While signs of DNA damage can accumulate in cell lines derived from ICF4 patients 25, key questions remain: what is the nature of the genomic instability in the ICF4 syndrome and by which molecular mechanism does LSH protect the genome? In this study, we investigated how genomic instability arises in LSH-deficient cells.
LSH has been implicated in DNA repair processes in response to ionizing radiation 24 and in promoting the efficiency of non-homologous end joining 25.Īn important hallmark of the ICF syndrome is genome instability with chromosomal aberrations in activated lymphocytes 12, 26. LSH is also known to influence DNA methylation level and to represses transcription of diverse repeat sequences, including ribosomal genes and retrotransposons 15, 22, 23. LSH is so far the only known factor involved in genome-wide incorporation, but it is currently unknown how LSH interacts with either variant 17, 20. A few factors have been found to remove macroH2A from its genomic location, such as ATRX which reduces macroH2A occupancy at telomeres, or FACT which removes macroH2A from transcriptionally active regions 20. Both variants confer nucleosome stability and promote chromatin compaction 18, 19, 21. The two histone variants macroH2A1 and macroH2A2 are encoded by two different genes and their products show a similar distribution pattern in the genome 18, 19, 20. LSH is an ATP-dependent remodeler that contributes to genome-wide incorporation of macroH2A1 and macroH2A2 17. ICF4 patients suffer from a myriad of symptoms, which are in part phenocopied by Lsh knockout mice, including immunodeficiency, neurologic defects, and reduced growth 12, 13, 14, 15, 16. Immunodeficiency, centromeric instability, facial anomalies (ICF) 4 is a severe human disease caused by mutations of the LSH/HELLS gene 12. Though it is hypothesized that chromatin compaction or relaxation modulates the accessibility of appropriate repair factors at stalled replication forks 1, 11, it remains to be determined what specific features of the chromatin environment are favorable for their assembly and how this prevents the degradation of nascent DNA. Some DNA repair promoting factors bind directly to DNA, while others interact with specific histone modifications 11. The pathological degradation of replication intermediates in the absence of BRCA1/2 proteins or of RAD51 leads to increased genomic instability. Under replication stress conditions which lead to fork stalling, BRCA1/2 proteins facilitate RAD51 filament formation which in turn prevents the degradation of newly synthesized DNA strands by nucleases 6, 7, 10. In the context of homology-directed DNA repair BRCA1 is counterbalanced by the repair factor 53BP1, and both factors may occupy sites of DNA damage in a mutually exclusive relationship 1, 9. Unresolved fork stalling, fork collapse, and degradation may lead to the generation of double-stranded DNA breaks (DSBs) and an increase in chromosomal aberrations 5.Ī multitude of proteins, including BRCA1, BRCA2, and RAD51, is required for replication fork protection as well as homology-directed repair of DSBs 5, 6, 7, 8, 9. Reversed forks can be restarted but also undergo resection by nucleases 6. Upon stalling, the replication machinery can dissociate, and fork reversal can occur 6. These loci share some features, such as the formation of secondary DNA structures, tightly bound protein complexes, packaging into heterochromatin, or increased risk of DNA polymerase slippage, and pose challenges to the replisome which can lead to replication stalling 4, 5. Regions in the human genome that are inherently difficult to replicate, include chromosome fragile sites, telomeres, and other loci with repeat sequences 2. Replication forks can encounter numerous obstacles that impede the replication process. Altogether, our results illuminate the mechanism underlying a human syndrome and reveal a critical role of LSH mediated chromatin remodeling in genomic stability.įaithful replication of DNA is critical for genome integrity 1, 2, 3. This is associated with perturbed histone modifications, including abnormal H4K20 methylation that is critical for BRCA1 enrichment and 53BP1 exclusion. The defect in RAD51 loading is linked to a disbalance of BRCA1 and 53BP1 accumulation at stalled forks. LSH or macroH2A deficiency leads to an impairment of RAD51 loading, a factor that prevents MRE11 and EXO1 mediated nascent DNA degradation. The protection of stalled forks is mediated by macroH2A, whose knockdown mimics LSH depletion and whose overexpression rescues nascent DNA degradation. Here, we demonstrate that LSH depletion results in degradation of nascent DNA at stalled replication forks and the generation of genomic instability. The Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is caused by mutations in LSH/HELLS, a chromatin remodeler promoting incorporation of histone variant macroH2A.
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