CHD7 and 53BP1 regulate distinct pathways for the re-ligation of DNA double-strand breaks

Chromatin structure is dynamically reorganized at multiple levels in response to DNA double-strand breaks (DSBs). Yet, how the different steps of chromatin reorganization are coordinated in space and time to differentially regulate DNA repair pathways is insufficiently understood. Here, we identify the Chromodomain Helicase DNA Binding Protein 7 (CHD7), which is frequently mutated in CHARGE syndrome, as an integral component of the non-homologous end-joining (NHEJ) DSB repair pathway. Upon recruitment via PARP1-triggered chromatin remodeling, CHD7 stimulates further chromatin relaxation around DNA break sites and brings in HDAC1/2 for localized chromatin de-acetylation. This counteracts the CHD7-induced chromatin expansion, thereby ensuring temporally and spatially controlled ‘chromatin breathing’ upon DNA damage, which we demonstrate fosters efficient and accurate DSB repair by controlling Ku and LIG4/XRCC4 activities. Loss of CHD7-HDAC1/2-dependent cNHEJ reinforces 53BP1 assembly at the damaged chromatin and shifts DSB repair to mutagenic NHEJ, revealing a backup function of 53BP1 when cNHEJ fails.

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April 2020
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Life sciences study design
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Sample size 1. Microscopy experiments aimed to acquire at least 100 cells per condition. In some cases, however, when fewer cells were acquired, the precise amount of analyzed cells is mentioned in the Figure legends. Analysis of >100 cells was sufficient to obtain normal distribution of the data and reliable mean. 2. Reporter assays were performed with acquisition of at least 150.000 cells per condition to obtain a clear GFP-positive cell population which accounted for ~2-5% of the total. 3. DSB repair junction analysis was done with ~100 sequences per condition which was sufficient to obtain enough wild type and mutated sequences for the normal distribution of the data. 4. Survival assays were performed with re-seeding cells in triplicate per condition to average out the variation between technical replicates.
Data exclusions No data was excluded.

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Experiments were performed at least in duplicate, but mostly in triplicate or more to asses the reproducibility. All attempts at reproduction were successful. Standard errors included in the graphs indicate the variation between replicates of each experiment.
Randomization Experiments were performed with cell lines. For each experiments, cells were randomly seeded for different treatments.

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Blinding has not been applied, also because it is not feasible for many of the approaches used in our manuscript, including IPs, western blot analysis and siRNA/inhibitor treatments.

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Cell lines were authenticated using Short Tandem Repeat (STR) analysis by ATCC services (100% match).

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All cell lines were routinely and regularly tested for mycoplasma and used only when non-contaminated.
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Flow Cytometry
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All plots are contour plots with outliers or pseudocolor plots. To analyze GFP and mCherry positive U2OS cells carrying the DSB reporters (EJ5-GFP, EJ2-GFP, DR-GFP), three initial gates were set in the following sequential plots: 1) SSC-A scatter (Y-axis) set out against FSC-A scatter (X-axis), allowing us to set gate P1 for living cells, 2) FSC-H scatter (Y-axis) set out against FSC-W scatter (X-axis), allowing us to set gate P2, and 3) SSC-H scatter (Y-axis) set out against SSC-W scatter (X-axis), allowing us to set gate P3. Gates P2 and P3 allowed us to exclude doublets. In a fourth plot (GFP-A on Y-axis and mCherry-A on X-axis), mCherry positive cells were scored using a gating based on mCherry negative control cells (gate P4). Subsequent gate P5 was positioned within Chery-positve population (within gate P4) to gate for GFP positive cells.
To analyze GFP positive U2OS cells transfected with pEGFP construct in random plamsmid integration assays, three initial gates were set in the following sequential plots: 1) SSC-A scatter (Y-axis) set out against FSC-A scatter (X-axis), allowing us to set gate P1 for living cells, 2) FSC-H scatter (Y-axis) set out against FSC-W scatter (X-axis), allowing us to set gate P2, and 3) SSC-H scatter (Y-axis) set out against SSC-W scatter (X-axis), allowing us to set gate P3. Gates P2 and P3 allowed us to exclude doublets. In a fourth plot (SSC-A on Y-axis and GFP-A on X-axis), GFP positive cells were scored using a gating based on GFP negative control cells (gate P4).
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