Feng Li

Replication stress (RS) is a major driver of genomic instability and a hallmark of cancer cells. Although dynamic heterochromatin remodeling has been implicated in RS response, the precise mechanisms remain unclear. The CHAMP1 complex, composed of CHAMP1, POGZ, HP1α, and the H3K9 methyltransferase SETDB1, is known to regulate heterochromatin assembly at multiple genomic sites. Interestingly, upon RS, the CHAMP1 complex is transiently recruited to stalled replication forks, where it facilitates H3K9me3 deposition and establishes a repressive chromatin environment. The complex is required for stabilization of replication forks, and it shields forks from MRE11-mediated degradation. The complex also reduces RS at specific chromosomal sites, such as the heterochromatin-rich telomeric sites in tumor cells which use the ALT pathway of telomere maintenance. Loss of the CHAMP1 complex results in increased micronuclei formation and heightened sensitivity to RS. Loss of the complex also leads to a compensatory increase in other pathways which reduce RS, such as the FA pathway and the ATR/CHK1 pathway. Notably, CHAMP1 deficiency induces synthetic lethality with FANCM inhibition in ALT-positive tumor cells, and the CHAMP1 complex is essential for the survival of CCNE1-amplified ovarian cancers. These findings uncover a heterochromatin-based mechanism of replication fork stabilization and suggest that CHAMP1 may represent a candidate therapeutic vulnerability in cancers with elevated RS.

Replication stress is a major driver of genomic instability and a hallmark of cancer cells. Although dynamic heterochromatin remodeling has been implicated in replication stress response, the precise mechanisms remain unclear. Here, we identify the CHAMP1 complex, composed of CHAMP1, POGZ, HP1α, and the H3K9 methyltransferase SETDB1, as a critical regulator of heterochromatin assembly at stalled replication forks. Upon replication stress, the CHAMP1 complex is recruited to stalled forks where it facilitates H3K9me3 deposition, creating a repressive chromatin environment that shields replication forks from MRE11-mediated degradation. The complex promotes the recruitment of the origin recognition complex (ORC) to sites of replication stress, such as the telomeric heterochromatin in alternative lengthening of telomeres (ALT)-positive tumor cells, thereby supporting efficient telomeric DNA replication. Loss of CHAMP1 disrupts ORC2 recruitment and impairs fork restart, leading to increased micronuclei formation and heightened sensitivity to replication stress. Notably, CHAMP1 deficiency induces synthetic lethality with FANCM inhibition in ALT-positive tumor cells, and the CHAMP1 complex is essential for the survival of CCNE1-amplified ovarian cancers. These findings uncover a chromatin-based mechanism of replication fork stabilization and suggest that CHAMP1 may represent a candidate therapeutic vulnerability in cancers with elevated replication stress.

The CHAMP1 complex, a little-known but highly conserved protein complex consisting of CHAMP1, POGZ, and HP1α, is enriched in heterochromatin though its cellular function in these regions of the genome remain unknown. Here we show that the CHAMP complex promotes heterochromatin assembly at multiple chromosomal sites, including centromeres and telomeres, and promotes homology-directed repair (HDR) of DNA double strand breaks (DSBs) in these regions. The CHAMP1 complex is also required for heterochromatin assembly and DSB repair in highly-specialized chromosomal regions, such as the highly-compacted telomeres of ALT (Alternative Lengthening of Telomeres) positive tumor cells. Moreover, the CHAMP1 complex binds and recruits the writer methyltransferase SETDB1 to heterochromatin regions of the genome and is required for efficient DSB repair at these sites. Importantly, peripheral blood lymphocytes from individuals with CHAMP1 syndrome, an inherited neurologic disorder resulting from heterozygous mutations in CHAMP1, also exhibit defective heterochromatin clustering and defective repair of DSBs, suggesting that a defect in DNA repair underlies this syndrome. Taken together, the CHAMP1 complex has a specific role in heterochromatin assembly and the enhancement of HDR in heterochromatin.

The CHAMP1 complex, a little-known but highly conserved protein complex consisting of CHAMP1, POGZ, and HP1α, is enriched in heterochromatin though its cellular function in these regions of the genome remain unknown. Here we show that the CHAMP complex promotes heterochromatin assembly at multiple chromosomal sites, including centromeres and telomeres, and promotes homology-directed repair (HDR) of DNA double strand breaks (DSBs) in these regions. The CHAMP1 complex is also required for heterochromatin assembly and DSB repair in highly-specialized chromosomal regions, such as the highly-compacted telomeres of ALT (Alternative Lengthening of Telomeres) positive tumor cells. Moreover, the CHAMP1 complex binds and recruits the writer methyltransferase SETDB1 to heterochromatin regions of the genome and is required for efficient DSB repair at these sites. Importantly, peripheral blood lymphocytes from individuals with CHAMP1 syndrome, an inherited neurologic disorder resulting from heterozygous mutations in CHAMP1 , also exhibit defective heterochromatin clustering and defective repair of local DSBs, suggesting that a defect in DNA repair underlies this syndrome. Taken together, the CHAMP1 complex has a novel role in heterochromatin assembly and the enhancement of HDR in heterochromatin.

Genome editing tools based on CRISPR–Cas systems can repair genetic mutations in situ; however, off-target effects and DNA damage lesions that result from genome editing remain major roadblocks to its full clinical implementation. Protein and chemical inhibitors of CRISPR–Cas systems may reduce off-target effects and DNA damage. Here we describe the identification of several lead chemical inhibitors that could specifically inhibit the activity of Streptococcus pyogenes Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 in cellulo. Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 in cellulo at low-micromolar concentration. Furthermore, microscale thermophoresis (MST) assays showed that SP24 might inhibit SpCas9 activity by interacting with both the SpCas9 protein and the SpCas9–gRNA ribonucleoprotein complex. Taken together, SP24 is a novel chemical inhibitor of SpCas9 which has the potential to enhance therapies that utilize SpCas9.

A critical determinant of DNA repair pathway choice is the HORMA protein REV7, a small abundant adaptor which binds to various DNA repair proteins through its C-terminal seatbelt domain. The REV7 seatbelt binds to the REV3 polymerase to form the Polymerase ζ complex, a positive regulator of translesion synthesis (TLS) repair. Alternatively, the REV7 seatbelt binds to SHLD3 in the Shieldin complex, a positive regulator of NHEJ repair. Recent studies have identified another novel REV7 seatbelt-binding protein, CHAMP1 (Chromosome Alignment-Maintaining Phosphoprotein, though its role in DNA repair is unknown. Here, we show that the REV7-CHAMP1 complex promotes homologous recombination (HR) repair by sequestering REV7 from the Shieldin complex. CHAMP1 competes directly with the SHLD3 subunit of the Shieldin complex for a limited pool of C-REV7, thereby inhibiting the REV7-mediated recruitment of the SHLD2 and SHLD1 effector subunits to DNA double strand breaks. CHAMP1 thereby channels DNA repair away from error-prone NHEJ and towards the competing error-free HR pathway. Similarly, CHAMP1 competes with the REV3 component of the POLζ complex, thereby reducing the level of mutagenic TLS repair. CHAMP1 interacts with POGZ in a heterochromatin complex further promoting HR repair. Importantly, in human tumors, CHAMP1 overexpression promotes HR, confers PARP inhibitor resistance, and correlates with poor prognosis. Thus, by binding to either REV3, SHLD3, or CHAMP1 through its seatbelt, the REV7 protein can promote either TLS repair, NHEJ repair, or HR repair respectively.

Pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs), ESCs derived by somatic cell nuclear transfer (ntESCs), and induced pluripotent stem cells (iPSCs) have unlimited capacity for self-renewal and pluripotency and can give rise to all types of somatic cells. In order to maintain their self-renewal and pluripotency, PSCs need to preserve their telomere length and homeostasis. In recent years, increasing studies have shown that telomere reprogramming is essential for stem cell pluripotency maintenance and its induced pluripotency process. Telomere-associated proteins are not only required for telomere maintenance in both stem cells, their extra-telomeric functions have also been found to be critical as well. Here, we will discuss how telomeres and telomere-associated factors participate and regulate the maintenance of stem cell pluripotency.

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