Figure 8.

Histone variant macroH2A1.2 and KDM5A promote transcriptional repression at DNA breaks. (A) Schematic of FokI-inducible DSB reporter cell system (Tang et al., 2013). Upon 4-OHT and Shield1 treatment, mCherry-FokI endonuclease induces DSBs upstream of a Dox-inducible reporter gene. Nascent transcription is visualized by YFP-MS2 protein binding to stem-loop structures in the mRNA. DSBs are visualized by mCherry-FokI localization to the LacO loci. (B) macroH2A1.2 is required for transcriptional repression following DSBs. Nascent transcription was analyzed at FokI-induced DSBs in siControl-, siKDM5A-, and simacroH2A1.2-treated cells. The presence of MS2 foci indicates loss of DSB-induced transcriptional repression. Arrows indicate the location of the LacO array. Scale bars, 5 µm. (C) Quantification of B; n = 2 with >100 cells analyzed per condition per replicate. Error bars represent SEM. P values were calculated using an unpaired Student’s t test (**, P < 0.01). (D) Model for KDM5A regulation by PARP1 and macroH2A1.2. Following DNA damage, PARP1 promotes PARylation and recruitment of KDM5A to DNA damage sites. KDM5A engages PAR chains through its C-terminal coiled-coil–containing PID. macroH2A1.2 also promotes KDM5A accrual at DSBs to facilitate DSB-induced transcriptional repression and HR repair.