Gadd45, a p53-responsive stress protein, modifies DNA accessibility on damaged chromatin

Abstract

This report demonstrates that Gadd45, a p53-responsive stress protein, can facilitate topoisomerase relaxing and cleavage activity in the presence of core histones. A correlation between reduced expression of Gadd45 and increased resistance to topoisomerase I and topoisomerase II inhibitors in a variety of human cell lines was also found. Gadd45 could potentially mediate this effect by destabilizing histone-DNA interactions since it was found to interact directly with the four core histones. To evaluate this possibility, we investigated the effect of Gadd45 on preassembled mononucleosomes. Our data indicate that Gadd45 directly associates with mononucleosomes that have been altered by histone acetylation or UV radiation. This interaction resulted in increased DNase I accessibility on hyperacetylated mononucleosomes and substantial reduction of T4 endonuclease V accessibility to cyclobutane pyrimidine dimers on UV-irradiated mononucleosomes but not on naked DNA. Both histone acetylation and UV radiation are thought to destabilize the nucleosomal structure. Hence, these results imply that Gadd45 can recognize an altered chromatin state and modulate DNA accessibility to cellular proteins.

FIG. 1

Gadd45 facilitates topoisomerase relaxing activity in the presence of core histones. Reactions were performed as described in Materials and Methods (“Nucleosome assembly on relaxed plasmid”). (A) Supercoiled plasmid DNA (0.2 μg, pBR322) (lane 1) was relaxed by 10 U of Top1 (lane 2) and incubated in the presence (+) or absence (−) of core histones (CH), Gadd45 (G45), or NAP-1. After incubation, the samples were deproteinized and run on a 1% agarose gel in the absence of chloroquine. The gel was then stained with ethidium bromide. The positions of forms I (S⁻, negatively supercoiled) and II (N, nicked circle; R, relaxed) of plasmid pBR322 are indicated. (B) Same as panel A but run in the presence of 40 μg of chloroquine per ml. (C) Plasmid relaxation assay as in panel A but performed in the absence of Top1. Amounts of Gadd45 were 0.5 and 1.0 μg (lanes 2 and 3); 0.5 μg of Gadd45 deletion mutants Δ72 (the resultant protein contains the first 71 amino acids) and Δ125 (mutant containing amino acids 1 to 124) were used (lanes 7 and 8). (D) Plasmid relaxation assay performed as for panel C in the absence (−) or presence (+) of 10 μM camptothecin, a Top1 inhibitor. (E) Increasing amounts (0.4 to 40 μg) of HeLa core histone were analyzed by Western blotting with human Top1 antibody. The proteins were blotted and hybridized as described in Materials and Methods. Positions of the protein markers and Top1 are indicated on the left. (F) Reactions similar to those in panel C, run in the presence of 40 μg of chloroquine per ml.

FIG. 2

Gadd45 interacts with core histones. (A) Plasmid relaxation assay performed as for Fig. 1A but in the absence of core histones. The assay was performed in the absence (lanes 1 to 7) or presence (lanes 8 to 14) of Gadd45 (1 μg). (B) Far-western analysis performed with 1 μg of normal (CH; lanes 2, 5, and 8) or hyperacetylated (Acet; lanes 3, 6, and 9) Drosophila core histones. Recombinant Gadd45 (rG; 15 ng) was used as a positive control (lanes 1, 4, and 7). The proteins were loaded on a 18% polyacrylamide gel, transferred to nitrocellulose, and processed as described in Materials and Methods. The blots were prehybridized in the absence (No Gadd45) or in the presence of 1 μg of recombinant Gadd45 protein per ml prior to Western blot analysis with Gadd45 polyclonal antibody. (C) Increasing amounts (0 to 100 ng) of Gadd45 were preincubated in the absence (−) or presence (+) of core histones (CH; 100 ng) before addition of a labeled oligonucleotide. The reaction products were run on a nondenaturing 4 to 20% polyacrylamide gel and exposed to X-ray film. The histone-DNA complex (shift), the double-stranded DNA probe (ds), and the residual single-stranded DNA (ss) are indicated. (D) The Top1 cleavage assay was performed in the presence of 10 μM camptothecin as described in Materials and Methods. A double-stranded DNA oligomer (30-mer) containing one Top1 site was labeled at the 3′ end of the sense strand and incubated in the presence (+) or absence (−) of core histones (CH; 0.4 μg), Gadd45 (0.5 μg), and Top1 (10 U). A 19-mer oligonucleotide fragment is released after cleavage by Top1 (lane 2, arrow).

FIG. 3

Evidence that Gadd45 can modulate topoisomerase sensitivity in vivo. (A) Wilcoxon rank sum test was used to determine whether individual compounds tested in the NCI anticancer drug screen were more active in cell lines with competent GADD45 induction (P = 1) or more active in cell lines with deficient GADD45 induction (P = 0). Results for a selection of COMPARE-identified topoisomerase II inhibitors (top) and for the remainder of the database of tested compounds (bottom) are presented. (B) Clonagenic survival after treatment with the Top1 inhibitor camptothecin was determined in the human RKO colon carcinoma line and its derivative RKO-AS45, which were stably transfected with vector alone and with vector containing GADD45 cDNA in the antisense orientation, respectively. Colonies were counted 2 weeks after treatment. Survival is expressed as the percentage of colonies obtained with untreated cells. (C) Survival analysis performed as in for panel B with VP-16, a Top2 inhibitor. (D) DNA-protein cross-links produced in RKO and RKO-AS45 cells after treatment with VP-16 as measured by alkaline elution.

FIG. 4

Gadd45 interacts with altered mononucleosomes. (A) Mobility shift assay performed on underacetylated (normal) mononucleosomes (lanes 1 to 4) or hyperacetylated mononucleosomes (lanes 5 to 11) in the presence of increasing amount (0 to 1 μg [lanes 1 to 4 and 5 to 8] or 25 to 100 μg [lanes 9 to 11]) of Gadd45. The binding conditions were as described in Materials and Methods. (B) Mobility shift assay as in panel D except that UV (7,500 J m⁻²)-irradiated mononucleosomes were used in the reactions. In lane 5, Gadd45 monoclonal antibody 30T-14 (Ab) was added to the reaction. (C) Mobility shift assay as in panel D but performed with an excess amount (0 to 100 μg) of Gadd45 on UV-irradiated mononucleosomes. In panels A to C, positions of free DNA, nucleosomes (Nucl), and a shifted band are indicated. (D) Mobility shift assay as in panel B but on free DNA.

FIG. 5

Gadd45 increases DNase I sensitivity on hyperacetylated mononucleosomes. (A) DNase I digestion pattern of naked DNA (Free; lanes 1 to 3), underacetylated (Norm; lanes 4 to 6), and hyperacetylated mononucleosomes (Acety; lanes 7 to 9) in the presence of increasing amounts of Gadd45 (0 to 50 ng). Digestion was performed as described in Materials and Methods. (B) Magnification of two typical bands from the DNase I digestion pattern shown in panel A. (C) Scanning analysis of band B, shown in panel A, performed with the program MacBAS version 2.0. The intensity of each individual band is expressed in pixels (PSL). (D) Average of three typical DNase I experiments. Increased DNase I sensitivity was analyzed on all bands; analysis of representative bands A to E, as indicated in panel A, is shown. The intensity of bands A to E was normalized to the total number of counts in each lane. The normalized number obtained in the absence of Gadd45 was assigned a DNase I sensitivity value of 100% (0% increased sensitivity). The increased DNase I sensitivity is expressed as the differences between the percentage of DNase I sensitivity obtained in the absence of Gadd45 and in the presence of 50 ng of Gadd45.

FIG. 6

Gadd45 protects UV-irradiated mononucleosomes from T4 Endo V digestion. Free DNA (Naked; lanes 1 to 4) and mononucleosomes (Nucleosomes; lanes 5 to 8) were UV (7,500 J m⁻²)-irradiated and incubated in the absence (−) or presence (+) of 50 ng of Gadd45 prior to the addition of T4 Endo V (10 ng). The reaction was performed as described in Materials and Methods. Bands protected by Gadd45 are indicated by brackets.