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Activation of the FA pathway mediated by phosphorylation and ubiquitination
Accepted Manuscript Title: Activation of the FA pathway mediated by phosphorylation and ubiquitination Authors: Masamichi Ishiai, Koichi Sato, Junya Tomida, Hiroyuki Kitao, Hitoshi Kurumizaka, Minoru Takata PII: DOI: Reference:
S0027-5107(17)30078-7 http://dx.doi.org/doi:10.1016/j.mrfmmm.2017.05.003 MUT 11594
To appear in:
Mutation Research
Received date: Revised date: Accepted date:
14-4-2017 4-5-2017 4-5-2017
Please cite this article as: Masamichi Ishiai, Koichi Sato, Junya Tomida, Hiroyuki Kitao, Hitoshi Kurumizaka, Minoru Takata, Activation of the FA pathway mediated by phosphorylation and ubiquitination, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesishttp://dx.doi.org/10.1016/j.mrfmmm.2017.05.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Special Section "Protein modifications in DNA damage repair and cancer" at Mutation Research
Activation of the FA pathway mediated by phosphorylation and ubiquitination Masamichi Ishiai1,6, Koichi Sato2,3,6, Junya Tomida1,4, Hiroyuki Kitao1,5 Hitoshi Kurumizaka2*, Minoru Takata1* 1
Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation
Biology Center, Kyoto University, Kyoto Japan 2
Laboratory of Structural Biology, Graduate School of Advanced Science and
Engineering, Waseda University, Tokyo, Japan 3
Present address: Hubrecht Institute–KNAW & University Medical Center Utrecht,
Utrecht, the Netherlands 4
Present address: Department of Epigenetics and Molecular Carcinogenesis, The
University of Texas MD Anderson Cancer Center, Smithville, TX 78957 5
Present address: Department of Molecular Cancer Biology, Graduate School of
Pharmaceutical Science, Kyushu University, Fukuoka, Japan 6
These authors contributed equally to this work.
* Correspondence should be addressed to H.K ([email protected]) or M.T. ([email protected])
Special Section "Protein modifications in DNA damage repair and cancer" at Mutation Research
Activation of the FA pathway mediated by phosphorylation and ubiquitination
Abstract Fanconi anemia (FA) is a devastating hereditary condition that impacts genome integrity, leading to clinical features such as skeletal and visceral organ malformations, attrition of bone marrow stem cells, and carcinogenesis. At least 21 proteins, when absent or defective, have been implicated in this disorder, and they together constitute the FA pathway, which functions in detection and repair of, and tolerance to, endogenous DNA damage. The damage primarily handled by the FA pathway has been assumed to be related to DNA interstrand crosslinks (ICLs). The FA pathway is activated upon ICL damage, and a hallmark of this activation is the mono-ubiquitination events of the key FANCD2-FANCI protein complex. Recent data have revealed unexpectedly complex details in the regulation of FA pathway activation by ICLs. In this short review, we summarize the knowledge accumulated over the years regarding how the FA pathway is activated via protein modifications.
1. Introduction Fanconi anemia (FA) is a devastating hereditary condition that compromises the genome integrity of small children, leading to clinical features such as skeletal and visceral organ malformations, attrition of bone marrow stem cells, and carcinogenesis. At least 21 proteins, when absent or defective, have been implicated in this disorder and they together constitute the FA pathway, which functions in detection and repair of, and tolerance to, endogenous DNA damage [1-4]. Sasaki et al. discovered that cells from FA patients display elevated numbers of chromosomal abnormalities upon mitomycin C treatment that induces interstrand crosslinks (ICLs) [5]. These include broken chromosomes and characteristic "radial" structures. Thus, the phenotypes in FA have been presumed to result from defects in ICL repair, and by extension the nature of the endogenous DNA damage handled by the FA pathway has been assumed to be related to ICLs. In hematopoietic stem cells, the endogenous DNA damage is likely induced by aldehyde metabolism (we consider this issue is outside the scope of this review)[2,6]. Alternatively, in FA patients, repeated activation of hematopoietic stem cells from dormancy due to stress such as infection [7], collision of replication and transcription machineries [8], or the inability to replicate large fragile genomic regions due to improper resolution of DNA:RNA hybrids [9] may all lead to DNA damage and contribute to the FA phenotype. The FA pathway consists of the upstream E3 ligase complex termed "the core FA complex" and the downstream FANCD2-FANCI complex (Figure 1A and B) [1-4]. Further
downstream
lies
the
structure-specific
nuclease
complex
SLX4/FANCP-XPF/FANCQ (this complex is thought to be responsible for incision of the ICL), the homologous recombination machineries including BRCA2/FANCD1 and RAD51/FANCR, and translesion synthesis (TLS) components such as REV7/FANCV. The core complex comprises eight FA proteins (i.e., FANCA-B-C-E-F-G-L-M) and other associated factors (FAAP100, FAAP24, FAAP20, MHF1 and MHF2). Although how the core complex senses the presence of DNA damage remains unclear, the detection events take place probably during S phase and can be related to stalled replication forks. The FANCM-FAAP24 sub-complex is required for loading of the rest of the core complex onto damaged chromatin, and the stalled replication fork itself or the action of FANCM translocase [10] exposes single-stranded DNA (ssDNA) that
accumulates the ssDNA binding RPA complex (Figure 1A). The checkpoint kinase ATR-ATRIP complex is recruited by RPA, and phosphorylates the S/TQ cluster residues of FANCI [11], which then leads to the triggering of FANCD2 mono-ubiquitination by the core complex, according to the previous understanding [11]. Mono-ubiquitination of FANCD2 is essential for assembly of the FANCD2 foci [12], chromatin localization of FANCD2 [13], and cell survival of DNA damage caused by ICL inducing agents. Thus, the mono-ubiquitination event of the key FANCD2-FANCI protein complex is considered as the hallmark of FA pathway activation [1-4]. FANCD2 in chromatin may provide a "landing pad" function that recruits other DNA repair proteins. Alternatively, it may have a function of its own. So far, no enzymatic motif has been identified; however, one report described a histone chaperone activity [14], which will be described below. In this short review, we will summarize the role of FANCD2 and FANCI in DNA repair as well as the activation mechanisms of the FANCD2-FANCI complex and the FA pathway, with a focus on protein modifications including phosphorylation, mono-ubiquitination, and de-ubiquitination.
2. FANCD2: the master coordinator of ICL repair FANCD2 is critical for ICL repair. In the absence of FANCD2 or FANCD2 mono-ubiquitination (due to the core complex defects), cells are highly sensitive to ICLs, accumulate higher levels of DNA damage markers, and arrest at G2 phase for a remarkably longer time than wild type cells [15]. A complete FANCD2 null mutation could be lethal in humans, since in all FANCD2 mutated cells derived from human FA patients, a small amount of residual FANCD2 protein has been invariably present [16]. Accumulated DNA damage or persistent DNA damage signaling drives the hematopoietic stem cells (HSCs) in FA patients and FA knockout mice to undergo cell death and senescence, leading to the HSC depletion and functional impairment [15]. So is there a single FANCD2 (and the FA pathway) function in cells? Perhaps not. Accumulating evidence suggests that several distinct functions in ICL repair and related functions are possible as summarized below. First, decreased homologous recombination (HR) repair of a chromosomal double strand break (DSB) induced by restriction enzyme I-SceI has been reported in
FA mutants generated in the chicken DT40 cell line [17,18] and in human FA cells [19]. Gene targeting efficiencies are also dramatically decreased [17]. The mechanisms of these defects are still not entirely clear but could be related to the FANCD2 function in end resection or in stabilizing RAD51 filaments (see below). On the other hand, levels of spontaneous sister chromatid exchange (SCE), which reflects the crossing over events during HR in the cell, are rather increased. The SCE levels are increased after MMC stimulation but only minimally compared to wild type cells [17]. This abnormal SCE phenotype is shared by various DT40 FA mutants [20,21] and mouse FANCM-deficient cells [22]. The increase in SCE levels may be explained by partial loss of BLM helicase activity, since the FA pathway and BLM can physically and functionally interact [21,23-25]. However, our understanding of these defects is still insufficient, and more study is needed. Second, FANCD2 can bind to core histones, and it can assemble nucleosomes in vitro (i.e., histone chaperone activity) [14]. Mobility of histone H3 in cells is reduced following MMC treatment in the absence of FANCD2. A patient-derived mutation in FANCD2 abolished the chaperone activity, and cells expressing this mutant are hypersensitive to cisplatin [14]. Thus, FANCD2 might participate in restoring chromatin during or after ICL repair. Third, FANCD2 has been shown to be critical for unhooking/incision of the ICL at both sides upon fork collision [26]. As pointed out above, the cutting activity itself depends on the SLX4-XPF complex, and FANCD2 and its ubiquitination are also needed for localizing SLX4-XPF at the ICL [27]. However, whether FANCD2 recruits SLX4-XPF through an interaction of its mono-ubiquitinated lysine with the ubiquitin-binding domains of SLX4 is a matter that is still unsettled [27-29]. Fourth, several groups have found that FANCD2 directly interacts and regulates the subnuclear localization of CtIP （for CtIP function, see an excellent review, such as [30]), which is important for ICL repair [31-33] and replication fork recovery [34]. CtIP recruited by FANCD2 promotes end resection upon unhooking of the ICL. The ssDNA generated by processing of the DSB is covered by RPA, which is replaced by RAD51/FANCR with help from mediators including BRCA2/FANCD1, RAD51C/FANCO and XRCC2/FANCU [35,36].
Fifth, FANCD2 has been suggested to work with key HR factor RAD51 as well as BRCA2 in protection of stalled replication forks from degradation mediated by MRE11 and DNA2 nucleases [37-40]. Perhaps related to this, it has been recently reported that the FANCD2-FANCI complex directly interacts with RAD51/FANCR and stabilizes
RAD51-DNA
filaments
[41].
Consistent
with
these
observations,
accumulation of RAD51 is mildly decreased by FANCD2 depletion. This reduction in RAD51 foci might also be influenced by the decreased recruitment of the end resection factor CtIP. Sixth, FANCD2 is phosphorylated by ATM kinase following ionizing radiation and participates in the S phase checkpoint that suppresses replication origin firing. This function has been observed with human [42] but not mouse FANCD2 [43]. It is unclear whether FANCI has a similar role, but FANCI appears to be involved in an ionizing radiation-induced G2/M DNA damage checkpoint [44]. To make the picture more complicated, FANCA is also required for the G2/M checkpoint in response to re-replication, together with ATR and BRCA1/FANCS [45]. Seventh, FANCD2 has been suggested to recruit factors like BLM[21,46], FAN1 [47,48], or DNA2 [49], which may further contribute to replication fork recovery and/or ICL repair. Eighth, it is becoming increasingly clear that FANCD2 and FANCI can have distinct functions. For example, FANCD2 can suppress dormant origin firing while FANCI promotes the process (discussed below) [50]. FANCI but not FANCD2 is important for the core complex to be recruited to chromatin (discussed below) [51]. A recent study revealed that FANCD2 deficient cells display higher sensitivity to replication stress induced by hydroxyurea treatment, but this function is independent of its mono-ubiquitination or FANCI [52]. The authors concluded that non-ubiquitinated FANCD2 together with RAD51 facilitates PCNA mono-ubiquitination and translesion synthesis. Collectively, all these distinct FANCD2-FANCI functions might coordinate the progression of DNA repair steps directed at stalled replication forks, ICLs, and related endogenous DNA damage.
3. FANCD2 mono-ubiquitination is key to its function for ICL repair
FANCD2 is mono-ubiquitinated in vivo in response to DNA damage or replication stress in a manner dependent on the core complex, ATR-ATRIP kinase, FANCI, and the E2 enzyme UBE2T/FANCT (Figure 1A) [1-4,53-55]. FANCD2 carrying a mutation of the mono-ubiquitination site lysine (K561 in human protein) can neither form foci nor relocalize to chromatin, and the exogenously expressed mutant protein cannot reverse the ICL sensitivity of FANCD2-deficient cells [12,42]. To examine the functional significance of FANCD2 mono-ubiquitination, chicken FANCD2 carrying a mutated mono-ubiqutination site was fused with ubiquitin (termed D2KR-Ub) [56]. Expression of D2KR-Ub was able to reverse the ICL sensitivity of FANCD2 deficient cells to near (albeit not 100%) wild type levels. Interestingly, D2KR-Ub protein could be found constitutively in chromatin; however, post-MMC foci formation could not be detected. These phenotypes may resemble USP1 deficient cells, in which constitutive FANCD2 ubiquitination, loss of FANCD2 foci, and mild ICL sensitivity have been observed [57-60]. It is still unclear how the ubiquitin on FANCD2 mediates the chromatin localization, though it seems plausible that the mono-ubiquitinated FANCD2-FANCI complex has a higher affinity to damaged chromatin than unmodified form. Of note, it has been discovered that FANCD2-FANCI complex is SUMOylated upon DNA damage and then polyubiqutinated by SUMO-targeted ubiquitin ligase RNF4. Thus SUMOylation-deSUMOylation regulates amount of FANCD2-FANCI complex at DNA damage sites, and defects in this control result in higher sensitivity to DNA damage [61].
4. FANCI phosphorylation is a trigger to mono-ubiquitinate FANCD2 It has been reported that phosphorylation of FANCI at its S/TQ cluster domain (Figure 1B) is a molecular switch to trigger FANCD2 mono-ubiquitination, mostly based on the evidence that substitution of alanine at all six clustered S/TQ sites near the mono-ubiquitination residue (the non-phosphorylatable mutant Ax6) abrogates FANCD2 ubiquitination [11]. Using Phostag reagent, which retards migration of phosphorylated proteins in SDS-PAGE gels, they found that only a small fraction of FANCI is (perhaps heavily) phosphorylated after ICL damage [62,63], suggesting that FANCI may be rapidly de-phosphorylated following FANCD2 mono-ubiquitination.
Furthermore, the phosphomimetic mutant Dx6 (six aspartic acid replacements of the same residues as Ax6) induces constitutive mono-ubiquitination of FANCD2 in a chicken DT40 FANCI knockout cell line. Enhanced interaction between FANCD2 and FANCI by phosphorylation (using the Dx6 mutant) was not detected [11], and we also failed to detect an increase in FANCL co-immunoprecipitation with Dx6 FANCI compared to the WT FANCI (our unpublished data). On the other hand, one report maintained that phosphorylated FANCI dissociates from FANCD2 in Xenopus egg extracts [64]. However, a structural study has suggested that the phosphorylation in the FANCI S/TQ cluster may somehow enhance the FANCD2-FANCI interaction and hinder de-ubiquitination by USP1, since the mono-ubiquitination sites of FANCD2 and FANCI are buried in the FANCD2-FANCI interface and therefore inaccessible to USP1 [65]. This idea is fascinating, and suggests that FANCI phosphorylation is actually functioning to suppress USP1 de-ubiquitinase access rather than triggering activation of the E3 ligase FANCL. Interestingly, a reconstituted in vitro FANCD2 ubiquitination reaction using purified recombinant factors (these include an E3 ligase subunit FANCL protein but not the whole core complex) is greatly stimulated by addition of FANCI and DNA [66]. The shape of DNA (ssDNA or dsDNA, Y-shape, or Holliday junction etc.) seems not to really matter; however, unstructured ssDNA or chromatinized DNA is ineffective [67]. Similar observations have been made using the purified whole core complex from DT40 cells [68] or the recombinant core complex from reconstituted insect cells [69]. These approaches have begun to define the fine structure and stoichiometry of the core complex and the distinct role of each component [68-70]. FANCI DNA binding is important for efficient FANCD2 mono-ubiquitination to proceed [66,67]. This suggests that DNA binding by FANCI induces a conformational change of the complex to allow E3 ligase to access ubiquitination sites. Crystallographic analysis of the FANCD2-FANCI complex from mice showed it has DNA binding grooves that nicely fit to two single/double stranded DNA structures crosslinked by an ICL [65]. One can argue that the FANCD2-FANCI complex should probably be DNA-bound to be efficiently mono-ubiquitinated. Indeed, small amounts of non-ubiquitinated FANCD2 (carrying the ubiquitination site mutation K561R) can be found in chromatin upon DNA damage in a manner dependent on the C-terminal
portion of FANCD2 (termed the Tower domain) [71]. Consistent with these notions, FANCI Dx6 displayed weaker activity in vitro to stimulate FANCD2 ubiquitination than WT FANCI [66], indicating that persistent phosphorylation of FANCI may lead to a conformation less open to FANCL access. FANCD2 mono-ubiquitination has been shown to be regulated by ATR-ATRIP kinase [72]. ATR kinase is recruited to an RPA complex bound on exposed ssDNA via binding of ATRIP, a subunit of ATR, with RPA. It was observed that FANCI phosphorylation is carried out by ATR-ATRIP kinase in a manner dependent on the core complex and FANCD2, suggesting that FANCI is phosphorylated in the FANCD2-FANCI complex [52,53]. Furthermore, the core complex components are found to facilitate efficient localization of ATR-ATRIP in chromatin [62]. It was also shown that FANCI phosphorylation does not require the RAD9-RAD1-HUS1 (9-1-1) complex, the RAD17-RFC complex, or TopBP1[62,63]. TopBP1 is the stimulator of ATR activity, since it has an ATR activation domain (AAD), while the 9-1-1 complex is the recruiter of TopBP1, and the RAD17-RFC complex is the loader of the 9-1-1 complex to a junction between ssDNA and dsDNA. This mode of ATR activation in the FA pathway seems distinct and non-canonical, because, for example, Chk1 phosphorylation by ATR depends on these protein complexes. Notably, additional or alternative mechanisms that lead to ATR activation have
been
reported.
The
ATR
enzymatic
activity
can
be
enhanced
by
autophosphorylation on the ATR Thr 1989 residue that engages TopBP1 via its BRCT domains [73]. Recent reports further identified an ATR activation pathway parallel to TopBP1 that is governed by ETAA1 protein. ETAA1 is recruited by RPA and functions as an alternative ATR activator, as it carries another version of the AAD [74,75]. It would be of great interest to test whether these alternative pathways might have any role in FANCI phosphorylation. In addition to FANCI, other components of the FA pathway are also known to be phosphorylated following DNA damage. Human FANCD2 is phosphorylated on Ser222 by ATM kinase and functions in the S-phase checkpoint [42]. FANCM is hyperphosphorylated in response to DNA damage [76,77]. Still other reports indicate that FANCA [78,79] and FANCG [80,81] are phosphorylated in the FA pathway.
5. Non-phosphorylated FANCI can contribute to ICL repair via dormant origin firing Relatively recently Tony Huang's group published a very interesting paper on FANCI phosphorylation [50]. They reported that FANCI can promote dormant replication origin firing, which is inhibited by FANCI phosphorylation as well as the presence of FANCD2. The replicative helicase MCM2-7 complex is loaded onto DNA during the licensing process in a large excess over what is required for normal replication to commence. During replication stress (i.e., difficulty in replication due to various causes), the replisome progression is impeded, and then to overcome this stress, dormant (normally silent) origins fire and new replisomes are assembled and begin to replicate the genome [82]. This is referred to as dormant origin firing. They found that FANCI interacts with the MCM helicase complex, and there is a reduction in the number of active origins in FANCI-depleted cells during replication stress. On the other hand, FANCD2 suppresses firing of dormant origins, and therefore this role of FANCI is independent of the canonical FA pathway. Their detailed analysis indicated that FANCI is needed for MCM phosphorylation by replication-linked kinase DDK （ Dbf4 -dependent kinase) to promote firing. The FANCI Ax6 mutant (termed 6SA in their paper), but not Dx6 (termed 6SD), was able to rescue dormant origin firing in FANCI-depleted human RPE cells upon mild replication stress. Thus ATR-mediated FANCI phosphorylation mediates FANCD2 mono-ubiquitination but actually inhibits dormant origin firing. Unmodified FANCI promotes dormant origin firing but is insufficient to facilitate replication fork restart or DNA repair mediated by the FA pathway. Very strikingly, post-MMC cell survival is also rescued by the Ax6 mutant but not by Dx6 in FANCI-depleted human RPE cells, indicating that the dormant origin firing is the predominant mode used by the RPE cell line to attempt cell survival in the presence of MMC [50].
6. FANCI functions to accumulate the core complex in chromatin More recent work from the Taniguchi lab has provided another perspective on FANCI function. They demonstrated that FANCI is required for foci formation of the core complex components including FANCA, FANCG, FANCC and others [51]. The foci
formation of the core complex components was identified in some reports [56,83], but the foci are generally very difficult to be observed, possibly because of lower expression levels. Taniguchi’s group developed a novel method to detect MMC-induced foci formation of the core complex components, and reported that FANCI as well as BRCA1/FANCS, ATR, and USP1 are required for the core complex foci formation. Interestingly, this role of FANCI is dependent on neither FANCI phosphorylation nor ubiquitination. Rather, ubiquitinated FANCI loses this activity, and therefore, the de-ubiquitinase USP1 is critical for core complex focus formation. How this occurs is mechanistically unclear. Of note, however, the FANCM-FAAP24 complex has been implicated in localizing the core complex to damaged chromatin [84]. Therefore, whether FANCI regulates FANCM is an important issue. Another recent paper developed a method in DT40 cells to visualize FANCM recruitment to stalled replication forks, and showed that FANCM foci formation is independent of the FANCD2-FANCI complex [24]. Thus the role of FANCI in the core complex foci formation is likely downstream of FANCM (Figure 1A). Taniguchi’s group also tested a series of FANCI S/TQ cluster mutants including Ax6 or Dx6 expressed in human cells [51]. In keeping with the results in the chicken DT40 cell line, FANCD2 foci and mono-ubiquitination were suppressed in Ax6 non-phosphorylatable mutants, while the Dx6 mutant partially restored D2 foci and FANCI ubiquitination. A bit surprisingly, non-phosphorylatable FANCI Ax6 could partially rescue ICL sensitivity in human FANCI deficient cells (in both transformed FANCI fibroblasts and FANCI-knockout HCT116 colon cancer cells, to a similar extent), while FANCD2 foci and ubiquitination were deficient. The FANCI Ax6 mutants supported FANCA foci formation, suggesting that FANCI-promoted core complex foci formation is independent of FANCI phosphorylation or ubiquitination, and serves an important function in ICL repair. It is currently unclear how the core complex functions in a manner independent of FANCD2 mono-ubiquitination; however, it has been suggested that the core complex can have a FANCD2-independent function [56] and it could be related to REV1 regulation [85]. These results highlight the difference between chicken and human systems, or even the difference between lymphocytes and fibroblasts. This paper [51] further describes an interesting difference between the patient-derived FANCI deficient fibroblasts and a FANCI knockout in the
HCT116 colon cancer cell line, in terms of effects of FANCI on FANCA chromatin binding. The amount of FANCA in chromatin is affected in the former cell line but not in the latter.
7. Role of USP1 and de-ubiquitination in the FA pathway The de-ubiquitinase complex USP1-UAF1 [86,87] counteracts the mono-ubiquitination of FANCD2-FANCI. For example, rapid removal of USP1 by the Auxin degron system [88] instantly induces FANCD2-FANCI mono-ubiquitination without DNA damage stimulation (our unpublished observation). Upon DNA damage, USP1 is downregulated by transcriptional shut-off [86], perhaps to facilitate FANCD2 mono-ubiquitination. USP1 suppression could be considered as a potential measure to reverse the FA phenotype. However, USP1 is actually required for normal levels of ICL tolerance [57,58] and USP1 knockout mice displayed an FA-like phenotype [58]. For example, a USP1 knockout in a FANCC deficient background can reverse the loss of FANCD2 mono-ubiquitination without enhancing post-MMC cell survival [68]. These results may suggest that coupled ubiquitination and de-ubiquitination of FANCD2 (i.e., recycling between chromatin and nucleoplasm) [87] is also an integral component of the DNA repair process. However, de-ubiquitination of the FANCD2-FANCI complex by USP1-UAF1 occurs after removal of DNA in vitro, suggesting that de-ubiquitination is the last step following completion of DNA repair [69]. Alternatively, in the absence of USP1, the whole cellular pool of FANCD2 is mono-ubiquitinated, which may result in deregulated FANCD2 recruitment in cells (e.g., failed recruitment of FANCD2 to required damage sites). Indeed, USP1 is required for DNA damage-induced FANCD2 foci formation [58-60] and FANCI de-ubiquitinated by USP1 is needed for efficient foci formation of the core complex [51]. In addition, USP1 may have a signaling role in maintenance of Chk1 phosphorylation [59]. In conclusion, the precise mechanism and functional significance of FANCD2 de-ubiquitination in DNA repair remains unclear. Interestingly, USP1 is also the enzyme responsible for removing ubiquitin from mono-ubiquitinated PCNA, which promotes switching from replicative polymerases to translesion polymerases [89].
8. Perspective
We have summarized our current knowledge about the function and activation mechanisms of the key FA complex FANCD2-FANCI. FANCD2-FANCI is regulated by intricate protein modifications during cell cycle progression, DNA damage responses, and replication stress. In brief, we suggest the following story regarding how FANCD2-FANCI is regulated by protein modifications. The FANCD2-FANCI complex may change their conformation upon binding with DNA, which is more open to ubiquitination by the core complex E3 ligase that is antagonized by USP1. Then FANCI phosphorylation by ATR probably limits access of USP1 (but not the core complex), leading to mono-ubiquitinated FANCD2-FANCI complex with a higher affinity to damaged chromatin. How USP1 eventually removes a ubiquitin from the FANCD2-FANCI complex which might be in a closed conformation to USP1 access is still a question. It is an interesting possibility that the FANCD2-FANCI complex may be somehow evicted from the damage sites after completion of repair and the DNA-free complex may be in a conformation subjected to de-ubiquitination [69]. Insights into mechanistic details of these steps would be provided by future structural and biochemical studies. It is particularly striking that the phosphorylation site mutants of FANCI affect MMC sensitivity in a very distinct manner (Table 1). Normally these inconsistencies are explained by the use of different systems (in this case, a difference in species); however, cell type differences might be also at play. Perhaps the choice of dormant origin firing or DNA repair through the FA pathway is very different among cell types. Alternatively, some unknown (and possibly cell-type specific) mechanisms may await to be discovered. It should be borne in mind that the defective FA pathway impacts bone marrow stem cells most severely compared to the other systems in the typical patient’s body.
Table 1. Comparison between the phenotypes of the cells expressing FANCI mutants. Cell line
FANCI
Ishiai et al.[11]
Chen et al. [50]
Castella et al. [51]
Chicken DT40
FANCI-depleted
FANCI-deficient
human HCT116
FANCI knock out (B
human RPE cells
F010191 human
FANCI knockout
lymphoma cell
(retinal pigment
fibroblasts
(colon cancer
line)
epithelium)
cell line)
Ax6
Dx6
K525R
Ax6
Dx6
K523R
Ax6
Dx6
K523R
Ax6
Dx6
K523R
N
R
R
N
R
N
N
PR
N
N
PR
N
-
-
-
R
N
PR
-
-
-
-
-
-
N
R
PR
R
N
N
PR
PR
PR
PR
R
PR
mutant FANCD2 Ub/foci Dormant origin firing MMC sensitivity
N, not rescued PR, partially rescued R, rescued
-, not examined Ub, mono-ubiquitination K525R or K523R is the mono-ubiquitination site mutant of chicken or human FANCI, respectively.
Figure 1. A simplified schematic view of the FA pathway (A) and FANCD2-FANCI proteins (B). See text for details.
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A RPA-ssDNA
ATR-ATRIP
ICL
FANCM-FAAP24 FANCI The core complex
Subnuclear focus
UBE2T/FANCT
FANCI-P FANCD2-Ub
USP1-UAF1 Core Histone RAD51
B
SLX4-XPF
CtIP
K561 Ub 1451 aa
FANCD2 K523 Ub FANCI
1328 aa S/TQ cluster Figure 1
S0027-5107(17)30078-7 http://dx.doi.org/doi:10.1016/j.mrfmmm.2017.05.003 MUT 11594
To appear in:
Mutation Research
Received date: Revised date: Accepted date:
14-4-2017 4-5-2017 4-5-2017
Please cite this article as: Masamichi Ishiai, Koichi Sato, Junya Tomida, Hiroyuki Kitao, Hitoshi Kurumizaka, Minoru Takata, Activation of the FA pathway mediated by phosphorylation and ubiquitination, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesishttp://dx.doi.org/10.1016/j.mrfmmm.2017.05.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Special Section "Protein modifications in DNA damage repair and cancer" at Mutation Research
Activation of the FA pathway mediated by phosphorylation and ubiquitination Masamichi Ishiai1,6, Koichi Sato2,3,6, Junya Tomida1,4, Hiroyuki Kitao1,5 Hitoshi Kurumizaka2*, Minoru Takata1* 1
Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation
Biology Center, Kyoto University, Kyoto Japan 2
Laboratory of Structural Biology, Graduate School of Advanced Science and
Engineering, Waseda University, Tokyo, Japan 3
Present address: Hubrecht Institute–KNAW & University Medical Center Utrecht,
Utrecht, the Netherlands 4
Present address: Department of Epigenetics and Molecular Carcinogenesis, The
University of Texas MD Anderson Cancer Center, Smithville, TX 78957 5
Present address: Department of Molecular Cancer Biology, Graduate School of
Pharmaceutical Science, Kyushu University, Fukuoka, Japan 6
These authors contributed equally to this work.
* Correspondence should be addressed to H.K ([email protected]) or M.T. ([email protected])
Special Section "Protein modifications in DNA damage repair and cancer" at Mutation Research
Activation of the FA pathway mediated by phosphorylation and ubiquitination
Abstract Fanconi anemia (FA) is a devastating hereditary condition that impacts genome integrity, leading to clinical features such as skeletal and visceral organ malformations, attrition of bone marrow stem cells, and carcinogenesis. At least 21 proteins, when absent or defective, have been implicated in this disorder, and they together constitute the FA pathway, which functions in detection and repair of, and tolerance to, endogenous DNA damage. The damage primarily handled by the FA pathway has been assumed to be related to DNA interstrand crosslinks (ICLs). The FA pathway is activated upon ICL damage, and a hallmark of this activation is the mono-ubiquitination events of the key FANCD2-FANCI protein complex. Recent data have revealed unexpectedly complex details in the regulation of FA pathway activation by ICLs. In this short review, we summarize the knowledge accumulated over the years regarding how the FA pathway is activated via protein modifications.
1. Introduction Fanconi anemia (FA) is a devastating hereditary condition that compromises the genome integrity of small children, leading to clinical features such as skeletal and visceral organ malformations, attrition of bone marrow stem cells, and carcinogenesis. At least 21 proteins, when absent or defective, have been implicated in this disorder and they together constitute the FA pathway, which functions in detection and repair of, and tolerance to, endogenous DNA damage [1-4]. Sasaki et al. discovered that cells from FA patients display elevated numbers of chromosomal abnormalities upon mitomycin C treatment that induces interstrand crosslinks (ICLs) [5]. These include broken chromosomes and characteristic "radial" structures. Thus, the phenotypes in FA have been presumed to result from defects in ICL repair, and by extension the nature of the endogenous DNA damage handled by the FA pathway has been assumed to be related to ICLs. In hematopoietic stem cells, the endogenous DNA damage is likely induced by aldehyde metabolism (we consider this issue is outside the scope of this review)[2,6]. Alternatively, in FA patients, repeated activation of hematopoietic stem cells from dormancy due to stress such as infection [7], collision of replication and transcription machineries [8], or the inability to replicate large fragile genomic regions due to improper resolution of DNA:RNA hybrids [9] may all lead to DNA damage and contribute to the FA phenotype. The FA pathway consists of the upstream E3 ligase complex termed "the core FA complex" and the downstream FANCD2-FANCI complex (Figure 1A and B) [1-4]. Further
downstream
lies
the
structure-specific
nuclease
complex
SLX4/FANCP-XPF/FANCQ (this complex is thought to be responsible for incision of the ICL), the homologous recombination machineries including BRCA2/FANCD1 and RAD51/FANCR, and translesion synthesis (TLS) components such as REV7/FANCV. The core complex comprises eight FA proteins (i.e., FANCA-B-C-E-F-G-L-M) and other associated factors (FAAP100, FAAP24, FAAP20, MHF1 and MHF2). Although how the core complex senses the presence of DNA damage remains unclear, the detection events take place probably during S phase and can be related to stalled replication forks. The FANCM-FAAP24 sub-complex is required for loading of the rest of the core complex onto damaged chromatin, and the stalled replication fork itself or the action of FANCM translocase [10] exposes single-stranded DNA (ssDNA) that
accumulates the ssDNA binding RPA complex (Figure 1A). The checkpoint kinase ATR-ATRIP complex is recruited by RPA, and phosphorylates the S/TQ cluster residues of FANCI [11], which then leads to the triggering of FANCD2 mono-ubiquitination by the core complex, according to the previous understanding [11]. Mono-ubiquitination of FANCD2 is essential for assembly of the FANCD2 foci [12], chromatin localization of FANCD2 [13], and cell survival of DNA damage caused by ICL inducing agents. Thus, the mono-ubiquitination event of the key FANCD2-FANCI protein complex is considered as the hallmark of FA pathway activation [1-4]. FANCD2 in chromatin may provide a "landing pad" function that recruits other DNA repair proteins. Alternatively, it may have a function of its own. So far, no enzymatic motif has been identified; however, one report described a histone chaperone activity [14], which will be described below. In this short review, we will summarize the role of FANCD2 and FANCI in DNA repair as well as the activation mechanisms of the FANCD2-FANCI complex and the FA pathway, with a focus on protein modifications including phosphorylation, mono-ubiquitination, and de-ubiquitination.
2. FANCD2: the master coordinator of ICL repair FANCD2 is critical for ICL repair. In the absence of FANCD2 or FANCD2 mono-ubiquitination (due to the core complex defects), cells are highly sensitive to ICLs, accumulate higher levels of DNA damage markers, and arrest at G2 phase for a remarkably longer time than wild type cells [15]. A complete FANCD2 null mutation could be lethal in humans, since in all FANCD2 mutated cells derived from human FA patients, a small amount of residual FANCD2 protein has been invariably present [16]. Accumulated DNA damage or persistent DNA damage signaling drives the hematopoietic stem cells (HSCs) in FA patients and FA knockout mice to undergo cell death and senescence, leading to the HSC depletion and functional impairment [15]. So is there a single FANCD2 (and the FA pathway) function in cells? Perhaps not. Accumulating evidence suggests that several distinct functions in ICL repair and related functions are possible as summarized below. First, decreased homologous recombination (HR) repair of a chromosomal double strand break (DSB) induced by restriction enzyme I-SceI has been reported in
FA mutants generated in the chicken DT40 cell line [17,18] and in human FA cells [19]. Gene targeting efficiencies are also dramatically decreased [17]. The mechanisms of these defects are still not entirely clear but could be related to the FANCD2 function in end resection or in stabilizing RAD51 filaments (see below). On the other hand, levels of spontaneous sister chromatid exchange (SCE), which reflects the crossing over events during HR in the cell, are rather increased. The SCE levels are increased after MMC stimulation but only minimally compared to wild type cells [17]. This abnormal SCE phenotype is shared by various DT40 FA mutants [20,21] and mouse FANCM-deficient cells [22]. The increase in SCE levels may be explained by partial loss of BLM helicase activity, since the FA pathway and BLM can physically and functionally interact [21,23-25]. However, our understanding of these defects is still insufficient, and more study is needed. Second, FANCD2 can bind to core histones, and it can assemble nucleosomes in vitro (i.e., histone chaperone activity) [14]. Mobility of histone H3 in cells is reduced following MMC treatment in the absence of FANCD2. A patient-derived mutation in FANCD2 abolished the chaperone activity, and cells expressing this mutant are hypersensitive to cisplatin [14]. Thus, FANCD2 might participate in restoring chromatin during or after ICL repair. Third, FANCD2 has been shown to be critical for unhooking/incision of the ICL at both sides upon fork collision [26]. As pointed out above, the cutting activity itself depends on the SLX4-XPF complex, and FANCD2 and its ubiquitination are also needed for localizing SLX4-XPF at the ICL [27]. However, whether FANCD2 recruits SLX4-XPF through an interaction of its mono-ubiquitinated lysine with the ubiquitin-binding domains of SLX4 is a matter that is still unsettled [27-29]. Fourth, several groups have found that FANCD2 directly interacts and regulates the subnuclear localization of CtIP （for CtIP function, see an excellent review, such as [30]), which is important for ICL repair [31-33] and replication fork recovery [34]. CtIP recruited by FANCD2 promotes end resection upon unhooking of the ICL. The ssDNA generated by processing of the DSB is covered by RPA, which is replaced by RAD51/FANCR with help from mediators including BRCA2/FANCD1, RAD51C/FANCO and XRCC2/FANCU [35,36].
Fifth, FANCD2 has been suggested to work with key HR factor RAD51 as well as BRCA2 in protection of stalled replication forks from degradation mediated by MRE11 and DNA2 nucleases [37-40]. Perhaps related to this, it has been recently reported that the FANCD2-FANCI complex directly interacts with RAD51/FANCR and stabilizes
RAD51-DNA
filaments
[41].
Consistent
with
these
observations,
accumulation of RAD51 is mildly decreased by FANCD2 depletion. This reduction in RAD51 foci might also be influenced by the decreased recruitment of the end resection factor CtIP. Sixth, FANCD2 is phosphorylated by ATM kinase following ionizing radiation and participates in the S phase checkpoint that suppresses replication origin firing. This function has been observed with human [42] but not mouse FANCD2 [43]. It is unclear whether FANCI has a similar role, but FANCI appears to be involved in an ionizing radiation-induced G2/M DNA damage checkpoint [44]. To make the picture more complicated, FANCA is also required for the G2/M checkpoint in response to re-replication, together with ATR and BRCA1/FANCS [45]. Seventh, FANCD2 has been suggested to recruit factors like BLM[21,46], FAN1 [47,48], or DNA2 [49], which may further contribute to replication fork recovery and/or ICL repair. Eighth, it is becoming increasingly clear that FANCD2 and FANCI can have distinct functions. For example, FANCD2 can suppress dormant origin firing while FANCI promotes the process (discussed below) [50]. FANCI but not FANCD2 is important for the core complex to be recruited to chromatin (discussed below) [51]. A recent study revealed that FANCD2 deficient cells display higher sensitivity to replication stress induced by hydroxyurea treatment, but this function is independent of its mono-ubiquitination or FANCI [52]. The authors concluded that non-ubiquitinated FANCD2 together with RAD51 facilitates PCNA mono-ubiquitination and translesion synthesis. Collectively, all these distinct FANCD2-FANCI functions might coordinate the progression of DNA repair steps directed at stalled replication forks, ICLs, and related endogenous DNA damage.
3. FANCD2 mono-ubiquitination is key to its function for ICL repair
FANCD2 is mono-ubiquitinated in vivo in response to DNA damage or replication stress in a manner dependent on the core complex, ATR-ATRIP kinase, FANCI, and the E2 enzyme UBE2T/FANCT (Figure 1A) [1-4,53-55]. FANCD2 carrying a mutation of the mono-ubiquitination site lysine (K561 in human protein) can neither form foci nor relocalize to chromatin, and the exogenously expressed mutant protein cannot reverse the ICL sensitivity of FANCD2-deficient cells [12,42]. To examine the functional significance of FANCD2 mono-ubiquitination, chicken FANCD2 carrying a mutated mono-ubiqutination site was fused with ubiquitin (termed D2KR-Ub) [56]. Expression of D2KR-Ub was able to reverse the ICL sensitivity of FANCD2 deficient cells to near (albeit not 100%) wild type levels. Interestingly, D2KR-Ub protein could be found constitutively in chromatin; however, post-MMC foci formation could not be detected. These phenotypes may resemble USP1 deficient cells, in which constitutive FANCD2 ubiquitination, loss of FANCD2 foci, and mild ICL sensitivity have been observed [57-60]. It is still unclear how the ubiquitin on FANCD2 mediates the chromatin localization, though it seems plausible that the mono-ubiquitinated FANCD2-FANCI complex has a higher affinity to damaged chromatin than unmodified form. Of note, it has been discovered that FANCD2-FANCI complex is SUMOylated upon DNA damage and then polyubiqutinated by SUMO-targeted ubiquitin ligase RNF4. Thus SUMOylation-deSUMOylation regulates amount of FANCD2-FANCI complex at DNA damage sites, and defects in this control result in higher sensitivity to DNA damage [61].
4. FANCI phosphorylation is a trigger to mono-ubiquitinate FANCD2 It has been reported that phosphorylation of FANCI at its S/TQ cluster domain (Figure 1B) is a molecular switch to trigger FANCD2 mono-ubiquitination, mostly based on the evidence that substitution of alanine at all six clustered S/TQ sites near the mono-ubiquitination residue (the non-phosphorylatable mutant Ax6) abrogates FANCD2 ubiquitination [11]. Using Phostag reagent, which retards migration of phosphorylated proteins in SDS-PAGE gels, they found that only a small fraction of FANCI is (perhaps heavily) phosphorylated after ICL damage [62,63], suggesting that FANCI may be rapidly de-phosphorylated following FANCD2 mono-ubiquitination.
Furthermore, the phosphomimetic mutant Dx6 (six aspartic acid replacements of the same residues as Ax6) induces constitutive mono-ubiquitination of FANCD2 in a chicken DT40 FANCI knockout cell line. Enhanced interaction between FANCD2 and FANCI by phosphorylation (using the Dx6 mutant) was not detected [11], and we also failed to detect an increase in FANCL co-immunoprecipitation with Dx6 FANCI compared to the WT FANCI (our unpublished data). On the other hand, one report maintained that phosphorylated FANCI dissociates from FANCD2 in Xenopus egg extracts [64]. However, a structural study has suggested that the phosphorylation in the FANCI S/TQ cluster may somehow enhance the FANCD2-FANCI interaction and hinder de-ubiquitination by USP1, since the mono-ubiquitination sites of FANCD2 and FANCI are buried in the FANCD2-FANCI interface and therefore inaccessible to USP1 [65]. This idea is fascinating, and suggests that FANCI phosphorylation is actually functioning to suppress USP1 de-ubiquitinase access rather than triggering activation of the E3 ligase FANCL. Interestingly, a reconstituted in vitro FANCD2 ubiquitination reaction using purified recombinant factors (these include an E3 ligase subunit FANCL protein but not the whole core complex) is greatly stimulated by addition of FANCI and DNA [66]. The shape of DNA (ssDNA or dsDNA, Y-shape, or Holliday junction etc.) seems not to really matter; however, unstructured ssDNA or chromatinized DNA is ineffective [67]. Similar observations have been made using the purified whole core complex from DT40 cells [68] or the recombinant core complex from reconstituted insect cells [69]. These approaches have begun to define the fine structure and stoichiometry of the core complex and the distinct role of each component [68-70]. FANCI DNA binding is important for efficient FANCD2 mono-ubiquitination to proceed [66,67]. This suggests that DNA binding by FANCI induces a conformational change of the complex to allow E3 ligase to access ubiquitination sites. Crystallographic analysis of the FANCD2-FANCI complex from mice showed it has DNA binding grooves that nicely fit to two single/double stranded DNA structures crosslinked by an ICL [65]. One can argue that the FANCD2-FANCI complex should probably be DNA-bound to be efficiently mono-ubiquitinated. Indeed, small amounts of non-ubiquitinated FANCD2 (carrying the ubiquitination site mutation K561R) can be found in chromatin upon DNA damage in a manner dependent on the C-terminal
portion of FANCD2 (termed the Tower domain) [71]. Consistent with these notions, FANCI Dx6 displayed weaker activity in vitro to stimulate FANCD2 ubiquitination than WT FANCI [66], indicating that persistent phosphorylation of FANCI may lead to a conformation less open to FANCL access. FANCD2 mono-ubiquitination has been shown to be regulated by ATR-ATRIP kinase [72]. ATR kinase is recruited to an RPA complex bound on exposed ssDNA via binding of ATRIP, a subunit of ATR, with RPA. It was observed that FANCI phosphorylation is carried out by ATR-ATRIP kinase in a manner dependent on the core complex and FANCD2, suggesting that FANCI is phosphorylated in the FANCD2-FANCI complex [52,53]. Furthermore, the core complex components are found to facilitate efficient localization of ATR-ATRIP in chromatin [62]. It was also shown that FANCI phosphorylation does not require the RAD9-RAD1-HUS1 (9-1-1) complex, the RAD17-RFC complex, or TopBP1[62,63]. TopBP1 is the stimulator of ATR activity, since it has an ATR activation domain (AAD), while the 9-1-1 complex is the recruiter of TopBP1, and the RAD17-RFC complex is the loader of the 9-1-1 complex to a junction between ssDNA and dsDNA. This mode of ATR activation in the FA pathway seems distinct and non-canonical, because, for example, Chk1 phosphorylation by ATR depends on these protein complexes. Notably, additional or alternative mechanisms that lead to ATR activation have
been
reported.
The
ATR
enzymatic
activity
can
be
enhanced
by
autophosphorylation on the ATR Thr 1989 residue that engages TopBP1 via its BRCT domains [73]. Recent reports further identified an ATR activation pathway parallel to TopBP1 that is governed by ETAA1 protein. ETAA1 is recruited by RPA and functions as an alternative ATR activator, as it carries another version of the AAD [74,75]. It would be of great interest to test whether these alternative pathways might have any role in FANCI phosphorylation. In addition to FANCI, other components of the FA pathway are also known to be phosphorylated following DNA damage. Human FANCD2 is phosphorylated on Ser222 by ATM kinase and functions in the S-phase checkpoint [42]. FANCM is hyperphosphorylated in response to DNA damage [76,77]. Still other reports indicate that FANCA [78,79] and FANCG [80,81] are phosphorylated in the FA pathway.
5. Non-phosphorylated FANCI can contribute to ICL repair via dormant origin firing Relatively recently Tony Huang's group published a very interesting paper on FANCI phosphorylation [50]. They reported that FANCI can promote dormant replication origin firing, which is inhibited by FANCI phosphorylation as well as the presence of FANCD2. The replicative helicase MCM2-7 complex is loaded onto DNA during the licensing process in a large excess over what is required for normal replication to commence. During replication stress (i.e., difficulty in replication due to various causes), the replisome progression is impeded, and then to overcome this stress, dormant (normally silent) origins fire and new replisomes are assembled and begin to replicate the genome [82]. This is referred to as dormant origin firing. They found that FANCI interacts with the MCM helicase complex, and there is a reduction in the number of active origins in FANCI-depleted cells during replication stress. On the other hand, FANCD2 suppresses firing of dormant origins, and therefore this role of FANCI is independent of the canonical FA pathway. Their detailed analysis indicated that FANCI is needed for MCM phosphorylation by replication-linked kinase DDK （ Dbf4 -dependent kinase) to promote firing. The FANCI Ax6 mutant (termed 6SA in their paper), but not Dx6 (termed 6SD), was able to rescue dormant origin firing in FANCI-depleted human RPE cells upon mild replication stress. Thus ATR-mediated FANCI phosphorylation mediates FANCD2 mono-ubiquitination but actually inhibits dormant origin firing. Unmodified FANCI promotes dormant origin firing but is insufficient to facilitate replication fork restart or DNA repair mediated by the FA pathway. Very strikingly, post-MMC cell survival is also rescued by the Ax6 mutant but not by Dx6 in FANCI-depleted human RPE cells, indicating that the dormant origin firing is the predominant mode used by the RPE cell line to attempt cell survival in the presence of MMC [50].
6. FANCI functions to accumulate the core complex in chromatin More recent work from the Taniguchi lab has provided another perspective on FANCI function. They demonstrated that FANCI is required for foci formation of the core complex components including FANCA, FANCG, FANCC and others [51]. The foci
formation of the core complex components was identified in some reports [56,83], but the foci are generally very difficult to be observed, possibly because of lower expression levels. Taniguchi’s group developed a novel method to detect MMC-induced foci formation of the core complex components, and reported that FANCI as well as BRCA1/FANCS, ATR, and USP1 are required for the core complex foci formation. Interestingly, this role of FANCI is dependent on neither FANCI phosphorylation nor ubiquitination. Rather, ubiquitinated FANCI loses this activity, and therefore, the de-ubiquitinase USP1 is critical for core complex focus formation. How this occurs is mechanistically unclear. Of note, however, the FANCM-FAAP24 complex has been implicated in localizing the core complex to damaged chromatin [84]. Therefore, whether FANCI regulates FANCM is an important issue. Another recent paper developed a method in DT40 cells to visualize FANCM recruitment to stalled replication forks, and showed that FANCM foci formation is independent of the FANCD2-FANCI complex [24]. Thus the role of FANCI in the core complex foci formation is likely downstream of FANCM (Figure 1A). Taniguchi’s group also tested a series of FANCI S/TQ cluster mutants including Ax6 or Dx6 expressed in human cells [51]. In keeping with the results in the chicken DT40 cell line, FANCD2 foci and mono-ubiquitination were suppressed in Ax6 non-phosphorylatable mutants, while the Dx6 mutant partially restored D2 foci and FANCI ubiquitination. A bit surprisingly, non-phosphorylatable FANCI Ax6 could partially rescue ICL sensitivity in human FANCI deficient cells (in both transformed FANCI fibroblasts and FANCI-knockout HCT116 colon cancer cells, to a similar extent), while FANCD2 foci and ubiquitination were deficient. The FANCI Ax6 mutants supported FANCA foci formation, suggesting that FANCI-promoted core complex foci formation is independent of FANCI phosphorylation or ubiquitination, and serves an important function in ICL repair. It is currently unclear how the core complex functions in a manner independent of FANCD2 mono-ubiquitination; however, it has been suggested that the core complex can have a FANCD2-independent function [56] and it could be related to REV1 regulation [85]. These results highlight the difference between chicken and human systems, or even the difference between lymphocytes and fibroblasts. This paper [51] further describes an interesting difference between the patient-derived FANCI deficient fibroblasts and a FANCI knockout in the
HCT116 colon cancer cell line, in terms of effects of FANCI on FANCA chromatin binding. The amount of FANCA in chromatin is affected in the former cell line but not in the latter.
7. Role of USP1 and de-ubiquitination in the FA pathway The de-ubiquitinase complex USP1-UAF1 [86,87] counteracts the mono-ubiquitination of FANCD2-FANCI. For example, rapid removal of USP1 by the Auxin degron system [88] instantly induces FANCD2-FANCI mono-ubiquitination without DNA damage stimulation (our unpublished observation). Upon DNA damage, USP1 is downregulated by transcriptional shut-off [86], perhaps to facilitate FANCD2 mono-ubiquitination. USP1 suppression could be considered as a potential measure to reverse the FA phenotype. However, USP1 is actually required for normal levels of ICL tolerance [57,58] and USP1 knockout mice displayed an FA-like phenotype [58]. For example, a USP1 knockout in a FANCC deficient background can reverse the loss of FANCD2 mono-ubiquitination without enhancing post-MMC cell survival [68]. These results may suggest that coupled ubiquitination and de-ubiquitination of FANCD2 (i.e., recycling between chromatin and nucleoplasm) [87] is also an integral component of the DNA repair process. However, de-ubiquitination of the FANCD2-FANCI complex by USP1-UAF1 occurs after removal of DNA in vitro, suggesting that de-ubiquitination is the last step following completion of DNA repair [69]. Alternatively, in the absence of USP1, the whole cellular pool of FANCD2 is mono-ubiquitinated, which may result in deregulated FANCD2 recruitment in cells (e.g., failed recruitment of FANCD2 to required damage sites). Indeed, USP1 is required for DNA damage-induced FANCD2 foci formation [58-60] and FANCI de-ubiquitinated by USP1 is needed for efficient foci formation of the core complex [51]. In addition, USP1 may have a signaling role in maintenance of Chk1 phosphorylation [59]. In conclusion, the precise mechanism and functional significance of FANCD2 de-ubiquitination in DNA repair remains unclear. Interestingly, USP1 is also the enzyme responsible for removing ubiquitin from mono-ubiquitinated PCNA, which promotes switching from replicative polymerases to translesion polymerases [89].
8. Perspective
We have summarized our current knowledge about the function and activation mechanisms of the key FA complex FANCD2-FANCI. FANCD2-FANCI is regulated by intricate protein modifications during cell cycle progression, DNA damage responses, and replication stress. In brief, we suggest the following story regarding how FANCD2-FANCI is regulated by protein modifications. The FANCD2-FANCI complex may change their conformation upon binding with DNA, which is more open to ubiquitination by the core complex E3 ligase that is antagonized by USP1. Then FANCI phosphorylation by ATR probably limits access of USP1 (but not the core complex), leading to mono-ubiquitinated FANCD2-FANCI complex with a higher affinity to damaged chromatin. How USP1 eventually removes a ubiquitin from the FANCD2-FANCI complex which might be in a closed conformation to USP1 access is still a question. It is an interesting possibility that the FANCD2-FANCI complex may be somehow evicted from the damage sites after completion of repair and the DNA-free complex may be in a conformation subjected to de-ubiquitination [69]. Insights into mechanistic details of these steps would be provided by future structural and biochemical studies. It is particularly striking that the phosphorylation site mutants of FANCI affect MMC sensitivity in a very distinct manner (Table 1). Normally these inconsistencies are explained by the use of different systems (in this case, a difference in species); however, cell type differences might be also at play. Perhaps the choice of dormant origin firing or DNA repair through the FA pathway is very different among cell types. Alternatively, some unknown (and possibly cell-type specific) mechanisms may await to be discovered. It should be borne in mind that the defective FA pathway impacts bone marrow stem cells most severely compared to the other systems in the typical patient’s body.
Table 1. Comparison between the phenotypes of the cells expressing FANCI mutants. Cell line
FANCI
Ishiai et al.[11]
Chen et al. [50]
Castella et al. [51]
Chicken DT40
FANCI-depleted
FANCI-deficient
human HCT116
FANCI knock out (B
human RPE cells
F010191 human
FANCI knockout
lymphoma cell
(retinal pigment
fibroblasts
(colon cancer
line)
epithelium)
cell line)
Ax6
Dx6
K525R
Ax6
Dx6
K523R
Ax6
Dx6
K523R
Ax6
Dx6
K523R
N
R
R
N
R
N
N
PR
N
N
PR
N
-
-
-
R
N
PR
-
-
-
-
-
-
N
R
PR
R
N
N
PR
PR
PR
PR
R
PR
mutant FANCD2 Ub/foci Dormant origin firing MMC sensitivity
N, not rescued PR, partially rescued R, rescued
-, not examined Ub, mono-ubiquitination K525R or K523R is the mono-ubiquitination site mutant of chicken or human FANCI, respectively.
Figure 1. A simplified schematic view of the FA pathway (A) and FANCD2-FANCI proteins (B). See text for details.
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A RPA-ssDNA
ATR-ATRIP
ICL
FANCM-FAAP24 FANCI The core complex
Subnuclear focus
UBE2T/FANCT
FANCI-P FANCD2-Ub
USP1-UAF1 Core Histone RAD51
B
SLX4-XPF
CtIP
K561 Ub 1451 aa
FANCD2 K523 Ub FANCI
1328 aa S/TQ cluster Figure 1