PARP-1 promotes the localized compaction of chromatin, but not naked DNA. (A) AFM topographical images, as described for Fig. 1C, of naked pFastbac1-cDNA plasmid DNA (left) purified recombinant PARP-1 protein (center), and PARP-1 plus naked pFastbac1-cDNA plasmid DNA (right). (B) (i) Enlargement of a scan probe oscillation amplitude AFM image from Fig. 1C showing an ACF-assembled chromatin array with a subsaturating amount (33%) of PARP-1 added. In ii through iv, the same image is marked with white circles to highlight unbound nucleosomes (ii) and supranucleosomal structures of increasing size, representing localized compaction (iii and iv).

The CAT is required for chromatin compaction by PARP-1. (A) Schematic diagram of the PARP-1 deletion mutants used in these studies. (B) PARP-1 chromatin binding assays as described in the legend to Fig. 3D using the PARP-1 mutants shown in panel A. (C) AFM images of ACF-assembled pFASTbac1-cDNA chromatin alone or with the addition of wild-type or mutant PARP-1, as indicated. Scan probe oscillation amplitude images (top) and topographical images (bottom) are as described for Fig. 1C. (D) Quantification of AFM images like those shown in panel C, as described in the legend to Fig. 1D. All determinations are based on at least 30 molecules derived from two independent preparations of chromatin. Each bar is the mean plus standard error of the mean.

The DBD and CAT are necessary and sufficient for chromatin compaction by PARP-1 but do not function in trans. (A) Schematic diagram of the PARP-1 deletion mutants used in these studies. (B) PARP-1 chromatin binding assays as described in the legend to Fig. 3D using the PARP-1 mutants shown in panel A. DBD-NBD indicates a single fusion peptide containing both domains, whereas DBD + CAT indicates separate polypeptides added in trans. Wt, wild type. (C) AFM images of ACF-assembled pFASTbac1-cDNA chromatin alone or with the addition of wild-type or mutant PARP-1, as indicated. Scan probe oscillation amplitude images (top) and topographical images (bottom) are as described for Fig. 1C. (D) Quantification of AFM images like those shown in panel C, as described in the legend to Fig. 1D. All determinations are based on at least 25 molecules derived from two independent preparations of chromatin. Each bar is the mean plus standard error of the mean.

Summary of binding, chromatin compaction, transcriptional repression, and automodification characteristics of PARP-1 mutants. (Left) Schematic diagrams showing the structural and functional domains of PARP-1, as well as a set of PARP-1 deletion and point mutants. The locations of the point mutations in DBD mut (C21G, C125G, and L139P) and CAT mut (E988K) are marked with Xs. The amino acid start points and end points for the mutants are shown. NLS, nuclear localization signal. (Right) The PARP-1 deletion mutants shown were expressed in E. coli and purified by nickel-NTA affinity chromatography (see Materials and Methods). The purified proteins were screened for four different activities, as indicated: (i) binding to chromatin, as determined by in vitro chromatin binding assays (Fig. 4B and 6B); (ii) compaction of chromatin, as determined by AFM imaging (Fig. 4C and D and 6C and D); (iii) repression of transcription, as determined by in vitro transcription assays (Fig. 7D); and (iv) automodification, as determined by an in vitro PARylation assay in the presence of [³²P]NAD⁺ (Fig. 5B). The data for DBD mut and CAT mut are from Kim et al. (15). Activity key: + indicates at least 75% of wild-type PARP-1 activity, − indicates less than 20% of wild-type PARP-1 activity, and +/− indicates ∼40 to 60% of wild-type PARP-1 activity.

PARP-1 compacts in vitro-assembled chromatin. (A) Overview of the preparation of S190-assembled chromatin samples for AFM imaging (see Materials and Methods). (B) (Left) Agarose gel electrophoresis of MNase-digested chromatin assembled using S190 and pFASTbac1-cDNA before and after purification by glycerol gradient and size exclusion chromatography (see Materials and Methods). M, 123-base pair ladder. (Right) SDS-PAGE analysis of purified baculovirus-expressed FLAG-tagged PARP-1 visualized by staining with Coomassie brilliant blue R-250. (C) AFM images of S190-assembled pFASTbac1-cDNA chromatin with increasing amounts of PARP-1, a saturating amount of PARP-1 plus 300 μM NAD⁺, or a saturating amount of H1. The amount of PARP-1 or H1 required to saturate compaction was determined empirically (see Materials and Methods). (Top) Scan probe oscillation amplitude images. The length scale is indicated by the white bars. (Bottom) Topographical images. The height scale is shown along the bottom of each image. (D) Quantification of AFM images like those shown in panel C. (Left) Average minimum radius of the circle needed to enclose an entire molecule of chromatin (black bars) and average number of discernible particles per molecule of chromatin (gray bars) under the conditions indicated. The values are plotted as percentages of chromatin with no PARP-1 or H1 added. (Right) Average height of nucleosomal arrays with or without saturating amounts of PARP-1 or H1. All determinations for PARP-1 are averages of at least 20 individual molecules of chromatin derived from three independently prepared batches of chromatin. All determinations for H1 are averages of at least 15 molecules of chromatin derived from two independent preparations of chromatin. Each bar is the mean plus standard error of the mean.

Histone tails are dispensable for PARP-1-mediated chromatin compaction. (A) Overview of the preparation of ACF-assembled tailless chromatin samples for AFM imaging (see Materials and Methods). (B) Agarose gel electrophoresis of MNase-digested chromatin assembled using recombinant ACF and pERE (see Materials and Methods). M, 123-base pair ladder. (C) Western blot for H2A and H2B illustrating the effective removal of histone tails by trypsin treatment. The digestion condition shown in lane 2 (i.e., complete, not excessive, digestion) was used for all studies with tailless chromatin. gH2A/B, globular (i.e., tailless) H2A and H2B. (D) PARP-1 chromatin binding assays. (Lanes 1 to 6) PARP-1 was subjected to small-scale size exclusion chromatography in the absence or presence of intact or tailless chromatin to assess its chromatin binding ability (see Materials and Methods). Two percent of the input (I) and 10% of the FT were analyzed by Western blotting for PARP-1. In this assay, unbound PARP-1 has a high residence time on the resin and thus is not evident in the FT. In contrast, chromatin-bound PARP-1 passes through the column with the FT. (Lanes 7 to 10) The effect of NAD⁺ on PARP-1 binding to chromatin was assayed in a similar manner. (E) AFM images of intact and tailless pFASTbac1-cDNA chromatin molecules alone, with a saturating amount of PARP-1, or with a saturating amount of PARP-1 plus NAD⁺, as indicated. Scan probe oscillation amplitude images (top) and topographical images (bottom) are as described for Fig. 1C. (F) Quantification of AFM images like those shown in panel E, as described in the legend to Fig. 1D. All determinations are based on at least 25 molecules derived from two independent preparations of chromatin. Each bar is the mean plus standard error of the mean.

Purification and enzymatic activities of PARP-1 mutants. The PARP-1 mutants shown in Fig. 4A and 6A were expressed in E. coli and purified by nickel-NTA affinity chromatography (see Materials and Methods). (A) Purified wild-type and mutant PARP-1 proteins were analyzed by SDS-PAGE with staining by Coomassie brilliant blue R-250. Molecular mass markers are shown. (B) Automodification activities of wild-type and mutant PARP-1 proteins. The automodification activities of the indicated PARP-1 proteins were determined in the presence of [³²P]NAD⁺, as described in Materials and Methods. Sheared DNA was added as an allosteric activator of PARP-1. The samples were analyzed by SDS-PAGE with detection of the ³²P signal by phosphorimager analysis.

Both the DBD and the CAT are required for efficient transcriptional repression by PARP-1. (A) Schematic diagram of chromatinized pERE, a plasmid template containing four EREs upstream of the adenovirus E4 core promoter. (B) Effect of wild-type (Wt) PARP-1 on ERα-dependent transcription with chromatin templates. pERE was assembled into chromatin using ACF and transcribed using a HeLa cell nuclear extract with or without ERα plus E2 and in the presence or absence of wild-type PARP-1, as indicated. The RNA products from the in vitro transcription reactions were analyzed by primer extension, and the data were quantified by phosphorimager analysis. All reactions were performed in duplicate, and the experiment was run at least four times to ensure reproducibility. (C) Quantification of in vitro transcription experiments like the one shown in panel B. Each bar is the mean plus standard error of the mean (SEM) from more than four independent experiments. (D) Effects of PARP-1 mutants on ERα-dependent transcription with chromatin templates. pERE was assembled into chromatin, transcribed in the presence or absence of wild-type or mutant PARP-1, and analyzed as described in the legend to panel B. The PARP-1 mutants are the same as those shown in Fig. 4, 5, and 6. The data are expressed as percentages of the transcriptional repression observed with wild-type PARP-1 (i.e., a percentage of “no repression” minus “repression by PARP-1”; see panel C). Each bar is the mean plus SEM from more than four independent experiments. Bars that are marked similarly with asterisks are statistically the same, while bars that are marked differently with asterisks are statistically different, as determined by analysis of variance; P < 0.05.