| Abstract |
Inborn mutations in genes of the Nucleotide Excision Repair (NER) pathway are known to be associated with syndromes showing clinical features with severe developmental abnormalities. However, little is known about the functional role of NER factors in mammalian development. Using a new knock-in mouse model expressing the NER structure-specific endonuclease XPF fused with a FTA-tag that gets specifically biotinylated by the bacterial ligase BirA, we performed pull-down experiments followed by mass spectrometry (MS) to isolate and characterize NER-associated protein complexes during postnatal murine development. We identified 140 XPF-bound core proteins that were common between three replicate experiments. By analyzing the MS data and verifying the interactions by co-immunoprecipitation, we were able to characterize a new protein complex that consists of ERCC1-XPF, CTCF, SMC1A, SMC3 and MBD2. Ablation of Ercc1 or exposure to DNA-damage agents causing intra-stand crosslinks (ICLs) triggers the localization of CTCF to heterochromatin regions in the nucleus and the dissociation of CTCF, SMC1A, SMC3 and ATRX from promoters and imprinting control regions (ICRs) of imprinted genes. In line with these findings, we also observe altered histone post-translational modification (PTMs) marks and the recruitment of RNAPII and basal transcription factors on promoters of imprinted genes. As a result Ercc1 depleted mice show aberrant developmental expression of a subset of imprinted genes without the DNA methylation status of their ICRs being affected. This response is cell-autonomous and requires signaling by ATM. We propose that ERCC1-XPF binds CTCF, SMC1A, SMC3 and MBD2 to form a complex required for the developmental silencing of imprinted genes and that persistent DNA damage signaling triggers chromatin changes that affect gene expression programs associated with NER developmental disorders.
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The role of NER factors in development and the physiology of the mouse
