In support of a critical role for the BRCA1 complex in chromosome end protection and in agreement with previous reports28, 29, we show that inhibition of BRCA1, RAP80 (also known as UIMC1) or BRCC3 results in partial loss of end protection (Supplementary Fig. 16). The identification of BRCC3 as a critical factor involved in TRF2-dependent telomere protection suggests that an important physiological function of the BRCA1 complex is to maintain genomic stability by aiding telomere-associated proteins in maintaining telomere integrity.
Mice and MEFs
Rosa26-CreERT2 mice (Jackson Laboratories) and mice carrying a conditional TRF2 construct1 were crossed to generate MEFs. MEFs were immortalized with pBabeSV40LT and treated 4-hydroxytamoxifen (0.6 μM) to induce Cre-mediated recombination.
Constructs, plasmids and viral infections
TRFc constructs were generated by PCR amplification using as templates pBabe-MYC-TRF1 or pBabe-MYC-TRF2 constructs (primers used are listed in Supplementary Table 2). pLDT-GFP-RNF8, pLDT-GFP-RNF168 and GFP-MDC1 were a gift from M. D. Weitzman. pOZ-FH-BRCC3 was obtained from Addgene. pcDNA-MYC-MRE11 and pcDNA-MYC-NBS1 were a gift from X. Wu.
Immunofluorescence, FISH and ChIP
Immunofluorescence, fluorescence in situ hybridization and chromatin immunoprecipitation (ChIP) experiments were performed as described previously2.