For this we used as being responsible for the observed survival (Fig

For this we used as being responsible for the observed survival (Fig.?1c). the level Lacosamide of DNA repair pathway choice. Our data are most consistent with an important role for H3K36me2 in more downstream steps of the DNA repair process. Moreover, Lacosamide we find additional H3K36me2-specific HMTs to contribute to NHEJ at deprotected telomeres, further emphasizing the importance of H3K36me2 in DNA repair. is deleted in human Wolf-Hirschhorn syndrome and dysregulated in multiple myeloma patients with a t(4;14) translocation, in which the translocation-dependent overexpression of MMSET drives oncogenic transformation [20C25]. Moreover, mRNA and protein levels are increased in multiple cancers [26, 27]. Interestingly, MMSET has been implicated in the repair of DNA lesions caused by various DNA-damaging sources [28C30]. Here, we describe a novel role for MMSET in controlling DNA repair at telomeres. We find that MMSET promotes Ligase4-dependent c-NHEJ at uncapped telomeres and thereby genomic instability, in a manner directly correlating with its ability to catalyze H3K36-dimethylation (H3K36me2). Since upstream control of NHEJ by ATM-signaling and 53BP1-mediated inhibition of DNA end-resection were unaffected by MMSET depletion, we hypothesize that MMSET, through catalyzing H3K36me2, affects the engagement or activity of factors acting downstream in NHEJ. Furthermore, we identified additional H3K36-methyltransferases that contribute to telomere-NHEJ. Altogether, this suggests an important role for H3K36me2 in the processing of dysfunctional telomeres. Results MMSET regulates telomere dysfunction-induced genomic instability To better understand how modification of chromatin affects recognition and processing of uncapped telomeres we set out to identify histone modifying enzymes that contribute to telomere-induced genomic instability. For this we used as being responsible for the observed survival (Fig.?1c). Multiple independent shRNAs targeting rescued telomere dysfunction-induced lethality to an extent correlating with MMSET levels (Fig.?1d, Supplementary Fig.?1A). Indeed, cells depleted of MMSET continued proliferating despite telomere uncapping (Fig.?1e). Moreover, complementation of MMSET-depleted cells with expression of full-length MMSET cDNA abolished the rescue of cell proliferation in conditions of telomere uncapping (Fig.?1e, Supplementary Fig.?1B, C), showing that this effect is specific for MMSET. Importantly, knockdown did not affect cell cycle distribution (Supplementary Fig.?1D, E), Tmem1 excluding disturbed cell cycle kinetics as potential factor in escape from genomic crisis. Together, these results identify MMSET as a novel regulator of telomere dysfunction-induced genomic instability. Open in a separate window Fig. 1 MMSET identified as a novel telomere-induced genomic instability regulator.a Experimental setup of the survival screen shown in (b). After infection with the retroviral shRNA-pools, cells were grown at the nonpermissive temperature (39?C) to induce telomere uncapping for 12 days and returned to 32?C for 14 days prior to staining with crystal violet. b Relative survival of TRF2ts MEFs infected with the indicated shRNA target gene pools (showed significantly reduced telomere fusion Lacosamide (Fig.?2d, e, Supplementary Fig.?2A). Telomeres terminate in G-rich 3 single-stranded DNA (ssDNA) overhangs that are lost during NHEJ-mediated ligation [15, 34]. In line with their reduction in chromosome fusions, MMSET-depleted cells retained telomeric G-overhangs after 48?h of telomere uncapping (Fig.?2f, g). Moreover, aneuploidy caused by missegregation of chromosomes that fused upon telomere uncapping, was partially alleviated in cells with reduced or inhibition (Supplementary Lacosamide Fig.?2B, C). Open in a separate window Fig. 2 MMSET induces NHEJ-mediated telomere fusion and G-overhang degradation. a Chromosome fusions in TRF2ts MEFs and LigIV?/? TRF2ts MEFs transduced with control or test: ns, not significant; *test: **test: *knockdown TRF2ts MEFs after 48?h at the nonpermissive temperature (37?C). h Chromosome fusions in TRF1F/FTRF2F/FKu70?/?p53?/? MEFs treated with DMSO or PARPi (Olaparib, 0.5?M), or transduced with control virus or shRNA targeting test: ns, not significant; **inhibition, suggesting that MMSET does not contribute to Ligase4-independent alt-NHEJ (Fig.?2a, c). To further address this, we used TRF1F/F;TRF2F/F;Ku70?/?;p53?/?;Cre-ERT2.