Fig. 2: The CHD6 N-terminus contains a PAR-interacting motif needed for relocalization to DNA damage.
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 illustrating nuclear localization signal (NLS), tandem chromodomains (CD1 + 2), catalytic subunit (ATPase), SANT, BRK domain, two regions of interest, and Hallerman-Streiff syndrome mutation domain (CHD6CT2). Sequence alignment of putative PAR-binding motif (PBM) and corresponding mutants. Panel b Workflow of laser micro-irradiation and analysis; this figure was created using Microsoft PowerPoint. Panel c Nuclear localization controls for CHD6ΔPBM (K→A)-GFP and CHD6ΔPBM (K→Q)-GFP. Panel d Cells expressing GFP-tagged CHD6ΔPBM (K→A) and CHD6ΔPBM (K→Q) were micro-irradiated, imaged over 20 minutes, and analyzed as in (b); n = 18-30. Panel e–f Cells expressing GFP-tagged wildtype or CHD6ΔPBM (K→A) were treated with DMSO, 2.5 nM PARPi or 5 μM PARG inhibitor (PARGi) for 1 h prior to micro-irradiation, imaged and quantified as in (b); n = 15–30. Panel g Purified, maltose binding protein (MBP)-tagged CHD6 fragments were slot-blotted on a nitrocellulose membrane and probed with 100 nM purified PAR. PAR binding was detected with the anti-PAR antibody 10H. Histone H1 and MBP were used as positive and negative PAR binding controls, respectively, while Sypro Ruby stain visualized all blotted proteins as a loading control. A Sypro Ruby stained SDS-PAGE of all proteins used in slot blot assay is shown below slot blots.
