA damage and promote homologous recombination, support cell survival and maintain genomic integrity.

importance (1). Chromatin cannot be a rigid and unchanging structure, however. It is highly dynamic in order to facilitate DNA replication, transcription, and repair.
Chromatin plasticity is a necessity, as without 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 post-translational modification (PTM) of histone tails. Nucleosome repositioning involves the physical sliding of nucleosomes along the DNA or their eviction. In histone exchange, histone variants are substituted for the canonical histones H2A, H2B, H3 or H4. For example, H2A can be substituted for the variant H2AX upon the formation of DNA double-strand breaks (DSBs) (2). Histone PTM is the addition of small molecules, such as acetyl-, methyl-, and phospho-groups, or small proteins, such as SUMO (small ubiquitin-like modifier) and ubiquitin to the tails of histones, which extend from the core nucleosome. These PTMs change chromatin structure in several ways, for example, by modulating the strength of histone-DNA interactions, and by facilitating the recruitment of chromatin reader proteins and/or chromatin remodeling complexes, which can lead to marked changes in chromatin structure and compaction. Single and combinatorial PTMs can have distinct signaling and cellular outcomes. Combinatorial marks add to the variability and complexity of chromatin recognition and plasticity (3,4). In this review, we will focus on one aspect of chromatin plasticity, namely histone PTM. Specifically, we will discuss the methylation of histone H4 lysine 20 and how this particular PTM has become increasingly recognized as a major determinant of DNA repair.

DNA Double-Strand Break Repair
DNA damage can arise as a result of endogenous agents, such as reactive oxygen species, a byproduct of normal cellular processes, or by exogenous means, such as exposure to UV light. DNA damage must be repaired in an efficient and timely manner in order to continue normal cellular processes like replication and transcription. While there are many types of DNA damage, here we will focus on DNA double-strand breaks (DSBs).
DSBs arise upon cellular exposure to ionizing radiation and as a consequence of replication fork collapse. DSBs can also arise transiently during DNA repair processes, including nucleotide excision repair and interstrand crosslink repair (5). Upon DSB formation, free ends of broken DNA are recognized by the MRN (MRE11-RAD50-NBS1) complex, which recruits the ATM (ataxia telangiectasia mutated) kinase (6,7).
ATM phosphorylates a histone variant called H2AX on serine 139, forming γH2AX (8,9). γH2AX was one of the first recognized histone PTMs, and has been extensively studied in relation to DSB repair (8). MDC1 (mediator of DNA damage checkpoint 1) recognizes γH2AX via its BRCT (BRCA1 C-Terminus) domain (10). MDC1 subsequently recruits additional molecules of ATM via its FHA (forkhead-associated) domain; ATM phosphorylates additional H2AX molecules thereby amplifying the γH2AX signal up to two megabases proximal to the DSB site (10-12) ( Figure 1A). As one of the first steps in DSB repair, H2AX phosphorylation is widely used as a marker for DSB formation.
DSBs are repaired by one of two ways: homologous recombination (HR) or nonhomologous DNA end joining (NHEJ). Homologous recombination is an error-free repair pathway that uses a homologous DNA sequence as a template to repair damaged   (33). The regulation of this process will be discussed in greater detail later in the review. Depletion of Brca1 results in increased NHEJ and decreased HR (34).
Depletion of both Brca1 and 53bp1, however, restores normal levels of HR in mice (25).
This, along with evidence that 53BP1 physically blocks end resection, indicates that BRCA1 may play a role in 53BP1 removal from DSB sites, allowing HR to proceed (33).
KMT5A depleted U2OS cells have increased cell cycle checkpoint activation, decreased cell cycle progression, and accumulate in S-phase, also in the absence of DNA damage (47).

KMT5B/C
The H4K20me2 mark has been shown to be involved in DNA repair. This histone mark is found throughout the nucleus, however it has been reported to be enriched at sites of DNA damage (48). Globally, Kmt5b and Kmt5c are responsible for dimethylation and trimethylation, respectively. (49). KMT5B/C has been shown to enzymatically catalyze dimethylation more efficiently than trimethylation in vitro (35,36,50). This suggests that additional proteins are necessary for efficient H4K20 trimethylation, or that another HMT catalyzes this reaction (35,49    acetylating H2AK15 (see H2AK15ac section), which also impacts 53BP1 binding to H4K20me2, discussed in more detail below (70) ( Figure 3A).

FANCD2
Our acetylation, and MBTD1 overexpression slightly increases H2AK15ac levels, however the requirement of MBTD1 for H2AK15ac needs to be more closely examined.
H2AK15ac appears most predominantly in G2/M phase, potentially overlapping with HR in early G2, and continuing to evict 53BP1 throughout mitosis (70). In general, histone acetylation is downregulated after DNA damage, however H2AK15 acetylation was shown to increase after DNA damage, indicating the role of H2AK15 acetylation in DNA damage repair (70). While the authors suggest this mark is associated with HR, regulation of this mark and MBTD1 in pathway choice has yet to be studied.

H2AK127Ub
As mentioned above, upon damage recognition, MRN complex recruitment, and H2AX phosphorylation, CtIP is recruited to DSBs during homologous recombination in order to catalyze end resection. In BRCA1-deficient cells, 53BP1 blocks end resection, and NHEJ takes place. However, in BRCA1-proficient cells, HR is favorable during S-phase (33  damage sites. MRN and CtIP then promote end resection, and HR moves forward (57).
This evidence is suggestive that SMARCAD1 binds to H2AK127Ub in order to reposition 53BP1, supporting previous work that shows that BRCA1 is involved in 53BP1 repositioning, and ultimate repair pathway choice ( Figure 4). Much remains to be determined about the dynamics, regulation and molecular function of this particular chromatin mark.

H4K16ac
In addition to catalyzing H2AK15 acetylation, TIP60 also catalyzes the acetylation of

Conclusions
H4K20me2 joins a growing list of histone PTMs that play a major role in the coordination of DNA repair processes (Table 1). Until recently, γH2AX was one of the few posttranslationally modified histones with a well-characterized role in the DNA damage response. However, the importance of chromatin plasticity and, in particular, histone PTMs for the orchestration of DNA repair has become increasingly well recognized. Many questions on the function and regulation of H4K20 methylation remain: For example, are the H4K20me writers and erasers differentially regulated in different tissue types? Do the different H4K20 methylation states have a role in the orchestration of loci-specific repair? In addition to H4K20 methylation, H3K9 and H3K27 methylation have also recently been shown to play key roles in the DNA damage response. Deciphering how the combinatorial modification of these marks and others coordinately contribute to DNA repair will be a considerable molecular challenge. While much remains to be answered, it is clear that the recognized roles for histone PTMs in the DNA damage response will continue to expand. As many of the writers, erasers, and readers of histone PTMs are druggable targets, a greater understanding of their homeostasis is highly likely to lead to the development of more targeted and effective combination cancer chemotherapy regimens.  which occurs within chromatin (9)(10)(11)(12). However, the mechanism(s) by which FANCD2 is tethered to chromatin, and whether FANCD2 displays specificity for particular histone PTMs, is unknown. Indeed, no reader domains have been identified for any of the FA proteins to date.
In this study, we describe the identification and characterization of a FANCD2 histonebinding domain (HBD) and embedded methyl-lysine-binding domain (MBD). We establish that the FANCD2 MBD can bind to mono-, di-, and tri-methylated H4K20 in vitro, and exhibits specificity for H4K20me2 in cellula. Knockdown of KMT5A, the histone methyltransferase responsible for H4K20 monomethylation, which primes Our studies uncover a novel and key mechanism by which FANCD2 is anchored to chromatin and functionally link this important human genetic disease to chromatin plasticity.

FANCD2 has a histone-binding domain and embedded methyl-lysine-binding domain.
A BLASTp search using short fragments of human FANCD2 uncovered amino acid sequence homology between FANCD2 and the Drosophila melanogaster p55 protein, a histone H4 binding protein and component of the NuRD, NuRF, and CAF1 nucleosome remodeling complexes (13-16) (Fig. S1A). This region of FANCD2 is highly evolutionarily conserved among vertebrates (Fig. S1A). Using an in silico molecular modeling approach, the histone H4 tail from the p55-H4 structure (PDB ID: 3C9C) was docked into murine Fancd2 (PDB ID: 3S4W) using AutoDock Vina (17), illustrating favorable predicted binding energies between the H4 tail and the histone-binding domain (HBD) (Fig. S1B). Further examination of the FANCD2 HBD uncovered a highly conserved putative methyl-lysine (Kme)-binding domain (MBD) with sequence homology to the methyl-binding chromodomains of HP1α, TIP60, and CBX8 ( Fig. 1A and B). A GST-tagged FANCD2-HBD/MBD fragment (amino acids 604-1194) was purified and, in a histone peptide array screen, bound to a H4 17-mer harboring unmodified, K20me1, K20me2, and K20me3 (data not shown). These findings were verified in an in vitro histone peptide pulldown assay ( Fig. S1C and D). Using a similar approach, a smaller GST-tagged FANCD2-MBD fragment (amino acids 1069-1142) bound to H4K20me1, H4K20me2, and H4K20me3, but not unmodified H4 or H3K27me3 (Fig. 1D). MBD-Kme binding involves the docking of Kme into an aromatic cage and the formation of cation-π interactions with delocalized electrons of aromatic residues (18). Highly conserved aromatic amino acids within the FANCD2 Kme binding cage include F1073, W1075, and F1078 (Fig. S1E). Using calf thymus histones, we observed a modest decrease in binding of a MBD-W1075A fragment to H4K20me2 and me3, compared to the wild-type MBD (Fig. S1F). Finally, using proximity ligation assay (PLA), we observed preferential binding of FANCD2 to H4K20me2 in cells, and stimulation of H4K20me binding upon MMC exposure ( Fig. 1E and S1G). Binding of 53BP1 to H4K20me was used as a positive control for our PLA assay (Fig. S1H). Taken together, our results demonstrate that FANCD2 interacts directly with methylated H4 via its HBD/MBD, and suggest that the chromatin recruitment of FANCD2 is mediated via an interaction between the HBD/MBD and H4K20, similar to that recently described for other important DNA repair proteins, e.g. 53BP1 and TIP60 (1-3, 19).  Fig. 2A), KMT5A knockdown resulted in a significant decrease in FANCD2 nuclear foci formation following exposure to MMC and aphidicolin (APH), a replicative DNA polymerase inhibitor ( Fig. 2B and C). Similar findings were observed for the nontransformed mammary epithelial line MCF10A ( Fig. S2A and B). In addition, similar to FA patient-derived cells, cells depleted of KMT5A exhibited increased basal and ICLinducible chromosome aberrations, including gaps, breaks and radial formations ( Fig. 2D and E). These results establish that H4K20 methylation is necessary for efficient activation of the FA pathway and ICL repair.

The FANCD2 MBD is required for efficient chromatin binding and nuclear foci
formation. To examine the functional importance of the FANCD2 HBD/MBD, we next  (Fig. 3A). These findings indicate that mutations in the HBD/MBD do not perturb overall protein structure or stability, or the propensity for monoubiquitination by the multi-subunit FA core complex ubiquitin ligase. Using a chromatin enrichment assay, similar to wild type FANCD2, the HBD/MBD mutants were capable of localizing to chromatin (Fig. S3A). However, the interactions between the HBD/MBD mutants and chromatin were more sensitive to increasing salt concentrations than wild type FANCD2, leading to release of the mutants at lower salt concentrations ( Fig. 3B and S3C). These results are suggestive of a reduced affinity of the mutants for chromatin. Moreover, similar to FANCD2-K561R and unlike wild type FANCD2, FANCD2-H1056A and FANCD2-W1075A failed to assemble into discrete nuclear foci following ICL exposure ( Fig. 4A and C). Consistent with previous studies showing a dependency between FANCD2 and FANCI nuclear foci formation (11,12), FANCI nuclear foci formation was also markedly impaired in cells expressing the HBD/MBD mutants (Fig. 4B). We next analyzed the interactions between FANCD2 and H4K20me in FA-D2 cells expressing wild type FANCD2 or the W1075A mutant using PLA. While wild type FANCD2 interacted strongly with H4K20me2 -and much less so with H4K20me1, H4K20me3, or H3K27me3 -this interaction was markedly impaired for the W1075A mutant (Fig. 4D). We simultaneously performed PLA with 53BP1 and H4K20me1, me2, me3, and H3K27me3 and observed a modest, yet statistically significant, increase in 53BP1 binding to H4K20me2 and me3 in FA-D2 cells expressing FANCD2-W1075A, compared to cells expressing wild type FANCD2 (Fig. S4). Taken together, our results indicate that FANCD2 preferentially binds to H4K20me2 in cells, and that binding is mediated by the HBD/MBD domain. In addition, our results suggest that decreased binding of H4K20me2 by FANCD2 may lead to increased H4K20me2 binding by 53BP1.

The FANCD2 HBD/MBD is required for efficient conservative ICL repair.
To  radial formations, compared with cells expressing wild type FANCD2 (Fig. 5D and   S5A). Stable expression of FANCD2-H1056A and -W1075A had no observable impact on cellular growth rate (Fig. S5C). Taken together, our findings indicate that H4K20me2 binding by the FANCD2 HBD/MBD is essential for the promotion of error-free conservative ICL repair, and link chromatin plasticity to activation of an important tumor suppressor pathway.

Discussion
In this study, we describe the identification and functional characterization of a methyllysine binding domain in the FANCD2 protein that exhibits specificity for H4K20me2.
Disruption of this domain results in a decreased affinity for chromatin and an inability to assemble into discrete nuclear foci, presumed sites of active ICL repair (10).
Consequently, cells expressing FANCD2 MBD mutants demonstrate evidence of persistent DSBs and an increased dependence on error-prone ICL repair pathways, including NHEJ. This, in turn, leads to increased sensitivity to ICL-inducible chromosome structural aberrations and cytotoxicity. A role for the FA proteins in suppressing erroneous NHEJ repair has previously been described (24). Consistent with an important role for the H4K20me2 chromatin mark in facilitating efficient activation of the FA pathway and ICL repair, depletion of the KMT5A H4K20 monomethyltransferase markedly reduced FANCD2 nuclear foci formation following ICL exposure. Mutation of the MBD and depletion of KMT5A did not, however, impact FANCD2 or FANCI monoubiquitination. This is consistent with several reports demonstrating the uncoupling of monoubiquitination from nuclear foci formation (10,(28)(29)(30)(31). Collectively, these studies indicate that monoubiquitination is necessary but not sufficient for nuclear foci formation, and that the ability of FANCD2 to assemble into nuclear foci is essential for effective ICL repair. Moreover, our findings indicate that one critical determinant of FANCD2 nuclear foci formation is the ability to interact directly with H4K20me2.

NaCl Extraction
Cells were plated at a density of 3

Conflict of Interest
The author(s) declare no competing financial interests.

Supplemental Information
Supplemental

APPENDIX-II
Supplemental information for Manuscript-II: "FANCD2 binding to H4K20me2 via a methyl-binding domain is essential for efficient DNA crosslink repair"   The Y-axis depicts the ratios of band intensities compared to the band from FA-D2 cells expressing wild type FANCD2 at the equivalent NaCl concentration.   In conclusion, these studies uncover important mechanistic insight into the molecular biology of HR and FA and suggest the existence of more FA genes linked to the regulation of RAD51 function.

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.

Funding
This work was supported by National Institutes of Health/National Heart, Lung and