The Role of CHAMP1 in Chromatin-Mediated DNA Damage Repair

Abstract

Defects in the replication stress response are major drivers of cancer development and present key targetable vulnerabilities that can be exploited for anti-cancer therapy. Recent studies have identified CHAMP1 as a novel DNA damage repair factor with roles in double-strand break repair and the replication stress response. Mutations in CHAMP1 are associated with the neurodevelopmental disorder CHAMP1 Syndrome. More recently, children with CHAMP1 Syndrome have developed leukemia, suggesting that CHAMP1 mutations are a potential cancer risk factor. CHAMP1 is part of two DNA damage repair complexes: CHAMP1-POGZ-REV7 (Complex I) and CHAMP1-POGZ-HP1α (Complex II). Complex I promotes homologous recombination by removing the Shieldin complex from the ends of double strand breaks and allowing DSB end resection to occur. Complex II enriches heterochromatin content through the recruitment of the methyltransferase SETDB1 to DNA damage sites. Increased heterochromatin at stalled forks is associated with proper fork stability and restart, demonstrating the importance of CHAMP1 in maintaining genomic integrity. Loss of CHAMP1 leads to increased sensitivity to DNA damaging agents and increased dependence on other DNA damage repair pathways, such as the DNA damage checkpoint and the Fanconi Anemia pathway. CHAMP1 is overexpressed in breast and ovarian cancer cells with high levels of replication stress, providing a molecular mechanism for the tolerance of replication stress. These new findings on the relationship of CHAMP1 with well-established DNA damage repair pathways, suggest that targeting CHAMP1 could present a new synthetic lethality opportunity for cancer cells with high levels of replication stress.

Part 1. INTRODUCTION

The replication stress response (RSR) is a critical DNA damage response (DDR) pathway that is essential for genome stability. Mutations in the RSR can lead to an increased risk of cancer development, e.g. mutations in the Fanconi Anemia (FA) genes, BRCA1 and BRCA2, are established risk factors for breast and ovarian cancer development (reviewed in [1, 2]). Cancer specific synthetic lethality therapies, such as PARP inhibitor (PARPi) treatment for BRCA1/s-mutated breast and ovarian cancers, have shown strong clinical efficacy by targeting defects in the DDR [3]. However, upregulation of the DNA damage response can cause cancer cell resistance to therapeutic drugs [4]. Therefore, improving our understanding of the RS response and the DNA damage response will help us identify new cancer risks and design more effective cancer specific therapies in the future.

CHAMP1 (Chromosome alignment phosphoprotein 1)–also known as C13orf8 and ZNF828–was first discovered for its role in chromosome alignment through a screen identifying proteins that interact with REV7 [5] (Figure 1). The CHAMP1 protein consists of multiple zinc-finger domains on both the N- and C-termini (N-ZNF and C-ZNF respectively), as well as additional conserved repeat motifs, including WK motif (SPxxWKxxP), SPE motif (consensus: PxxSPExxK), and FPE motif (FPExxK) (Figure 2A) [5, 6]. The C-ZNF and FPE domains are required for CHAMP1 localization to spindle fibers and centromeres [5]. Crystal structures show that the REV7 interaction with CHAMP1 is mediated through the WK motifs on CHAMP1 [5, 6]. However, the CHAMP1-REV7 interaction does not seem to be required for CHAMP1 localization to chromatin [6]. Loss or mutation of CHAMP1 results in chromosome misalignment, likely due to defective localization of kinetochore-associated proteins CENP-E and CENP-F [5]. The kinase motor CENP-E and its non-motor relative CENP-F are key to connecting unattached kinetochores to centromeres through microtubules for chromosome alignment along the spindle equator [7, 8]. De novo heterozygous mutations in CHAMP1 cause CHAMP1 Syndrome, a rare neurodevelopmental disorder. Recent studies have identified CHAMP1 protein as a DDR factor with important role in HR, heterochromatin assembly, and the RSR. CHAMP1 functions in two distinct DNA damage repair complexes: CHAMP1-POGZ-REV7 (Complex I) and CHAMP1-POGZ-HP1α (Complex II) (Figure 2B–C) [9–11].

Figure 1. Model of the known functions of CHAMP1.

CHAMP1 has multiple biological functions which include neuron development, muscle cell development, chromosome alignment, promoting heterochromatin formation, and promoting DSB repair through homologous recombination. Mutations in CHAMP1 are associated with CHAMP1 Syndrome and muscle development. CHAMP1 is found elevated in breast and ovarian cancer cells with high levels of CCNE1 expression.

Figure 2. CHAMP1 is a DNA damage repair factor.

a) Model of the known functional domains of CHAMP1. CHAMP1 contains N-terminal and C-terminal zinc-fingers: N-ZNFs and C-ZNFs. The SPF and FPE motifs are essential for chromosome alignment. The WK motif binds to REV7.

b) Model of CHAMP1-POGZ-REV7. The “seatbelt” loop on the C-terminal end of REV7 binds to the WK motif on CHAMP1.

c) Model of CHAMP1-POGZ-HP1α and SETDB1 bound to H3K9me3. POGZ binds to the N-terminal end of CHAMP1 and HP1α binds to the C-terminal end of CHAMP1. The C-terminal end of HP1α bind to the HPZ domain of POGZ. HP1α forms a dimer through its chromoshadowdomain and the chromodomain of HP1α binds to tri-methylated lysine 9 on histone H3 (H3K9me3).

CHAMP1 is Part of Two DDR Complexes

Recent work has shown that CHAMP1 is part of two DNA damage repair complexes with roles in DSB repair and RS. CHAMP1-POGZ-REV7 (Complex I) (Figure 2B) has been shown to promote repair of DSBs through HR by removing REV7 from the Shieldin complex from the ends of DSBs [10]. REV7 is the key partner of CHAMP1 in Complex I. While POGZ immunoprecipitates (IP) with REV7 [11, 13], the CHAMP1-POGZ interaction is not required for the CHAMP1-REV7 interaction [11]. REV7–also known as MAD2L2, MAD2B, or FANCV–is an evolutionarily conserved protein first identified in S. cerevisiae [14]. REV7 is a member of the HORMA family of proteins and contains a “seatbelt” motif that mediates protein-protein binding interactions (reviewed in [15]). REV7 is an abundant cellular protein with many known binding partners, including REV3, as part of the translesion polymerase Pol ζ in the FA pathway [2, 16], and SHLD3 of the Shieldin complex–discussed further below [17].

Subsequent work on CHAMP1 in DNA damage repair demonstrated that CHAMP1 forms a second complex with CHAMP1-POGZ-HP1α (Complex II) (Figure 2C). This complex enriches heterochromatin at DSB sites and at stalled replication forks [9, 11]. Key components of Complex II are POGZ and HP1α (Figure 2C). POGZ (Pogo Transposable Element Derived with ZNF Domain), like CHAMP1, is an autism risk gene that has been shown to regulate chromatin accessibility in mice [18]. Mutations in POGZ are linked to the neurodevelopmental disease known as White Sutton Syndrome which has similar phenotypes to CHAMP1 Syndrome [19–21]. Heterochromatin protein 1 (HP1) is an evolutionarily conserved transcriptional repressor that binds to methylated lysine 9 residues on histone H3 (H3K9me) (reviewed in [22]). There are three HP1 isoforms found in humans: HP1α, HP1β, and HP1γ [22]. HP1 consists of two primary domains: a chromo domain (CD) that binds to H3K9me and a chromo shadow domain (CSD) that facilitates HP1 dimerization [22]. CHAMP1 and POGZ were enriched at DSBs caused by IR and laser micro-irradiated induced DSBs [10, 23]. In addition, all three HP1 isomers were found to colocalize with POGZ [23].

There is evidence that CHAMP1-POGZ-REV7 (Complex I) and CHAMP1-POGZ-HP1α (Complex II) might exist as distinct complexes. REV7, POGZ, and HP1α were all detected when CHAMP1 was immunoprecipitated [11]. However, HP1α was not detected by Western blot when REV7 was pulled down, and REV7 was not detected when GFP-HP1α was pulled down [11, 13]. While it is possible that Complex I and Complex II might be mutually exclusive, it is also possible that the indirect interaction of REV7 with HP1α through CHAMP1 is not preserved in the IP. When 293T cells were exposed to ionizing radiation (IR), the CHAMP1-HP1α interaction was increased, even for CHAMP1 mutants incapable of binding with REV7 [11]. However, although both CHAMP1 complexes appear to function as separate complexes, both are likely to coexist in the cell, since HP1α and REV7 were detected when CHAMP1 was immunoprecipitated from whole cell lysates [11].

Part 2. CHAMP1-POGZ-REV7 (Complex I) Promotes DSB Repair Through Homologous Recombination

CHAMP1 promotes DSB repair through HR by removing the Shieldin complex from the ends of DSBs and exposing the ends of DSBs to resection machinery (Figure 3) [10, 24]. In DSB repair, resection is a key determining factor in repair pathway choice (reviewed in [25]). The Shieldin complex represses end resection at DSBs by protecting the ends of DSBs from resection machinery such as EXO1 and Sgs1/DNA2 [26, 27]. Knockdown of CHAMP1 by siRNA treatment in U2OS cells showed a significant reduction in ssDNA as measured by the Single Molecule Analysis of Resection Tracks (SMART) assay [10, 28]. In addition, loading of RAD51 onto ssDNA was significantly reduced in CHAMP1 knockout cells [10, 29], further indicating a decline in HR activity. Loss of POGZ significantly reduced the number of RAD51 and BRCA1 foci at DSBs in U2OS cells [23]. The CHAMP1-REV7 interaction is mediated by the WK motif of CHAMP1 and appears to be transient within the DDR. One hour after exposing U2OS cells to IR, there is an increase in the percentage of cells with CHAMP1-POGZ-REV7 colocalization, up to 80%, followed by a sharp decrease in CHAMP1-REV7 2–4 h after IR exposure [10]. These findings further support a model in which CHAMP1 facilitates HR by increasing access of resection machinery to the ends of DSBs through the removal REV7 from the Shieldin complex.

Figure 3. CHAMP1-REV7 promotes resection at stalled replication forks.

The Shieldin complex (SHLD1, SHLD2, REV7, and SHLD3) binds to the ends of DSBs to protect them from resection. CHAMP1-POGZ removes REV7 from the Shieldin complex to form the CHAMP1-REV7 complex (Figure 2B). Removal of the Shieldin complex allows for resection machinery to access the ends of the DSB and facilitate end resection.

Part 3. CHAMP1-POGZ-HP1α (Complex II) Promotes Heterochromatin Assembly at DNA Damage Sites

Complex II increases heterochromatin–marked by trimethylation of histone H3 (H3K9me3)–at DNA damage sites in highly specialized areas such as stalled replication forks, centromeres, and DSBs at telomeres (Figure 4A–B) [11]. This increase in H3K9me3 at stalled forks is important for fork stability and restart [9, 30]. Heterochromatin enrichment at DNA damage sites through Complex II was first studied at DSBs caused by IR and at DSBs made at telomeres using the TRF1-FokI system in U2OS cells [11] and was later shown to also occur at stalled replication forks in RPE^p53−/− cells [9]. Complex II specifically recruits the H3K9 methyltransferase SETDB1 to damage sites for heterochromatin enrichment at DSBs and stalled replication forks [9, 11]. This was shown to be independent of Complex I, as a CHAMP1 mutant that does not bind to REV7 (CHAMP1–2A) was sufficient for H3K9me3 enrichment at DSBs [11].

Figure 4. CHAMP1-POGZ-HP1α promotes H3K9me3 at DNA damage sites.

A) At double strand breaks CHAMP1-POGZ-HP1α recruits SETDB1 to increase local H3K9me3 content at the double strand break. This correlates with RAD51 foci formation at double strand breaks.

B) SETDB1, SUV39h1, and G9a (EHMT2) increase H3K9me3 at stalled replication forks to promote fork stability and fork restart. SETDB1 activity at stalled replication forks is dependent on recruitment through CHAMP1-POGZ-HP1α. CHAMP1 activity protects stalled replication forks from degradation by MRE11 and promotes RAD51 loading at stalled forks.

In addition to SETDB1, it has been shown that the H3 methyltransferases G9a (EHMT2) and SUV39h1 increase H3K9me3 at stalled forks as part of the RSR (Figure 4B) [30]. Deletion or drug inhibition of G9a or SUV39h1 prevented damage-dependent increases in H3K9me3 at stalled forks [30]. PICh (Proteomics of Isolated Chromatin Segments) analysis–an IP mass spectrometry technique [31]–revealed that SETDB1 is enriched at telomeric DSBs in a CHAMP1 and POGZ dependent manner [11]. In contrast, G9a and SUV39h1 were not enriched at the telomere probes following TRF1-FokI induction [11], suggesting that SETDB1 might be the primary methyltransferase that responds to either DSBs or telomere specific damage. In addition, RAD51 foci formation at TRF1-FokI induced DSBs at telomeres and stalled replication forks was significantly reduced in CHAMP1 knockout, N-terminal deletion, C-terminal deletion and 2A mutants [9, 11]. H3K9me3 enrichment at stalled forks is required for RAD51 loading, fork stability, and fork restart [9, 30], but it is currently unknown through what molecular mechanism this occurs through.

The Complex II dependent enrichment of H3K9me3 at stalled forks prevented MRE11-dependent fork degradation. Inhibition of SETDB1, G9a, or SUV39h1 caused stalled forks to become unstable, resulting in fork degradation [9, 30]. While loss of CHAMP1 did not affect MRE11 recruitment to stalled forks, pharmacological inhibition of MRE11 nuclease activity with mirin, was indeed able to rescue the fork degradation defect manifested by CHAMP1 KO [9, 32]. Increased MRE11-dependent fork degradation in CHAMP1 mutants may be caused partly by impaired RAD51 recruitment to stalled forks [33].

Part 4. Dynamic Regulation of H3K9me3 at Stalled Forks Affects Fork Stability and Restart

Recruitment of Complex II and H3K9me3 enrichment at stalled replication forks affects the recruitment of DNA damage repair proteins to stalled forks. While recruitment of BRCA1 to DSBs is dependent on POGZ, the POGZ-366–848 mutant protein, which is still capable of binding to HP1 proteins, was sufficient in restoring BRCA1 recruitment to DSBs [23]. It is unknown how the H3K9me3 content at DSBs or formation of Complex II is affected in the POGZ-366–848 mutant. This mutant may be capable of recruiting SETDB1 since the POGZ-SETDB1 interaction is mediated by the centromere binding B-DB and DDE-1/Transposase domains located downstream of the HP1 binding site on POGZ [34]. If SETDB1 recruitment to DNA damage sites is impaired in the POGZ-366–848 mutant, H3K9me3 enrichment at stalled forks could still be facilitated by the other H3K9me3 methyltransferases. The importance of H3K9me3 at stalled forks for protein recruitment was shown in an iPOND (isolation of proteins on nascent DNA) study done in murine cells. When G9a was inhibited, there was less FANCD2, ATRX/DAXX, RAD51C/D, and BRCA1 at stalled forks [30], suggesting that H3K9me3 is a requirement for the recruitment of these DDR factors to DNA damage sites.

However, the recruitment of some proteins was found to be increased at stalled forks when G9a was inhibited. The Primase and DNA Directed Polymerase (PRIMPOL) is a translesion polymerase that can facilitate replication restart at stalled forks, but leaves ssDNA gaps behind the fork (reviewed in [35]). PRIMPOL levels were found to be increased at stalled forks when G9a was inhibited [30]. In addition, ssDNA gaps were increased when G9a was inhibited, suggesting that H3K9me3 at stalled forks blocks PRIMPOL access and that PRIMPOL and G9a act separately in the RSR. Since PRIMPOL inhibition enhanced the defect in fork restart in G9a inhibited cells [30], repression of PRIMPOL might also sensitize CHAMP1 mutants to replication stress and present a potential synthetic lethality interaction.

The H3K9 demethylases Jumonji domain-containing protein 1A (JMJD1A)/Lysine (K)-Specific Demethylase 3A (KDM3a) was found to be enriched at stalled forks when G9a was inhibited. Demethylation of H3K9me3 is carried out by the KDM3 and KDM4 family of demethylases and mutations in these proteins is associated with greater risk of cancer development (reviewed in [36]). KDM3a was shown to be recruited to stalled replication forks in a G9a independent manner [30]. siRNA knockdown of KDM3a restored damage-dependent H3K9me3 enrichment at stalled forks and fork stability when G9a was inhibited [30]. However, fork restart was impaired when KDM3a was inhibited [30]. It is unknown how loss of KDM3a affects protein recruitment to stalled forks. Mass spectrometry to identify changes in protein recruitment to stalled forks ± KDM3 or KDM4 will provide insights into how demethylation of H3K9 fits into the RSR. Interestingly, inhibition of KDM3a conferred resistance to PARPi treatment and replication stress in G9a inhibited cells [30]. Improving our understanding of how precise control of H3K9 methylation affects sensitivity of cells to cancer therapies such as PARPi treatment will increase the efficacy of these treatments through combined drug therapies.

PART 5. The Relationship Between the CHAMP1 Pathway and the Replication Stress Response

CHAMP1 appears to play a key role in the DDR to DSBs and in the tolerance of replication stress [9]. While originally identified as a DDR factor through its interaction with the FA protein REV7, the CHAMP1 pathway and the FA pathway appear to have parallel functions in the RSR. In U2OS CHAMP1 depleted cells, PICh data indicated that FA pathway proteins and ATR/CHK1 pathway proteins were enriched at telomeres [9, 11]. We hypothesize that the increased recruitment of the FA proteins to telomeres under replication stress may compensate for the loss of the CHAMP1 pathway and therefore loss of heterochromatin assembly. Furthermore, in RPE^p53−/− cells, depletion of CHAMP1 results in a dependence on enzymatic activity of FANCM suggesting that CHAMP1 and FANCM might function in separate pathways and that loss of one pathway results in an increased dependence on the other pathway [9]. In summary, CHAMP1 levels or CHAMP1 pathway activity may be elevated in FA pathway mutant cells, suggesting that there is an inverse relationship between the FA pathway and the CHAMP1 pathway for cell survival

The replication checkpoint is activated in response to replication stress and stalled replication forks (reviewed in [37]). ATM and ATR are key kinases that are activated in response to the replication stress response (reviewed in [38]). CHAMP1 knockout cells exhibit elevated phosphorylation of CHK1 and KAP1 [9], suggesting increased activation of DNA damage checkpoint signaling [9]. Since fork stability and fork restart are both impaired in CHAMP1 deficient cells, checkpoint signaling might be increased in these cells due to an impaired ability to resolve replication stress. The increased activation of the DNA damage checkpoint in CHAMP1 knockout cells suggests that the cells will have increased sensitivity to ATM or ATR inhibitors.

PART 6. CHAMP1 Clinical Implications and Cancer Risk

Pathogenic de novo heterozygous mutations in CHAMP1 result in a rare neurodevelopmental disorder known as CHAMP1 syndrome, which is characterized by intellectual disability and severe developmental delay. While pleiotropic in nature, CHAMP1 syndrome patients can exhibit additional clinical manifestations including hypotonia, facial dysmorphisms, and various gastrointestinal abnormalities [39]. Large-scale genome sequencing studies identified CHAMP1, along with POGZ, as novel genetic determinants of developmental disorders, including autism spectrum disorder [40–42]. While generally associated with developmental delay, recent work has shown that CHAMP1 is involved in DNA repair and that its deregulation can lead to genomic instability. A 2021 study reported that CHAMP1 syndrome may cause acute myeloid leukemia (AML), suggesting an increased risk of developing cancer, perhaps due to impaired DNA repair processes [12]. Notably, CHAMP1 patient-derived primary lymphocytes exhibit increased sensitivity to replication stress inducing drugs, including IR, PARPi, and cisplatin, which is consistent with their elevated baseline level of replication stress [43–46]. Similar to the CHAMP1 knockout, inhibition of the methyltrasnferases SETDB1, G9a, and SUV39h1 all showed increased sensitivity to PARPi treatment [9, 30]. On the other hand, the upregulation of CHAMP1 expression confers a resistance to PARPi therapy. Notably, CHAMP1 is found to be highly overexpressed in ovarian and breast cancers that exhibit high levels of replication stress due to CCNE1 amplification [9, 47]. We hypothesize that these cancer cells select for higher levels of CHAMP1 which can thereby compensate for their elevated replication stress. Accordingly, siRNA-mediated knockdown of CHAMP1 impaired CCNE1-amplified cell viability [9].

Similar to CHAMP1 syndrome, de novo heterozygous mutations in POGZ also lead to a rare neurodevelopmental disorder known as White-Sutton Syndrome (WSS). WSS, characterized by autism spectrum disorder, intellectual disability, and developmental delay, exhibits similar clinical phenotypes to CHAMP1 syndrome. That said, POGZ has also been implicated in HR-mediated repair and heterochromatin assembly at DNA damage sites through its interactions with CHAMP1, REV7, and HP1α [11, 23]. Concurrently, POGZ has been implicated in several cancer types. For example, POGZ was identified as a potential prognostic biomarker in osteosarcoma, and knockdown of POGZ results in a decrease in cellular growth [48]. POGZ has also been linked to triple-negative breast cancer (TNBC) as a driver of tumor progression [49]. Although the long-term cancer risk associated with CHAMP1 syndrome and WSS remain unclear, the emerging role of CHAMP1 and POGZ in the DDR and replication stress management, highlights the possibility for increased cancer susceptibility. Altogether, this also suggests that the CHAMP1 complexes and their subsequent pathway components may serve as potential targets for anti-cancer therapy.

PART 7. Summary and Conclusion

CHAMP1 is a newly-identified DNA damage repair factor involved in DSB repair and the RSR. By binding to REV7 and antagonizing the Shieldin complex, CHAMP1 can enhance DSB end resection and increase homologous recombination repair activity. Through interactions with POGZ, HP1a, and SETDB1, CHAMP1 can increase H3K9me3 levels and heterochromatin assembly at certain chromosomal loci and thereby alleviate replication stress. CHAMP1-REV7 may also have a role in RAD51 loading at stalled replication forks, since it has been shown to promote RAD51 loading during DSB repair. Understanding the relationship between these two CHAMP1 functions may provide further insight into how CHAMP1 regulates DNA damage repair.

CHAMP1 is a potential target for future cancer therapies. Knockdown of CHAMP1 in breast cancer cells with high levels of replication stress–which have increased expression of CHAMP1–resulted in an even stronger enhancement of replication stress and an enhancement of cell killing. Furthermore, primary lymphocytes derived from patients with CHAMP1 Syndrome (de novo heterozygous mutations of CHAMP1) exhibited elevated replication stress and increased sensitivity to hydroxyurea and PARPi, Overexpression of CHAMP1 confers a resistance to PARPi. This suggests that CHAMP1 expression might help predict a patient’s response to PARPi. Additionally, inhibitors of the CHAMP1/POGZ/HP1a/SETDB1 pathway may increase drug efficacy when combined with PARPi. Further work is required in order to characterize the relationship between the CHAMP1 pathway and the FA pathway in DNA damage repair. Future insights into these relationships could uncover potential synthetic lethality between CHAMP1 and FA proteins offering promising therapeutic opportunities for cancer treatment.

Highlights.

• CHAMP1 is a DNA damage repair factor involved in double strand break repair and replication stress

• CHAMP1 is part of 2 DNA damage repair complexes: CHAMP1-POGZ-REV7 (Complex I) and CHAMP1-POGZ-HP1α (Complex II)

• Loss of CHAMP1 causes increased sensitivity to inhibition of the Fanconi Anemia pathway and the DNA damage checkpoint

• CHAMP1 is overexpressed in some breast and ovarian cancer cells with high levels of replication stress

Data Availability

No data was used for the research described in the article.