CDCA7 and HELLS suppress DNA:RNA hybrid-associated DNA damage at pericentromeric repeats

Abstract

Immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disorder that is caused by mutations in either DNMT3B, ZBTB24, CDCA7, HELLS, or yet unidentified gene(s). Previously, we reported that the CDCA7/HELLS chromatin remodeling complex facilitates non-homologous end-joining. Here, we show that the same complex is required for the accumulation of proteins on nascent DNA, including the DNMT1/UHRF1 maintenance DNA methylation complex as well as proteins involved in the resolution or prevention of R-loops composed of DNA:RNA hybrids and ssDNA. Consistent with the hypomethylation state of pericentromeric repeats, the transcription and formation of aberrant DNA:RNA hybrids at the repeats were increased in ICF mutant cells. Furthermore, the ectopic expression of RNASEH1 reduced the accumulation of DNA damage at a broad range of genomic regions including pericentromeric repeats in these cells. Hence, we propose that hypomethylation due to inefficient DNMT1/UHRF1 recruitment at pericentromeric repeats by defects in the CDCA7/HELLS complex could induce pericentromeric instability, which may explain a part of the molecular pathogenesis of ICF syndrome.

Chromatin remodeling in replication‐uncoupled maintenance DNA methylation and chromosome stability: Insights from ICF syndrome studies

“…Therefore, we asked whether the DNA hypomethylation of pericentromeric repeats in ZBTB24 KO, CDCA7 KO, and HELLS KO cells causes chromosome instability via similar process. Consistent with the hypomethylation state of the repeats, the transcription and formation of aberrant DNA:RNA hybrids at the repeats were significantly increased in ZBTB24 KO, CDCA7 KO, and HELLS KO cells (Unoki et al., 2019 , 2020 ). Since it is reported that DNA:RNA hybrids and R‐loops are poor substrates for DNMT1 (Ross et al., 2010 ), this may accelerate loss of methylation at the repeats in these KO cells.…”

“…Second, we identified the proteins that are less accumulated on newly synthesized DNA strands in the absence of CDCA7 by isolation of proteins on nascent DNA (iPOND)‐MS/MS analysis using asynchronized wild‐type and CDCA7 KO HEK293 cells. We found that the accumulation of DNMT1 and UHRF1 on newly synthesized strands was reduced by approximately 50% and 40%, respectively, in CDCA7 KO cells (Unoki et al., 2020 ), suggesting that the CDCA7/HELLS complex is required for access of the DNMT1/UHRF1 complex to approximately half of newly synthesized strands. Considering that the CDCA7/HELLS complex is plausibly required for replication‐uncoupled maintenance DNA methylation (Figure 1b ), this result is reasonable.…”

“…This can cause DNA damage, possibly by collisions between the R-loops and DNA replication forks. As DNA damage associated with R-loop formation is preferentially repaired by transcription-associated HR, and some ICF types have defects in NHEJ as a backup system of HR, the centromeric instability represented by multiradial chromosomes in ICF cells (see Figure 2) could result from unresolved Holliday junctions generated by aberrant HR between pericentromeric repeats in different chromosomes UNOKI (DDX21) and SPT16 homolog facilitates chromatin remodeling subunit (SUPT16H), the latter of which forms the FACT heterodimeric chaperone with structure-specific recognition protein 1 (SSRP1), interact with CDCA7, and are less accumulated on newly synthesized DNA strands in the absence of CDCA7 (Unoki et al, 2019(Unoki et al, , 2020. As DDX21 is involved in R-loop resolution, and the FACT complex prevents Rloop formation by organizing chromatin structure (Herrera-Moyano et al, 2014;Song et al, 2017), we hypothesized that these proteins recruit the CDCA7/HELLS complex to near Rloops in a feed-forward amplification manner.…”

“…Our recent IP‐MS/MS and iPOND‐MS/MS analyses (described above) revealed that DExD‐Box Helicase 21 (DDX21) and SPT16 homolog facilitates chromatin remodeling subunit (SUPT16H), the latter of which forms the FACT heterodimeric chaperone with structure‐specific recognition protein 1 (SSRP1), interact with CDCA7, and are less accumulated on newly synthesized DNA strands in the absence of CDCA7 (Unoki et al., 2019 , 2020 ). As DDX21 is involved in R‐loop resolution, and the FACT complex prevents R‐loop formation by organizing chromatin structure (Herrera‐Moyano et al., 2014 ; Song et al., 2017 ), we hypothesized that these proteins recruit the CDCA7/HELLS complex to near R‐loops in a feed‐forward amplification manner.…”

“…Cell division cycle associated 7 (CDCA7) and helicase lymphoid specific (HELLS), which compose a chromatin remodeling complex possessing nucleosome‐sliding activity (Jenness et al., 2018 ), are two of such factors. Because both proteins are mutated in a subset of patients with immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome (Thijssen et al., 2015 ), who exhibit centromeric/pericentromeric DNA hypomethylation and chromosome instability, the insights from recent studies regarding these proteins highlight the importance of chromatin remodeling in DNA methylation at centromeric/pericentromeric regions for chromosome stability (Unoki et al., 2019 , 2020 ). In this review article, the latest knowledge on maintenance DNA methylation is summarized, and the possible roles of ICF‐syndrome‐related proteins in maintenance DNA methylation and chromosome stability are discussed.…”