Fig. 3: The central SANT and AT-hook of CHD6 comprises a putative DNA binding domain important for PAR-responsive CHD6 localization to DNA damage. | Nature Communications

Fig. 3: The central SANT and AT-hook of CHD6 comprises a putative DNA binding domain important for PAR-responsive CHD6 localization to DNA damage.

From: CHD6 has poly(ADP-ribose)- and DNA-binding domains and regulates PARP1/2-trapping inhibitor sensitivity via abasic site repair

Fig. 3

For all statistical analysis in this figure, 2-way ANOVAs followed by Tukey post hoc tests were carried out. ns = not significant (p > 0.05), *p < 0.05. ** p < 0.01. ***p < 0.001.****p < 0.0001. Source data are provided as a Source Data file. All line graphs of microirradiation data are means normalized to t = 0 min, ± SEM, and full statistical test outcomes are in Supplemental Table 1. Panel a Schematic of CHD6 domains and corresponding conservation score determined by Jalview 2.10 (per109). Panel b Structural models of Region of Interest 2 (green, referred to as DBD1) and ‘CHD6CT2’ (beige, referred to as DBD2) of CHD6 based on homology modeling to S. cerevisiae CHD1, with superposition (right) to emphasize overlapping SANT domains and unique AT-hook structure in DBD1. Panel c Models mapping DBD1 and DBD2 mutations. Panel d Cells expressing mutants from (c) were micro-irradiated, imaged over 20 minutes; n = 30. Panel e Cells expressing wildtype or ΔDBD1 CHD6 were exposed to DMSO or 5 μM PARGi for 1h before micro-irradiation; n = 30. Panel f Purified, maltose binding protein (MBP)-tagged CHD6 fragments were slot-blotted, probed with 100 nM purified PAR and immunoblotted for PAR. Histone H1 and MBP were used as positive and negative controls, respectively. Panel g Workflow for delayed PARP (or DMSO) inhibition experiment. Panel h Cells were exposed to PARPi and/or H2O2, then immunoblotted for PAR and actin. Panel i Cells expressing wildtype and CHD6 ΔDBD1 were treated with DMSO or 2.5 nM PARPi per the workflow in (g), and analyzed as in Fig. 2b; n = 30. Panels j–k Cells expressing wildtype or indicated mutants were assayed for micro-irradiation relocalization; n = 28–30. For panel k, CHD6 ΔDBD1 + ΔPBM was additionally treated with 5 μM PARGi for 1 h prior to micro-irradiation and analyzed as in Fig. 2b; n = 18–30. Panel l Schematic of KillerRed DNA damage recruitment assay. Panel m: mCherry-tagged KillerRed was induced in U2OS 2-6-3 cells expressing indicated proteins and imaged for mCherry (red) and GFP (green).

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