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Division of Pharmacology, The Ohio State University College of Pharmacy (J.L.R., H.H., S.F.W., D.G.H.)
the Dorothy M. Davis Heart and Lung Research Institute (D.G.H.), Columbus, Ohio
Abstract
Engagement of integrin cell adhesion receptors in mouse lung endothelial cells induces global sensitivity of DNA to nuclease digestion, reflecting alterations in chromatin structure. These structural changes may contribute to the antigenotoxic effects of integrin engagement in lung endothelium. Because histone acetylation and poly(ADP-ribosyl)ation modulate chromatin structure, we investigated the effects of 1 integrin engagement with antibody on these post-translational modifications and the presence of histones at discrete DNA sequences in the mouse lung endothelial cell genome using chromatin immunoprecipitation. Integrin engagement increased acetylation of core histone H3. The presence of acetylated histone H3 at intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) promoters, and a nonpromoter sequence was also increased. As with integrin engagement, the histone deacetylase inhibitor trichostatin A caused global hypersensitivity of DNA to nuclease digestion and induced acetylation of histone H3 and its coimmunoprecipitation with VCAM-1 and ICAM-1 promoters and nonpromoter DNA. In contrast to acetyl-histone H3, the association of linker histone H1 with specific DNA sequences was either reduced or unaffected by integrin engagement and trichostatin A. Although integrin engagement and trichostatin A treatment did not affect histone H1 poly(ADP-ribosyl)ation, deletion of poly(ADP-ribose) polymerase-1 increased core histone H3 acetylation and increased its level at the iNOS promoter while decreasing the amount of histone H1. The results suggest that integrin engagement, as well as trichostatin A and PARP-1 deletion, regulate chromatin structure via core histone H3 acetylation and reduced linker histone H1-DNA association.
Integrins are a family of heterodimeric cell surface receptors composed of subunits that mediate adhesion of cells to each other, extracellular matrix, and other ligands. Integrins mediate signal transduction, and antibodies to specific subunits promote endothelial survival (Meredith et al., 1993; Aplin et al., 1998). Integrin signaling modulates DNA damage and anticancer drug action in various cell types (Buckley et al., 1999; Hazlehurst et al., 2001). Engagement of 1 integrins with antibodies or peptide ligands inhibited rapid DNA strand breakage caused by bleomycin in mouse lung endothelial cells (MLEC) (Hoyt et al., 1997; Jones et al., 2001). Integrins also inhibit DNA strand breaks caused by bacterial endotoxin and the topoisomerase inhibitor etoposide. This ability to inhibit DNA damage caused by numerous agents might be due to a common mechanism, such as an alteration in the nucleus or stimulation of DNA repair (Hoyt et al., 1996a,b). Engagement of 1 integrins indeed alters MLEC chromatin structure, as indicated by an induction of global hypersensitivity of DNA to exogenous nucleases (Jones et al., 2001; Huang et al., 2003).
DNA digestion by nucleases is restricted by DNA-protein interactions in chromatin. Chromatin includes nuclear DNA organized in compact packages with proteins. The fundamental repeating unit of chromatin is the nucleosome, which contains 146 to 165 bp of DNA wrapped around histone proteins. The nucleosome consists of two each of the core histones H2A, H2B, H3, and H4. Linker histone H1 binds nucleosomes where the coil of DNA enters and exits the core particle, and it attaches to DNA between nucleosomes, offering further stabilization of the chromatin fibers (Hansen, 2002).
Chromatin structure is regulated by post-translational modifications of histones and through chromatin remodeling complexes. Modifications of histones include acetylation, poly(ADP-ribosyl)ation, methylation, phosphorylation, ubiquitination, and biotinylation (Hansen, 2002). Acetylation of core histones and poly(ADP-ribosyl)ation of linker histone enhance digestibility of cellular DNA with nucleases (Simpson, 1978; Perez-Lamigueiro and Alvarez-Gonzalez, 2004).
Electrostatic effects of acetylation and poly(ADP-ribosyl)ation alter histone-DNA and histone-protein interactions in the nucleus, contributing to nuclease hypersensitivity. Acetylation of N-terminal tails of core histones neutralizes positive charge on lysines and disrupts interaction with negative phosphates in the backbone of DNA (Hansen, 2002). Poly-(ADP-ribosyl)ation, largely mediated by poly(ADP-ribose) polymerase-1 (PARP-1), occurs mainly on linker histone H1. Negatively charged ADP-ribose polymers interact poorly with anionic DNA, increasing accessibility of proteins to DNA (D'Amours et al., 1999; Ame et al., 2004). These post-translational modifications may contribute to integrin-mediated alterations in chromatin.
Because acetylation and poly(ADP-ribosyl)ation regulate histone interactions with DNA and other proteins, we hypothesized that integrin-mediated nuclease hypersensitivity is due to one or both of these histone modifications. Integrin actions on acetylation of core histone H3 and poly(ADP-ribosyl)ation of linker histone H1 were measured and compared with the effect of raising histone acetylation with the histone deacetylase inhibitor trichostatin A (TSA) and with PARP-1 knockout. Colocalization of these histones with specific DNA sequences in the genome of live cells was assessed by chromatin immunoprecipitation (ChIP). The results suggest that integrin engagement, trichostatin A, and PARP-1 knockout each increase histone H3 acetylation and alter the association of histone H1 with DNA.
Materials and Methods
Reagents. Endothelial cell growth supplement, heparin, phenylmethylsulfonyl fluoride, goat anti-rat IgG, phenol-chloroform-isoamyl alcohol, and Bradford reagent were purchased from Sigma Chemical Co. (St Louis, MO). Fetal bovine serum was purchased from Hyclone Laboratories (Logan, UT). Rat anti-mouse 1 integrin antibody was from BD PharMingen (San Diego, CA). Mouse anti-histone H1, rabbit anti-histone H3, and rabbit anti-acetyl lysine 9 and 14 histone H3 were purchased from Upstate Biotechnologies Inc. (Charlottesville, VA). Rabbit anti-poly(ADP-ribose) was obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Mouse anti-PARP-1 C2eC10 antibody was obtained from Trevigen Inc. (Gaithersburg, MD). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat antirabbit were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Gamma Bind Plus Sepharose was from Amersham Biosciences (Uppsala, Sweden). Bovine serum albumin and digoxigenin-11-dUTP were purchased from Roche Applied Science (Indianapolis, IN). DNA polymerase I was from New England Biolabs (Beverly, MA). Salmon sperm DNA, proteinase K, deoxynucleotides, platinum Taq DNA polymerase, DNase I, and trypsin were purchased from Invitrogen (Carlsbad, CA). Tris-base, sodium chloride, EDTA, Na3VO4, NaF, Tween 20, formaldehyde, SDS, agarose, and ethidium bromide were from Fisher Scientific (Fair Lawn, NJ). Triton X-100 was purchased from Pierce (Rockford, IL).
Cell Culture. Murine lung microvascular endothelial cells (MLEC) were isolated from wild-type (+/+) and PARP-1 knockout (-/-) mice as described previously (Jones et al., 2001).
Treatment of Cells. Integrin engagement with anti-1 Integrin antibody was performed as described previously (Hoyt et al., 1996a,b). PARP-1 +/+ and -/- MLEC were plated onto 75-cm2 flasks and, 24 h later, rinsed once with phosphate-buffered saline (PBS) and treated with 0 or 1 e of anti-1 integrin antibody/ml for 1 h at 37°C. Goat anti-rat IgG was added to all flasks for another 4 h of incubation at 37°C.
Cell Lysis. MLEC were washed three times with ice-cold PBS and lysed with lysis buffer containing 1% Triton X-100, 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5 mM Na3VO4, 50 mM NaF, 10 e/ml aprotinin, 10 e/ml leupeptin, 10 e/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride. Extracts were sonicated, and protein concentration was determined by the Bradford assay.
Immunoprecipitation. For immunoprecipitation of histone H1, 200 e of protein was incubated with 2 e of anti-histone H1 antibody overnight at 4°C. A 30% slurry of protein G Sepharose was added to each sample and rocked gently at 4°C for another 1 h. The antibody-antigen-Sepharose pellet was washed three times in ice-cold lysis buffer and two times in 10 mM Tris, pH 7.4. The washed pellet was reconstituted in protein loading buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 5% -mercaptoethanol, and 0.003% bromphenol blue) and heated for 10 min at 95°C. Proteins were separated on a 4 to 20% Tris-glycine polyacrylamide gel.
Total Protein Preparation. For total protein analysis, 10 e of protein was incubated with protein loading buffer and incubated for 10 min at 95°C. Proteins were separated on 4 to 20% Tris-glycine polyacrylamide gels.
Immunoblotting. After transfer, the blots were blocked with 3% nonfat dry milk in Tris-buffered saline buffer and Tween 20 (TTBS; 0.1% Tween 20, 10 mM Tris, pH 7.5, and 150 mM NaCl) and probed with primary antibody for 1 h at room temperature. After blots were washed, appropriate HRP-conjugated secondary antibody was added and incubated for 1 h. Proteins were visualized using enhanced chemiluminescent system and captured on X-ray film.
Chromatin Immunoprecipitation. After treatment, MLEC were fixed by addition of formaldehyde to a concentration of 1% directly in the culture medium for 10 min at 37°C. Cells were washed three times with PBS and scraped in ice-cold collecting buffer (PBS with 10 e/ml leupeptin, 10 e/ml aprotinin, 10 e/ml pepstatin A, and 1 mM phenylmethanesulfonyl fluoride). Collected cells were centrifuged at 2000 rpm for 4 min at 4°C. Pellets were resuspended in ice-cold lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris pH 8.1) and incubated on ice for 10 min. Chromatin was sheared to 1-kilobase fragments by sonication (four pulses, 20 s per pulse, at one-sixth maximum power) followed by addition of dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris, pH 8.1, and 167 mM NaCl). Immunoprecipitation of target histone protein was performed with the addition of 10 e of anti-Histone H1 or 10 e of anti-acetyl histone H3 (Lys 9 and 14) with gentle rocking overnight at 4°C. Immune complexes were collected for 1 h on Gamma Bind Plus Sepharose with 20 e/ml salmon sperm DNA. Immune complexes were sequentially washed for 4 min on ice with elution buffer A (0.1% SDS, 2 mM EDTA, 20 mM Tris, pH 8, 150 mM NaCl, and 1% Triton X-100), elution buffer B (0.1% SDS, 2 mM EDTA, 20 mM Tris, pH 8, 500 mM NaCl, and 1% Triton X-100), elution buffer C (0.25 M LiCl, 1% Nonidet P-40, 1 mM EDTA, 10 mM Tris, pH 8, and 1% sodium deoxycholate), and two washes with 10 mM Tris and 1 mM EDTA, pH 8. Immune complexes were eluted with 1% SDS in 0.1 M NaHCO3 and incubated at 65°C overnight to reverse DNA-protein cross-links. After proteinase K digestion (20 e, 1 h, 45°C) DNA was recovered with phenol-chloroform-isoamyl alcohol extraction and precipitated in ethanol. DNA was resuspended in 13.5 e of a mixture of 10 mM Tris and 1 mM EDTA and serially diluted to 1/12, 1/24, 1/48, and 1/96 of immunoprecipitated DNA for polymerase chain reactions (PCR) used to detect the relative level of specific DNA sequences recovered with the immunoprecipitated protein. Input samples (1% of volume after sonication) were reverseeCcross-linked and processed in the same way as immunoprecipitated DNA. The following promoter specific primers were used. inducible nitric-oxide synthase (iNOS): sense, 5'-CAACTATTGAGGCCACACAC-3'; antisense, 5'-AACCAGTGACACTGTGTCC-3'; 1133-bp product. VCAM-1: sense, 5'-CATTCTGCATCAACGTCC-3'; antisense, 5'-AAGTACCGTTGAGGCTCC-3'; 830-bp product. ICAM-1: sense: 5'-CTTGGATCGCTGCTTCAT-3'; antisense, 5'-AGCGGAGCTCAGCACTA-3'; 592-bp product. As a probe of a nonpromoter DNA, primers for a region upstream of the iNOS promoter were generated: sense, 5'-GTGTCACACCACAGAGCTGC-3'; antisense, 5'-GTGTCTCTGCTCCTCCATCC-3'; 984-bp product. The PCR conditions were as follows: 5 eof10x Taq buffer, 1.2 to 1.8 mM MgCl2, 200 e deoxyribonucleotides, 0.6 e primer mix, 0.05 units of platinum Taq DNA polymerase and H2O to a final volume of 50 e. PCR conditions were as follows: 94°C for 90 s; 35 cycles at 94°C for 30 s, 54eC66°C (depending on primer set) for 30 s and 72°C for 60 s; final elongation at 72°C 10 min. Samples were run on a 1% agarose gel and bands, digitally imaged, analyzed with Image J software (ver. 1.22d; http://rsb.info.nih.gov/ij/), and the integrated intensity of bands was calculated. The average pixel intensity and number of pixels in a band were multiplied to determine the total signal intensity. The ratio of antibody bound DNA samples was divided by the input sample for each dilution. The ratios were averaged for each treatment condition and indicate the relative presence of histones on specific DNA sequences.
DNase I Digestion and in Situ Nick Translation. After treatment of six wells per condition, cells in 96-well plates were fixed by addition of formaldehyde directly into the medium to a concentration of 1% for 10 min at 37°C. Cells were washed three times with PBS and made permeable by incubation overnight in 70% ethanol at -20°C. Cells were washed three times with PBS at 4°C. The relative sensitivity of nuclear DNA to digestion with exogenous nucleases was used to detect global alterations in chromatin structure (Hewish and Burgoyne, 1973; Hoyt et al., 1996a,b). Cells were rinsed in ISNT buffer (2.5 mM MgCl2, 50 mM Tris, pH 7.8, 10 mM -mercaptoethanol, and 10 e/ml bovine serum albumin) and then digested for 25 min at room temperature with 0 or 20 units/ml DNase I in ISNT buffer. The cells were rinsed three times with PBS. DNA breaks were then labeled by ISNT with digoxigenin-dUTP (Hoyt et al., 1997). Cells were incubated at 37°C for 80 min with ISNT buffer containing 16 e concentrations of dGTP, dATP, and dCTP, 16 e digoxigenin-dUTP, and 2 U/ml DNA polymerase I. Labeling was stopped by rinsing with PBS. Wells were blocked with 10% goat serum/TTBS for 1 h, incubated for 1 h with a 1:1000 dilution of mouse anti-digoxigenin in 10% goat serum/TTBS, washed three times with TTBS, and incubated for 1 h with 1:1000 dilution of goat anti-mouse HRP antibody. Amplex red reagent and hydrogen peroxide were added to each well according to manufacturer's instructions (Molecular Probes, Eugene, OR). Fluorescence was detected in a fluorescent microplate reader with excitation at 562 nm, emission at 599 nm, and a cutoff at 590 nm. Relative fluorescence units were obtained at 15 min in the linear phase of fluorescence. DNA content in each well was determined by addition of 25 ug/ml 33342 Hoechst with excitation at 355 nm and emission at 465 nm. Relative fluorescence units were normalized to DNA content in each well.
Statistical Testing. Data were analyzed by Student's t test or by analysis of variance with Bonferroni correction for multiple comparisons (Snedecor and Cochrane, 1980).
Results
Previous studies demonstrated that engagement of 1 integrins with antibody caused a global increase in sensitivity of MLEC DNA to digestion with nucleases (Jones et al., 2001). To determine the mechanisms underlying nuclease hypersensitivity, specific components of the chromatin architecture were probed for distinct post-translational modifications. Poly(ADP-ribosyl)ation and acetylation generally cause decondensation of chromatin, rendering DNA more susceptible to digestion with nucleases (Simpson, 1978; Realini and Althaus, 1992).
Linker histone H1 is the predominant histone modified with poly(ADP-ribose), which relaxes chromatin. Furthermore, a role for PARP-1 in integrin action was suggested by observations that integrin engagement failed to inhibit drug-induced DNA breakage in PARP-1 knockout cells and that knockouts were hypersensitive to DNase I (Jones et al., 2001; Huang et al., 2003). Therefore, we assessed poly(ADP-ribosyl)ation of histone H1 and its association with PARP-1 and DNA by immunoprecipitation of histone H1 from wild-type and PARP-1 knockout MLEC. PARP-1 coimmunoprecipiated with histone H1 in untreated wild-type cells, reflecting a basal association between the two proteins, and trichostatin A and integrin engagement did not affect this interaction. Furthermore, poly(ADP-ribosyl)ation of PARP-1 and of histone H1 in H1 immunoprecipitates was unaffected by trichostatin A or integrin engagement (data not shown). Histone H1 levels in PARP-1 knockout MLEC were equal to those in wild-type cells and, as expected, no poly(ADP-ribosyl)ation or association of PARP-1 with histone H1 was detected in the knockouts (not shown). The results indicate that integrin engagement and trichostatin A did not affect poly(ADP-ribosyl)ation of the 2 main nuclear substrates of PARP-1.
We next measured the effect of integrin engagement on histone acetylation. Wild-type MLEC were treated with integrin antibody and proteins were extracted for Western blotting. Figure 1A demonstrates a significant increase in acetyl-histone H3 in 1 integrin antibody-treated MLEC.
The effect of raising histone acetylation with a broad-spectrum histone deacetylase inhibitor, trichostatin A, was compared with integrin engagement. As expected, trichostatin A caused a concentration-dependent increase in global acetylation of histone H3 (Fig. 1B). Trichostatin A also increased the sensitivity of nuclear DNA to digestion with DNase I (Fig. 2).
Chromatin immunoprecipitation was used to measure local alterations in histone-DNA colocalization in live cells. We determined the effect of integrin engagement on the level of acetyl-histone H3 at the ICAM-1, VCAM-1, and iNOS promoter DNA sequences, near the start codons of these genes, where specific transcription factors are known to bind. Histone coimmunoprecipitation with a region that was 4763 base pairs 5' of the iNOS promoter sequence, which is characterized by a lack of consensus sites for transcription factors, was also measured. Integrin engagement increased the coimmunoprecipitation of acetyl-histone H3 with ICAM-1 and VCAM-1 promoters, and with the sequence upstream of the iNOS promoter (Fig. 3 AeCC). There was no effect on acetyl-histone H3 coimmunoprecipitation with the iNOS promoter sequence (Fig. 3D).
Because integrin engagement increased global and local histone H3 acetylation, we next determined the effect of trichostatin A on acetyl-histone H3 chromatin immunoprecipitation. As seen with 1-integrin engagement, trichostatin A increased the level of acetyl-histone H3 coimmunoprecipitated with VCAM-1 and ICAM-1 promoters and with the iNOS upstream sequence (Fig. 4, AeCC). Also as with integrin engagement, acetyl-histone H3 coimmunoprecipitated with iNOS promoter DNA was not affected by trichostatin A (Fig. 4D).
The effects of trichostatin A and integrin engagement on association of linker histone H1 with DNA was determined because reduced association of histone H1 with DNA may render DNA susceptible to nuclease digestion (Hansen, 2002). The histone H1 content of MLEC, measured by Western blotting, was not affected by 1-integrin antibody or trichostatin A (not shown). Integrin engagement decreased the level of linker histone H1 associated with the VCAM-1 and iNOS promoters (Fig. 5). Trichostatin A treatment reduced the association of histone H1 with the ICAM-1 and VCAM-1 promoters, and with the iNOS upstream sequence, but not with the iNOS promoter (Fig. 6).
Acetyl-histone H3 levels were greatly elevated in PARP-1 knockout relative to wild-type MLEC (Fig. 7). Furthermore, as with integrin engagement and treatment of wild-type cells with trichostatin A, coimmunoprecipitation of acetyl-histone H3 with VCAM-1 and iNOS promoter DNA and with DNA 5' of the iNOS promoter was increased in knockouts (Fig. 8, BeCD). In contrast, coimmunoprecipitation of ICAM-1 promoter DNA was reduced in PARP-1 knockout cells (Fig. 8A). As with integrin engagement and trichostatin A treatment, the amount of histone H1 at the iNOS promoter was decreased (Fig. 9). These data indicate that acetylation of the core histone H3 is increased in response to deletion of PARP-1 and that there were changes in histone-DNA coimmunoprecipitation that were generally similar, but not identical, to those caused by integrin engagement or the histone deacetylase inhibitor.
Discussion
We found previously that 1 integrin engagement increased the sensitivity of MLEC DNA to digestion by nucleases, indicating that chromatin structure is altered (Jones et al., 2001; Huang et al., 2003). In this study, we investigated the impact of integrin engagement on poly(ADP-ribosyl)ation and histone acetylation because both post-translational modifications regulate chromatin structure (Peterson and Cote, 2004).
Poly(ADP-ribosyl)ation of histones and other nuclear proteins dramatically increases negatively charged polymers of ADP-ribose in the vicinity of DNA breaks resulting in relaxation of chromatin structure (Perez-Lamigueiro and Alvarez-Gonzalez, 2004). This action of PARP-1 is thought to aid in the recruitment and access of DNA repair proteins to sites of DNA breaks (D'Amours et al., 1999; Parsons et al., 2005). We found previously that integrin-mediated suppression of bleomycin- and LPS-induced DNA strand breakage was prevented in PARP-1 knockout MLEC (Jones et al., 2001; Huang et al., 2003). Therefore, we hypothesized that integrin engagement might activate PARP-1 to generate polymers of ADP-ribose on nuclear proteins that would enhance nuclease sensitivity, recruit repair proteins, and ultimately suppress DNA damage. However, we found here that integrin engagement had no affect on poly(ADP-ribose) formation on the major PARP-1 targets, histone H1 and PARP-1. The requirement for PARP-1 in integrin-mediated suppression of drug-induced DNA breakage may be due to the direct role of PARP-1 protein in facilitating DNA repair rather than through modification of chromatin.
Acetylation of lysines neutralizes positive charge and reduces interaction of amino tails with negatively charged DNA (Hansen, 2002). In fact, raising acetylation with inhibitors of histone deacetylases causes nuclease hypersensitivity (Simpson, 1978). As expected, inhibition of histone deacetylases with trichostatin A in the present study increased the level of acetyl-histone H3 (Fig. 1B) and enhanced nuclease sensitivity (Fig. 2). Although integrin engagement did not affect poly(ADP-ribosyl)ation, it increased histone H3 acetylation (Fig. 1A) and nuclease sensitivity (Jones et al., 2001). The results suggest that acetylation of histone H3 may contribute to the effect of integrin engagement on chromatin.
It is interesting that PARP-1 knockouts, which are hypersensitive to nuclease digestion (Jones et al., 2001), were also highly acetylated on histone H3 (Fig. 8). Therefore, acetylation may also contribute to chromatin alterations in these cells. However, deletion of PARP-1 may raise nuclease sensitivity independent of acetylation. In vitro degradation of an oligonucleotide substrate by cellular nucleases was increased in PARP-1-depleted and PARP-1 knockout cell extracts, suggesting that PARP-1 directly protects DNA (Parsons et al., 2005). PARP-1 may also indirectly protect DNA by recruiting DNA binding proteins such as nuclear factor B (Chang and Alvarez-Gonzalez, 2001), and by association with nucleosomes, which promotes compaction (Kim et al., 2004). Acetylation and the absence of PARP-1 molecules together may produce the nuclease hypersensitivity seen in the knockouts.
Increased histone H3 acetylation in integrin antibody-treated and PARP-1 knockout cells may result from activation of histone acetyl transferases and/or reduction or inhibition of histone deacetylases. With respect to PARP-1, fibroblasts from knockout embryos had decreased expression of a number of histone acetyl transferases, but their activity was not measured (Ota et al., 2003). The activities of histone acetyl transferases and deacetylases need to be assessed in PARP-1 knockout and integrin antibody-treated MLEC. Acetylation may also increase in response to other histone post-translational modifications, such as phosphorylation or ubiquitination, which can affect the susceptibility of histones to acetyl transferases and deacetylases (Berger, 2001).
Having assessed the global chromatin characteristics, we measured histone presence at promoter and nonpromoter DNA to begin examining local variations in DNA-protein interactions in integrin antibody-treated, trichostatin A-treated, and PARP-1 knockout MLEC. Chromatin immunoprecipitation revealed increased relative presence of acetyl-histone H3 at promoter and nonpromoter DNA targets (Figs. 3, 4, and 8). Exceptions to this general trend at the ICAM promoter in PARP-1 knockouts, and the iNOS promoter in trichostatin A-treated and integrin antibody-treated MLEC, suggest that additional factors modulate the presence of acetyl-histone H3 at specific DNA targets. Nevertheless, increases in acetyl-histone H3 are consistent with global nuclease hypersensitivity in these experimental conditions.
Trichostatin A, 1 integrin engagement, and PARP-1 knockout generally reduced the level of linker histone H1 associated at the promoter and nonpromoter DNA targets (Figs. 5, 6, and 9). As an exception, trichostatin A did not reduce histone H1 association with iNOS promoter DNA, suggesting that additional factors affect histone H1 association at particular DNA targets. In any case, reduction in histone H1-DNA association in treated and knockout MLEC is also consistent with global nuclease hypersensitivity.
This study focused on the chromatin structural alterations caused by integrin engagement. The results suggest that acetylation of histone H3 may contribute to the opening of chromatin in response to integrin signals. In several other experimental systems, histone acetylation is associated with enhanced DNA repair (Ramanathan and Smerdon, 1989; Bird et al., 2002; Hasan and Hottiger, 2002; Peterson and Cote, 2004). Whether integrin-mediated acetylation of histone H3 inhibits drug-induced DNA damage or enhances repair remains to be determined.
In summary, integrin engagement, trichostatin A, and PARP-1 knockout increased acetylation of histone H3 and caused nuclease hypersensitivity in MLEC. Consistent with these global effects, the levels of acetyl-histone H3 present at several DNA targets were increased, whereas histone H1 association was generally reduced. The results suggest that integrin engagement, along with trichostatin A and PARP-1 deletion, regulate chromatin structure via core histone H3 acetylation and linker histone H1 dissociation.
Acknowledgements
We thank Csaba Szabo (Inotek Corporation, Beverly, MA) for providing PARP-1 knockout mice. The technical assistance of Rostislav I. Likhotvorik is also greatly appreciated.
doi:10.1124/mol.104.010876.
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