PARP-1 binds H4K20me1, H3K4me1, H3K36me1, H3K9me1, and H3K27me1 in vitro and in vivo.

(A) Illustration of the MODified histone peptide assay (Active Motif) used to determine PARP-1 binding to histone modifications. The histone peptide array (Top, left), comprising 19mer peptides with single or up to four concurrent histone modifications, was employed to investigate PARP-1’s binding affinity for histone modifications and to assess the impact of adjacent modified peptides on PARP-1 binding. This array was first blocked, then incubated with PARP-1 protein (Top, right). Subsequently, it was stained with a PARP-1 antibody and a horseradish peroxidase (HRP)-conjugated secondary antibody (Bottom, right). Visualization of PARP-1 binding was done through enhanced chemiluminescence detection and captured on X-ray film (Bottom, left). See Methods for a full description. (B) Signal intensity on modified histone peptide array based on incubation with PARP-1 protein. (C) Average intensities of PARP-1 binding to single histone peptides. (D-E) Reproducibility and specificity of spot intensities from modified histone peptide array duplicates. (D) Scatter plot showing the correlation of the average intensities of duplicate arrays. Intensities of PARP-1 binding to all peptides and spots containing single H4K20me1, H3K4me1, H3K36me1, H3K9me1 and H3K27me1 (key) are shown. Pearson’s correlation coefficient (r) is 0.92. (E) Bar chart showing top 8 histone modifications with the highest specificity for PARP-1 binding. The specificity factor was calculated by dividing the average intensity of spots that contain the modified histone peptide by the average intensity of spots that do not contain the peptide. (F) Spearman correlation of PARP-1, H4K20me1, H3K4me1, H3K36me1, H3K9me1, H3K9me2/3 and H3K27me1 peaks in Drosophila third-instar larvae based on fraction of overlap. (G) Heatmaps showing k-means clustering-generated occupancy of PARP-1, H4K20me1, H3K4me1, H3K36me1, H3K9me1, H3K9me2/3 and H3K27me1 normalized ChIP-seq signals in third-instar larvae at Drosophila genes. ChIP-seq signals are sorted in descending order. The upper plots show the summary of the signals.

PARP-1 colocalizes with H3K4me1 and H4K20me1 histone marks in polytene chromosomes.

(A) Immunofluorescent staining of Drosophila salivary gland chromosomes showing colocalization of PARP-1 with H3K4me1 and H4K20me1. White lines indicate areas of the colocalization-quantification shown in (B). (B-C) Quantification of fluorescence intensity of PARP-1, H3K4me1 and H4K20me1 at Drosophila polytene chromosomes in panel A. (B) Images show distribution of PARP-1 fluorescence intensity with H3K4me1 (top) and H4K20me1 (bottom) fluorescence intensity. (C) Images represent a scatterplot showing PARP-1 fluorescence intensity with H3K4me1 (top) and H4K20me1 (bottom) fluorescence intensity. Pearson correlation coefficients (r) are respectively 0.58 (top) and 0.71 (bottom).

PARP-1 and H4K20me1 are required for the repression of metabolic genes and activation of developmental genes at co-enriched genes.

(A) Scatterplot plot showing correlation of differentially expressed genes (DEGs) in parp-1C03256and pr-set720 (Pearson’s r = 0.79). (B) Heatmap showing the normalized read counts of DEGs in both parp-1C03256 and pr-set720. Normalized read counts are shown as row z-scores. (C-D) Dot plots showing transcriptional changes of genes co-enriched with PARP-1 and H4K20me1 in parp-1C03256 and pr-set720compared to WT at promoters (C) and gene bodies (D). (E) Summary of DEGs in parp-1C03256 and pr-set720 and both mutants that were co-enriched with PARP-1 and H4K20me1. (F) Gene ontology of upregulated (left) and downregulated (right) DEGs in both parp-1C03256 and pr-set720 mutants that were co-enriched with PARP-1 and H4K20me1.

Dynamic H4K20me1 enrichment regulates the expression of heat-shock genes during heat shock.

(A) Expression of hsp22, hsp23, hsp68, hsp70 and hsp83 in WT, parp-1C03256 and pr-set720third-instar larvae before and after 30 mins heat shock. Data shown are from 3-5 biological replicates. ***P < 0.001, **P < 0.01, *P < 0.05 (Unpaired t-test; Two-tailed). Integrative Genome Viewer (IGV) tracks showing normalized ChIP-seq tracks of PARP-1 and H4K20me1 in third-instar larvae at (B) hsp22, hsp68, hsp68, hsp70 and (C) hsp22, hsp83 before heat shock. Mononucleosome ChIP-qPCR in WT showing enrichment of H4K20me1 at (D) hsp70, hsp22, hsp68 and (E) hsp23 and hsp83 before heat shock (NHS), after 15 mins heat shock and 30 mins heat shock. Primers used spanned the hsp70 locus and the gene bodies of hsp22, hsp23, hsp68, hsp70 and hsp83. Data shown are from 3 biological replicates.

Mono-methylated histones controls PARP-1 binding along gene body to regulate transcription.

(A) PR-SET7/H4K20me1 is required for holding PARP-1 in chromatin during heat shock (HS). PARP-1 (green) protein recruitment to hsp70 locus (Arrows) in salivary gland polytene nuclei in wild type (WT) and pr-set720 mutant third instar larvae. A single salivary gland polytenized nucleus of wandering third instar larvae is shown for each genotype. Wild type genotype - UASt::PARP-1-EYFP, GAL4[Mz1087.hx]; pr-set7 mutant genotype – UASt::PARP-1-EYFP, GAL4Mz1087.hx; pr-set720. (B) Equal amounts of lysates from the wild-type expressing PARP-1-EYFP (WT) and pr-set720mutants expressing PARP-1-EYFP (pr-set720) grown at 22°C or heat shocked at 37°C for one hour at the third-instar larvae stage were subjected to immunoblot analysis using mouse anti-pADPr (10H), anti-H4K20me1 and anti-histone H4 (loading control) antibodies. (C) Model of PARP-1 regulation by histone modifications. PARP-1 binds to a nucleosome that carries the H2A variant (H2Av) at the promoter region. Upon developmental triggers or heat shock-induced phosphorylation of H2Av, PARP-1 is activated (11). Activated PARP-1 fosters a transcription start site (TSS) that is more accessible, thereby enabling the binding of RNA-Polymerase II (Pol II) and initiation of transcription (26). Following this, the distribution of PARP-1 is further enhanced by active mono-methylated forms of H4K20, H3K4, H3K36, or H3K9. Each of these histone modifications can interchangeably facilitate this function. The spreading of PARP-1 to the gene body contributes to the loosening of chromatin in this region (7, 16, 17, 49). Consequently, this facilitates the transition of Pol II into a productive elongation phase, leading to the generation of a mature transcript.