Volume 61, Issue 3, Pages 474-485 (February 2016) Analysis of Chromatin ADP-Ribosylation at the Genome-wide Level and at Specific Loci by ADPr-ChAP  Giody Bartolomei, Mario Leutert, Massimiliano Manzo, Tuncay Baubec, Michael O. Hottiger  Molecular Cell  Volume 61, Issue 3, Pages 474-485 (February 2016) DOI: 10.1016/j.molcel.2015.12.025 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 61, 474-485DOI: (10.1016/j.molcel.2015.12.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 The RNF146 WWE Specifically Pulls Down In Vivo ADP-Ribosylated Proteins Cross-Linked to DNA (A) PAR immunofluorescence (red) of unstimulated or H2O2-treated cells fixed in either methanol/acetic acid (MeOH/AcOH), 1% formaldehyde, or 4% formaldehyde. DAPI (blue) was used for nuclear staining. (B) PAR and ARTD1 western blotting analysis of chromatin prepared for ADPr-ChAP as described in the Experimental Procedures from A549 left untreated or treated with H2O2 and/or ABT-888. (C) qPCR analysis using chromatin prepared for ADPr-ChAP as described in the Experimental Procedures from A549 cells treated with H2O2 and/or ABT-888 and subjected to ChIP using the 10H monoclonal anti-PAR antibody. Data are represented as mean ± SD. (D) Schematic overview of the ADPr-ChAP method. (E) Western blot analysis of in vitro ADP-ribosylated ARTD1 and histones (i.e., H3) subjected to pull-down with wild-type (wt) or mutated (mut) WWE and Af1521. The original image was edited by removing one lane (indicated by dotted line). (F) Western blot analysis of PAR and ARTD1 using chromatin from A549 cells treated with H2O2 and/or ABT-888 prepared for ADPr-ChAP as described in the Experimental Procedures, and subjected to pull-down with WT or mut WWE. See also Figures S1 and S2. Molecular Cell 2016 61, 474-485DOI: (10.1016/j.molcel.2015.12.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Genome-wide Analysis of ADP-Ribosylation after H2O2 Treatment Reveals a Strong Correlation to Nucleosome Abundance (A) Box plots indicating ADP-ribosylation in H2O2 treated cells at regulatory segments of the A549 genome partitioned based on chromatin modifications and genomic features. Shown are log2 enrichments of ADP-ribosylation over input chromatin. (B) Average density plots indicate depletion of ADP-ribosylation at the TSS of transcribed genes. H3K36me3 was used to separate the genes according to transcriptional activity. Total histone H3 measurements resemble distribution of ADP-ribosylation along genes. (C) Genome-wide correlation between ADP-ribosylation and nucleosome abundance as measured by histone H3, depicted by direct comparison. Shown are log2-transformed reads per 1 kb windows spanning chromosome 19. (D) Promoters of active genes based on H3K9 acetylation (ON) are depleted of ADP-ribosylation. rnd., a random selection of TSS (N = 5,000), independent of H3K9 acetylation. (E) Cross-correlation matrix based on log2-enrichments at 1 kb tiles covering chromosome 19 and using various ChIP and ChAP-seq samples obtained from A549 cells. Correlations are calculated based on Spearman's rank coefficient. (F) Direct comparison of H3K9 acetylation and H3K9me3 with ADP-ribosylation after H2O2 treatment based on genome-wide log2-enrichments. (G) WWE ChAP measurements after H2O2 at LTR, LINE, SINE, and satellite repeats indicate enrichment of ADP-ribosylation at a subset of families. Box plots represent median (line), inter-quartile range (IQR) (box), and IQR∗1.5 (whisker). See also Figure S3. Molecular Cell 2016 61, 474-485DOI: (10.1016/j.molcel.2015.12.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Implementation of the APD-Ribose-Specific Chromatin Affinity Precipitation Method (A) qPCR analysis at the GAPDH TSS, IL1B promoter and αSAT regions using chromatin prepared for ADPr-ChAP as described in the Experimental Procedures from A549 cells treated with or without H2O2 and subjected to pull-down with WT or mut WWE. (B) Chromatin from shMock and shARTD1 cells stimulated with H2O2 in the absence or presence of ABT-888 was analyzed as in (A). (C) ChIP analysis of ARTD1 at the GAPDH TSS, IL1B promoter, and αSAT regions. (D) qPCR analysis of formaldehyde-fixed extracts from cells treated with H2O2 and enriched for ARTD1 by ChIP or ADPr-ChAP/ChIP. (E) Western blotting analysis for PAR and ARTD1 of chromatin from H2O2-treated cell enriched by histone H3 ChIP followed by DNase digestion, reverse-crosslinking, and pull-down with WT or mut WWE. (F) qPCR analysis using chromatin from A549 cells pre-treated without or with ABT-888 and subsequently left untreated (−), or treated for 10 min with H2O2 and analyzed immediately (10′), after recovery for an additional 20 min (30′), or 80 min (90′) prepared for ADPr-ChAP as described in the Experimental Procedures and enriched by ADPr-ChAP with WWE. All data in bar graphs are represented as mean ± SD. See also Figures S4 and S5 and Table S1. Molecular Cell 2016 61, 474-485DOI: (10.1016/j.molcel.2015.12.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 H2O2-Induced Chromatin ADP-Ribosylation Is Associated with Heterochromatic Histone Marks (A–F) ChIP analysis of histone H3 (A), H3ac (B), and H3k9me3 (C) at the GAPDH TSS, IL1B promoter, and αSAT regions. qPCR analysis of formaldehyde-fixed extracts from cells treated with H2O2 and enriched for histone H3 (D), H3Ac (E), or H3K9me3 (F) by ChIP or ADPr-ChAP/ChIP. (G) MNase-based CHART-PCR of cells treated with H2O2 in the absence or presence of ABT-888. All data in bar graphs are represented as mean ± SD. See also Figure S5. Molecular Cell 2016 61, 474-485DOI: (10.1016/j.molcel.2015.12.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Adipogenesis-Induced ADP-Ribosylated Proteins Localize at the PPRE of a Subset of PPARγ (A) Western blot analysis of PAR, ARTD1, and histone H3 using formaldehyde-fixed extracts from undifferentiated (d0) or differentiated (d7) adipocytes enriched by ADPr-ChAP with WWE. (B) qPCR analysis at the PPRE of the PPARγ target genes Ap2, Adipoq, and Cd36 of material enriched as in (A), cells inhibited with ABT-888 for 12 hr were included in the analysis. ChAP data are normalized over input, expressed as fold change over d0 and further normalized over Krt19 signal. (C) ARTD1 and PPARγ occupancy at the PPREs of Ap2 and Cd36 on d0 and d7. (D) qPCR analysis after ChIP and ADPr-ChAP/ChIP of ARTD1 and PPARγ at the PRREs of Ap2 and Cd36 on d7. All data in bar graphs are represented as mean ± SD. See also Figure S5. Molecular Cell 2016 61, 474-485DOI: (10.1016/j.molcel.2015.12.025) Copyright © 2016 Elsevier Inc. Terms and Conditions