Extended Data Fig. 5: HPF1-dependent chromatin relaxation relies on trans ADP-ribosylation of histones rather than PARP1 auto-modification.

(a) Western blot displaying ADPr signals, stained with a pan-ADPr antibody, in WT and HPF1^KO cells expressing HPF1 WT or HPF1 E284A and treated or not with H₂O₂. H2B and Tubulin were used as loading controls. (b) Western blot displaying ADPr signals, stained with a pan-ADPr antibody, in U2OS WT, PARP1^KO and PARP1/HPF1 double knockout cells expressing mCherry tagged PARP1 WT, PARP1 3SA or PARP1 LW/AA and treated or not with H₂O₂. H3 and Tubulin were used as loading controls. (c) Representative images of the recruitment of mCherry-tagged HPF1 WT or R239A to sites DNA damage induced by 405 nm laser irradiation in U2OS HPF1^KO cells. Scale bar, 5 μm. (d) Recruitment kinetics of mCherry-tagged HPF1 WT or HPF1 R239A mutant at sites of DNA damage in U2OS HPF1^KO cells. Data from d are a representative of 3 independent replicates where data were collected from 15–16 cells per condition. (e) Western blot displaying ADPr signals, stained with a pan-ADPr antibody, in WT cells overexpressing YFP-HPF1 or not with or without ATP depletion (ATPi) after H₂O₂ damage. H3 and Tubulin were used as loading controls. (f) Chromatin relaxation 120 s post irradiation at 405 nm in U2OS WT or ALC1^KO cells overexpressing HPF1 or not. Data from f are a representative of 3 independent replicates where data were collected from 16–18 cells per condition. (g) Western blot analysis confirming knockout status of ALC1^KO cells. (h) Chromatin relaxation 120 s post DNA damage in U2OS WT or CHD7^KO cells overexpressing HPF1 or not. Data from h are a representative of 3 independent replicates where data were collected from 15–18 cells per condition. (i) Western blot analysis confirming knockout status of CHD7^KO cells.

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