Ol HeLa cells (6.9 ?10-7), indicating that SETD2/H3K36me3 depletion causes a mutator phenotype. When precisely the same analysis was performed in hMSH6deficient shSETD2-DLD-1 and manage DLDL-1 cells, the mutation frequency basically remained unchanged (Figure 5B). This result suggests that SETD2 depletion only alters the mutation frequency in hMSH6-competent cells, that is consistent with the notion that H3K36me3 recruits hMutS. Interestingly, the mutation frequency in SETD2/H3K36me3depleted HeLa cells is 10-fold decrease than that in hMSH6-deficient DLD-1 cells, that is likely because of both the efficiency of SETD2 depletion plus the huge distinction inside the totalNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCell. Author manuscript; readily available in PMC 2014 April 25.Li et al.Pagepassage number between these two cells. Also, H3K36me3 might not be the only mechanism for hMSH6 recruitment.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptTo figure out if SETD2/H3K36me3 modulates the enzymatic function(s) of MMR proteins, the in vitro MMR activity of SETD2/H3K36me3-depleted HeLa cells was examined. Consistent with our model (see below), these cells aren’t defective in MMR in vitro (Figure 5C). This observation indicates that SETD2 and H3K36me3 are certainly not physically involved in MMR, and that depletion of SETD2/H3K36me3 will not alter the expression or function of MMR genes. Cancer cells deficient in SETD2 show MSI and fail to recruit hMutS to chromatin Recent studies identified SETD2 as a tumor suppressor for clear cell renal cell carcinoma (ccRCC) (Dalgliesh et al.Buy913642-78-1 , 2010; Duns et al., 2010; Gerlinger et al.Buy31420-52-7 , 2012; Varela et al.PMID:25105126 , 2011), but the mechanism linking SETD2 deficiency to ccRCC tumorigenesis remains unknown. We hypothesize that defective MMR may perhaps contribute to tumorigenesis in SETD2deficient ccRCC patients. To test this hypothesis, we screened many ccRCC cell lines for SETD2 mutations and identified a SETD2-deficient ccRCC cell line, UOK143, which carries A5197 G and T5306 C mutations, top to N1734D and S1769P amino acid substitutions in SETD2, respectively (Figure S4A). Western blot analysis shows that UOK143 cells express undetectable amounts of SETD2, that is readily detected within the SETD2-proficient ccRCC cell line, UOK121 (Figure 6A). As expected, the volume of H3K36me3 is substantially decrease in UOK143 cells than in UOK121 cells (Figure 6A). We then analyzed the distribution of hMSH6 and H3K36me3 in these ccRCC cells. H3K36me3 was barely detectable in UOK143 cells by immunofluorescence, but was somewhat much more abundant in UOK121 cells (Figure 6B). Correspondingly, in S-phase UOK121 cells, hMSH6 foci had been abundant and partially ( 70 ) colocalized with H3K36me3, whilst considerably fewer and smaller hMSH6 foci had been observed in S-phase UOK143 cells (Figures 6B and 6C), which can be comparable to what was observed in SETD2/H3K36me3-depleted HeLa (Figure 4B) and DLD-1 (Figure 3E) cells. As noted previously, these outcomes suggest that H3K36me3 facilitates localization of hMSH6 (hMutS) to chromatin. To identify if the failure to recruit hMutS confers an MMR-deficient phenotype to UOK143 cells, we examined MSI in UOK143 and UOK121 cells. The results revealed no MSI in UOK121 cells and that UOK143 cells exhibited mono- and di-nucleotide repeat instability, as all subclones, except clone 1, exhibit either new repeat species or deletion of a microsatellite marker (Figure 6D). To rule out the possibility tha.
