Hit and run versus long-term activation of PARP-1 by its different domains fine-tunes nuclear processes

Abstract

Poly(ADP-ribose) polymerase 1 (PARP-1) is a multidomain multifunctional nuclear enzyme involved in the regulation of the chromatin structure and transcription. PARP-1 consists of three functional domains: the N-terminal DNA-binding domain (DBD) containing three zinc fingers, the automodification domain (A), and the C-terminal domain, which includes the protein interacting WGR domain (W) and the catalytic (Cat) subdomain responsible for the poly(ADP ribosyl)ating reaction. The mechanisms coordinating the functions of these domains and determining the positioning of PARP-1 in chromatin remain unknown. Using multiple deletional isoforms of PARP-1, lacking one or another of its three domains, as well as consisting of only one of those domains, we demonstrate that different functions of PARP-1 are coordinated by interactions among these domains and their targets. Interaction between the DBD and damaged DNA leads to a short-term binding and activation of PARP-1. This "hit and run" activation of PARP-1 initiates the DNA repair pathway at a specific point. The long-term chromatin loosening required to sustain transcription takes place when the C-terminal domain of PARP-1 binds to chromatin by interacting with histone H4 in the nucleosome. This long-term activation of PARP-1 results in a continuous accumulation of pADPr, which maintains chromatin in the loosened state around a certain locus so that the transcription machinery has continuous access to DNA. Cooperation between the DBD and C-terminal domain occurs in response to heat shock (HS), allowing PARP-1 to scan chromatin for specific binding sites.

Keywords: PARP-1; PARP-1 regulation; drosophila; poly(ADP-ribose); protein domains.

Fig. 1.

N- and C-terminal PARP-1 domains contribute to PARP-1 protein localization genome-wide in vivo. (A) Domains of PARP-1: The N-terminal DNA-binding domain (DBD) containing Zn fingers: ZI, ZII, ZIII (ZF domains 1, 2, and 3), the automodification domain (A), the only domain of PARP-1 known to accept pADPr, the WGR domain (W), and the C-terminal catalytic domain (C). The PARP signature (PS) is an evolutionarily conserved PARP-1 catalytic site in the Cat domain. The dotted blue line indicates known interactions between the domains induced by interactions with damaged DNA (21). The red arrow indicates automodification of PARP-1. (B) Structure of recombinant-transgenic PARP-1 constructs for in vivo experiments. (C) Localization of deletional recombinant isoforms of PARP-1 in salivary gland polytene chromosomes. Green is the fluorescence of proteins fused to YFP, red is DNA. All isoforms of PARP-1 carrying ZI and ZII demonstrate colocalization with DNA resulting in the yellow color in the overlay. All isoforms without ZI and ZII localized in active open chromatin only, resulting in the separation of red and green in the overlay. (D) PARP-1 deletional isoform activity assay in vivo. PARP-1 deletional isoforms were expressed in the parg^27.1; parp-1^C03256 mutant flies. All isoforms containing the Cat domain restored pADPr accumulation. (E) Both DNA- and C-terminal domains contributed to PARP-1 protein dynamic binding to chromatin in vivo. Comparative analysis of fluorescent recovery after photobleaching (FRAP) assay for recombinant protein is shown, including ZI-II-YFP, ZIII-A-W-Cat-YFP, full-length PARP-1-YFP, and H4-YFP. Data for the FRAP experiment show the average based on 10 replicates.

Fig. 2.

C-terminal domains of PARP-1 are responsible for PARP-1 targeting to the TSS of the hsp70 locus, whereas DNA-binding domains target PARP-1 to areas outside of the promoter region. (A–D) The comparison of recombinant protein distribution within the hsp70 locus, ChIP assay before and after HS: PARP-1-YFP (A); ZIII-A-W-Cat-YFP (B); W-Cat-YFP (C); and ZI-ZII-YFP (D). (E) PARP-1 isoforms with C-terminal domains rescue transcription activation at the hsp70 locus following HS in parp-1^C03256 mutants. The level of hsp70 mRNA was recorded before and after 30 min of HS treatment using quantitative RT PCR. (F) PARP-1 isoforms with C-terminal domains rescue histone H3 displacement from hsp70 locus, following HS in parp-1^C03256 mutants. ChIP assay compares amounts of H3 histone in the promoter region of the hsp70 locus in wild-type and parp-1^C03256 mutant animals expressing full-length PARP-1 (PARP-1-YFP) or PARP-1 deletional isoforms (ZI-ZII, ZIII-A-W-Cat, W-Cat) before (−) and after (+) 30 min of HS treatment. All error bars are based on the average of triplicates.

Fig. 3.

Histone H4-mediated PARP-1 activation is dependent on H4 binding to the C-terminal W-CAT domains of PARP-1. (A) Composition of deletional recombinant PARP-1 isoforms for in vitro experiments. His, 6 histidine tag. (B) In vitro binding assay (15): DNA, histone H4 and a mutant form of H4, H4^G23G61 were each covalently coupled to CnBr beads and preincubated with a solution containing each deletional isoform of PARP-1. Following precipitation of beads, pellet (P) and solution (S) were subjected to PAGE and Western blot. The presence of PARP-1 isoforms in pellet and solution was detected on Western blot using anti–6XHis-tag antibody. Histone H3 was used as a positive control, which interacts with DNA and both forms of histone H4. IgG was used as a nonspecific protein-binding control. (C) In vitro activation assay. Sepharose beads with covalently attached DNA or H4 were preincubated with PARP-1 deletional isoforms and then mixed with NAD. The accumulation of pADPr was detected on Western blot using an anti-pADPr antibody. Input: the Coomassie Brilliant Blue stained PAGE gel shows the quantities of PARP-1 isoforms loaded in each reaction. (D) Diagram illustrating interactions between PARP-1 deletional isoforms and their targets (DNA and histones): ZIII-A-W-Cat (Top) interacting with H4-coupled beads only, ZI-ZII (Bottom) interacts with DNA-coupled beads but not with H4.

Fig. 4.

Regulation and targeting of enzymatic activity of PARP-1 by histones and DNA. (A–B) PARP-1 modifies either itself via the A domain (A) or the linker histone H1 in vitro. (A) Binding-activation assay: full-length PARP-1 and deletional isoform W-Cat were separately incubated with H4-bound sepharose beads, washed, and mixed with NAD and other histones (H1, H2A, H2B, H3, and H2Av) to induce pADPr production. The supernatant (S) was removed and the pellet (P) was washed. The absence of the A domain in the W-Cat isoform precludes automodification of PARP-1. Histones were added as potential substrates for the poly(ADP ribosyl)ation assay. Accumulation of pADPr was detected on Western blot using an anti-pADPr antibody (10H). Input: the Coomassie Brilliant Blue stained PAGE gels show the amounts of PARP-1, W-Cat, and histones loaded in each reaction. (B) Diagram illustrating the activity of full-length PARP-1 and PARP-1 deletional isoforms via the A domain and modification of H1. (C–D) Upon activation and automodification, PARP-1 loses interaction with DNA but remains bound to histone H4. (C) Interactive-activity assay of PARP-1 with sepharose beads coupled to either nicked DNA or H4. PARP-1 was incubated separately with either nicked DNA- or H4-coupled beads, washed, and mixed with NAD to trigger pADPr production. The solution was removed and the pellet was washed twice (W1, W2). The distribution of pADPr between fractions was measured after PAGE on a Western blot using an anti-pADPr antibody. (D) Diagram illustrating that automodified PARP-1 dissociates from DNA but remains bound to H4.

Fig. 5.

Model of PARP-1 dual binding to chromatin. (A) Full-length PARP-1 has two chromatin-binding regions: N-terminal ZI and ZII bind to DNA nonspecifically, allowing PARP-1 to scan chromatin by “walking” along DNA and the C-terminal domain which recognizes and binds to the H2Av-bearing nucleosome specifically. (B) The C-terminal domain recognizes specific epitopes of the H2Av-bearing nucleosome. (C) The ZI-ZII deletional isoform binds to DNA nonspecifically and does not interact with the nucleosome.