Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Poly(ADP-ribose) Polymerase 1 Is Inhibited by a Histone H2A Variant, MacroH2A, and Contributes to Silencing of the Inactive X Chromosome

2007; Elsevier BV; Volume: 282; Issue: 17 Linguagem: Inglês

10.1074/jbc.m610502200

ISSN

1083-351X

Autores

Dmitri A. Nusinow, Inmaculada Hernández‐Muñoz, Thomas G. Fazzio, Girish M. Shah, W. Lee Kraus, Barbara Panning,

Tópico(s)

Plant Virus Research Studies

Resumo

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is involved in modulating chromatin structure, regulation of gene expression, and sensing DNA damage. Here, we report that PARP-1 enzymatic activity is inhibited by macroH2A, a vertebrate histone H2A variant that is enriched on facultative heterochromatin. MacroH2A family members have a large C-terminal non-histone domain (NHD) and H2A-like histone domain. MacroH2A1.2 and PARP-1 interact in vivo and in vitro via the NHD. The NHD of each macroH2A family member was sufficient to inhibit PARP-1 enzymatic activity in vitro. The NHD of macroH2A1.2 was a mixed inhibitor of PARP-1 catalytic activity, with affects on both catalytic activity and the substrate binding affinity of PARP-1. Depletion of PARP-1 by RNA interference caused reactivation of a reporter gene on the inactive X chromosome, demonstrating that PARP-1 participates in the maintenance of silencing. These results suggest that one function of macroH2A in gene silencing is to inhibit PARP-1 enzymatic activity, and this may affect PARP-1 association with chromatin. Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is involved in modulating chromatin structure, regulation of gene expression, and sensing DNA damage. Here, we report that PARP-1 enzymatic activity is inhibited by macroH2A, a vertebrate histone H2A variant that is enriched on facultative heterochromatin. MacroH2A family members have a large C-terminal non-histone domain (NHD) and H2A-like histone domain. MacroH2A1.2 and PARP-1 interact in vivo and in vitro via the NHD. The NHD of each macroH2A family member was sufficient to inhibit PARP-1 enzymatic activity in vitro. The NHD of macroH2A1.2 was a mixed inhibitor of PARP-1 catalytic activity, with affects on both catalytic activity and the substrate binding affinity of PARP-1. Depletion of PARP-1 by RNA interference caused reactivation of a reporter gene on the inactive X chromosome, demonstrating that PARP-1 participates in the maintenance of silencing. These results suggest that one function of macroH2A in gene silencing is to inhibit PARP-1 enzymatic activity, and this may affect PARP-1 association with chromatin. In eukaryotic cells DNA is packaged into chromatin, and this packaging impacts all DNA-templated processes, including transcription. Regulated changes in chromatin structure are crucial to establish and maintain the diverse expression profiles that characterize the hundreds of cell types in multicellular organisms (1Kornberg R.D. Lorch Y. Cell. 1999; 98: 285-294Abstract Full Text Full Text PDF PubMed Scopus (1421) Google Scholar). The nucleosome is the structural unit of chromatin and comprises 147 bp of DNA wrapped around a histone octamer, which is composed of two copies each of the four core histones H2A, H2B, H3, and H4.Chromatin structure can be affected through the action of ATP-dependent chromatin remodeling enzymes or by the covalent modification of histone proteins, creating binding sites for additional regulatory proteins (1Kornberg R.D. Lorch Y. Cell. 1999; 98: 285-294Abstract Full Text Full Text PDF PubMed Scopus (1421) Google Scholar, 2Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7536) Google Scholar). In addition, chromatin structure can be modulated through the binding of effector proteins to nucleosomes. Poly(ADP-ribose) polymerase 1 (PARP-1) 5The abbreviations used are: PARP-1, poly(ADP-ribose) polymerase 1; NHD, non-histone domain; GFP, green fluorescent protein; mH2A, macroH2A; BRCT, BRCA1 C-terminal region; BAL, B-aggressive lymphoma; PBS, phosphate-buffered saline; Xi, inactive X chromosome; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; HA, hemagglutinin; RNAi, RNA interference; 5-Aza-dC, 5-aza-2′-deoxycytidine; TSA, trichostatin A; shRNA, short hairpin RNA. 5The abbreviations used are: PARP-1, poly(ADP-ribose) polymerase 1; NHD, non-histone domain; GFP, green fluorescent protein; mH2A, macroH2A; BRCT, BRCA1 C-terminal region; BAL, B-aggressive lymphoma; PBS, phosphate-buffered saline; Xi, inactive X chromosome; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; HA, hemagglutinin; RNAi, RNA interference; 5-Aza-dC, 5-aza-2′-deoxycytidine; TSA, trichostatin A; shRNA, short hairpin RNA. is an example of a nucleosome binding protein that can affect chromatin structure (3Kim M.Y. Mauro S. Gevry N. Lis J.T. Kraus W.L. Cell. 2004; 119: 803-814Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 4Kim M.Y. Zhang T. Kraus W.L. Genes Dev. 2005; 19: 1951-1967Crossref PubMed Scopus (645) Google Scholar). PARP-1 is the prototypical member of a family of PARP proteins, which catalyze the transfer of ADP-ribose units from donor nicotinamide adenine dinucleotide (NAD+) molecules to target proteins (5Ame J.C. Spenlehauer C. de Murcia G. BioEssays. 2004; 26: 882-893Crossref PubMed Scopus (1205) Google Scholar). PARP-1 functions as a structural component of chromatin, modulator of chromatin structure, and a sensor of DNA damage through its intrinsic enzymatic activity (4Kim M.Y. Zhang T. Kraus W.L. Genes Dev. 2005; 19: 1951-1967Crossref PubMed Scopus (645) Google Scholar, 6Rouleau M. Aubin R.A. Poirier G.G. J. Cell Sci. 2004; 117: 815-825Crossref PubMed Scopus (170) Google Scholar, 7Hassa P.O. Haenni S.S. Elser M. Hottiger M.O. Microbiol. Mol. Biol. Rev. 2006; 70: 789-829Crossref PubMed Scopus (562) Google Scholar). In the absence of NAD+, PARP-1 binds to nucleosomes, compacts chromatin, and inhibits transcription in vitro (3Kim M.Y. Mauro S. Gevry N. Lis J.T. Kraus W.L. Cell. 2004; 119: 803-814Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). Furthermore, catalytically inactive PARP-1 maintains silencing of heterochromatic retrotransposons in Drosophila (8Tulin A. Stewart D. Spradling A.C. Genes Dev. 2002; 16: 2108-2119Crossref PubMed Scopus (172) Google Scholar). However, at physiological concentrations of NAD+, PARP-1 is enzymatically active and does not bind nucleosomes (3Kim M.Y. Mauro S. Gevry N. Lis J.T. Kraus W.L. Cell. 2004; 119: 803-814Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). Despite this, PARP-1 binds chromatin in vivo and is implicated in transcriptional silencing, suggesting that modulation of PARP-1 activity in vivo may be a mechanism that is employed to direct changes in chromatin structure.Chromatin structure can also be regulated by replacement of core histones with histone variants. MacroH2A1 and macroH2A2 are vertebrate-specific variants that replace H2A in an estimated three percent of nucleosomes (9Pehrson J.R. Fried V.A. Science. 1992; 257: 1398-1400Crossref PubMed Scopus (278) Google Scholar). MacroH2A family members consist of a histone domain that is highly similar to H2A and a large C-terminal non-histone domain (NHD). The NHD domain is related to a family of proteins that includes a class of ADP-ribose processing enzymes and NAD+ metabolite binding proteins (10Martzen M.R. McCraith S.M. Spinelli S.L. Torres F.M. Fields S. Grayhack E.J. Phizicky E.M. Science. 1999; 286: 1153-1155Crossref PubMed Scopus (354) Google Scholar, 11Karras G.I. Kustatscher G. Buhecha H.R. Allen M.D. Pugieux C. Sait F. Bycroft M. Ladurner A.G. EMBO J. 2005; 24: 1911-1920Crossref PubMed Scopus (382) Google Scholar, 12Kustatscher G. Hothorn M. Pugieux C. Scheffzek K. Ladurner A.G. Nat. Struct. Mol. Biol. 2005; 12: 624-625Crossref PubMed Scopus (244) Google Scholar). MacroH2A1 consists of two isoforms, macroH2A1.1 and macroH2A1.2, that are produced by alternative splicing and differ by only 30 amino acids in the NHD. All three forms of macroH2A are enriched in regions of silent chromatin, such as the inactive X chromosome (Xi) and senescence-associated heterochromatin (13Costanzi C. Pehrson J.R. Nature. 1998; 393: 599-601Crossref PubMed Scopus (476) Google Scholar, 14Chadwick B.P. Willard H.F. Hum. Mol. Genet. 2001; 10: 1101-1113Crossref PubMed Scopus (141) Google Scholar, 15Costanzi C. Pehrson J.R. J. Biol. Chem. 2001; 276: 21776-21784Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 16Zhang R. Poustovoitov M.V. Ye X. Santos H.A. Chen W. Daganzo S.M. Erzberger J.P. Serebriiskii I.G. Canutescu A.A. Dunbrack R.L. Pehrson J.R. Berger J.M. Kaufman P.D. Adams P.D. Dev. Cell. 2005; 8: 19-30Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 17Changolkar L.N. Pehrson J.R. Mol. Cell. Biol. 2006; 26: 4410-4420Crossref PubMed Scopus (50) Google Scholar). Depletion of macroH2A1 in female cells causes reactivation of genes on the Xi, demonstrating a role in the maintenance of silent chromatin (18Hernandez-Munoz I. Lund A.H. van der Stoop P. Boutsma E. Muijrers I. Verhoeven E. Nusinow D.A. Panning B. Marahrens Y. van Lohuizen M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7635-7640Crossref PubMed Scopus (245) Google Scholar). MacroH2A1 is also required for silencing of the IL-8 gene in a cell-type specific fashion, indicating that it regulates gene expression at individual loci as well as larger domains (19Agelopoulos M. Thanos D. EMBO J. 2006; 25: 4843-4853Crossref PubMed Scopus (76) Google Scholar).Both the H2A-like domain and the NHD of macroH2A are implicated in regulation of gene expression. When the macroH2A1 H2A-like domain is incorporated into nucleosomes, it interferes with SWI/SNF nucleosome remodeling in vitro (20Angelov D. Molla A. Perche P.Y. Hans F. Cote J. Khochbin S. Bouvet P. Dimitrov S. Mol. Cell. 2003; 11: 1033-1041Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). The NHD associates with histone deacetylases in vivo, interferes with the binding of NF-κB in vitro, and also inhibits the initiation of transcription (20Angelov D. Molla A. Perche P.Y. Hans F. Cote J. Khochbin S. Bouvet P. Dimitrov S. Mol. Cell. 2003; 11: 1033-1041Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 21Perche P.Y. Vourc'h C. Konecny L. Souchier C. Robert-Nicoud M. Dimitrov S. Khochbin S. Curr. Biol. 2000; 10: 1531-1534Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 22Chakravarthy S. Gundimella S.K. Caron C. Perche P.Y. Pehrson J.R. Khochbin S. Luger K. Mol. Cell. Biol. 2005; 25: 7616-7624Crossref PubMed Scopus (136) Google Scholar, 23Doyen C.M. An W. Angelov D. Bondarenko V. Mietton F. Studitsky V.M. Hamiche A. Roeder R.G. Bouvet P. Dimitrov S. Mol. Cell. Biol. 2006; 26: 1156-1164Crossref PubMed Scopus (110) Google Scholar), suggesting that macroH2A regulates transcription at multiple levels. Here, we provide evidence for a mechanism by which macroH2A can regulate chromatin structure and gene expression. We find that macroH2A associates with PARP-1 through the NHD in vivo and in vitro and that macroH2A blocks PARP-1 enzymatic activity in vitro. We also demonstrate that the NHD of macroH2A can promote recruitment of PARP-1 to the Xi. Finally, knockdown of PARP-1 results in reactivation of a gene on the Xi, consistent with a role for PARP-1 in maintenance of heterochromatic silencing. Together, these data suggest that transcriptional repression by macroH2A may be mediated through recruitment of PARP-1 and inhibition of its enzymatic activity.EXPERIMENTAL PROCEDURESHEK293 Cell Culture and Immunopurification—5 × 108 293 cells or 293-histone-green fluorescent protein (GFP) cells were washed in phosphate-buffered saline (PBS) and resuspended in lysis buffer (50 mm Tris, pH 7.4, 250 mm NaCl, 1 mm EDTA, 0.1% Triton X-100, 10 mm sodium butyrate, 1 mm phenylmethylsulfonyl fluoride, 1× phosphatase inhibitor mixtures I and II (Sigma), 0.5 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.7 μg/ml pepstatin). The resulting lysate was sonicated and centrifuged to remove debris, and the extract was adjusted to 1.8 mg ml-1. GFP-tagged histones were affinity purified from the supernatant from 293-histone-GFP cells using 2 μg of rabbit polyclonal anti-GFP antibody (Abcam) per mg of cell extract. Endogenous macroH2A1 was purified from the supernatant of 293 cells using 20 μg of rabbit polyclonal antibodies that recognize macroH2A1.1 and macroH2A1.2 (24Chu F. Nusinow D.A. Chalkley R.J. Plath K. Panning B. Burlingame A.L. Mol. Cell. Proteomics. 2006; 5: 194-203Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Immunoprecipitations were performed at 4 °C for 2 h and captured on protein-A-coated magnetic beads (Dynal), and the beads were washed three times in room temperature lysis buffer. Construction of H2A-GFP and macroH2A1.2-HD-GFP was performed by PCR of sequences from cDNAs generated from HEK293 cells and cloning into a backbone originating from pBOS-H2B-GFP (Clontech), generating a construct that expresses either H2A or macroH2A-HD tagged with GFP on the C terminus. After sequencing to ensure integrity, plasmids were transfected into HEK293 cells using FuGENE 6 (Roche Diagnostics) and stable lines were selected for using Blasticidin S (Invitrogen). Western blotting was performed according to standard procedures. Antibodies used were mouse C-2–10 anti-PARP (Trevigen) 1:1000, rabbit anti-PARP-1 antibody directed against the DNA-binding region 1:2000 (3Kim M.Y. Mauro S. Gevry N. Lis J.T. Kraus W.L. Cell. 2004; 119: 803-814Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar), rabbit anti-macroH2A1 antibody 1:1000 (24Chu F. Nusinow D.A. Chalkley R.J. Plath K. Panning B. Burlingame A.L. Mol. Cell. Proteomics. 2006; 5: 194-203Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), rabbit anti-macroH2A1 antibody 1:2000 (Upstate), rabbit anti-EZH2 1:400 (25Pasini D. Bracken A.P. Jensen M.R. Lazzerini Denchi E. Helin K. EMBO J. 2004; 23: 4061-4071Crossref PubMed Scopus (656) Google Scholar), mouse anti-HA.11 (Covance) 1:1000, and mouse JL-8 anti-GFP (Clonetech) 1:2000.Mass Spectrometry—In gel digestion and mass spectrometry (ProtTech, Inc.) identified 12 peptides, comprising 17.85% coverage of PARP-1.Chromosome Spreads—Cells were exposed to 1 μg/ml colchicine (Sigma) for 4 h at 37 °C, and mitotic cells were harvested by washing off plate. Cells were then washed twice in PBS, swollen in 0.075 m KCl for 30 min at 37 °C. Approximately 10,000 cells were spun down onto ethanol washed slides at 1300 rpm for 10 min in a Shandon Cytospin. Cells were then incubated in KCM buffer (120 mm KCl, 20 mm NaCl, 10 mm Tris-HCl, pH 8.0, 0.5 mm EDTA, and 0.1% Triton X-100) for 8 min, fixed in 2% paraformaldehyde diluted in 1x PBS for 10 min, and washed in PBS + 0.2% Tween 20, then processed for immunofluorescence as described (26Plath K. Fang J. Mlynarczyk-Evans S.K. Cao R. Worringer K.A. Wang H. de la Cruz C.C. Otte A.P. Panning B. Zhang Y. Science. 2003; 300: 131-135Crossref PubMed Scopus (943) Google Scholar). Anti-PARP-1 catalytic domain rabbit antibody was used at 1:200 (3Kim M.Y. Mauro S. Gevry N. Lis J.T. Kraus W.L. Cell. 2004; 119: 803-814Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar) and was detected with 1:200 Texas Red anti-rabbit secondary (Jackson ImmunoResearch Laboratories). All microscopy was performed on an Olympus BX-60 Fluorescence microscope. Images were captured using a Hamamatsu ORCA-ER CCD camera and Openlab Digital Darkroom software (Improvision).Protein Purification—All macroH2A non-histone domain GST/His6 constructs were generated by PCR cloning and insertion into pGEX-4T bacterial expression construct, followed by sequencing to ensure integrity of plasmids. BL21-DE3pLysS (Stratagene) bacteria transfected with constructs were induced with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside after growth to an absorbance of A600 0.6 for 3 h and harvested. Cells were lysed in EQ buffer (50 mm sodium Phosphate, pH 7.0, 300 mm NaCl, 1 mm PMSF, 1 μg/ml Aprotinin, 1 μm Leupeptin, and 0.1% Nonidet P-40). Lysates were clarified by centrifugation, loaded onto Talon Metal affinity beads, washed with 20 column volumes of EQ buffer, then 5 column volumes of high salt EQ buffer (50 mm sodium phosphate, pH 7.0, 1 m NaCl, 1 mm PMSF, 1 μg/ml aprotinin, 1 μm leupeptin, and 0.1% Nonidet P-40) to ensure removal of contaminating DNA, washed in 10 column volumes of EQ buffer with 20% glycerol, and then eluted with buffer EQoff (50 mm sodium phosphate, pH 7.0, 300 mm NaCl, 1 mm PMSF, 150 mm imidazol, 20% glycerol, and 0.1% Nonidet P-40). Fractions containing eluted proteins were then bound to glutathione resin in EQ buffer with 2 mm DTT added, washed in GST wash buffer (50 mm Tris-HCl, pH 8.0, 250 mm NaCl, 1 mm EDTA, 1 mm PMSF, and 1 mm DTT). Proteins were eluted in GST wash buffer plus 10 mm glutathione, then dialyzed against GST dialysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm PMSF, 1 mm DTT, 20% glycerol). FLAG-tagged human PARP-1 was expressed and purified from bacculovirus infected SF9 insect cells as described previously (3Kim M.Y. Mauro S. Gevry N. Lis J.T. Kraus W.L. Cell. 2004; 119: 803-814Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar).GST IP Protocol of Recombinant and in Vitro Transcribed Proteins—PARP-1 in vitro transcription templates were generated using PCR primers that allowed for the addition of a T7 promoter to the 5′ end and introduction of a HA epitope to the C terminus of the PARP-1 fragment. Templates were added to a TNT T7 Quick for PCR DNA (Promega) in vitro transcription and translation kit to generate HA-tagged proteins. 500 nm GST bait constructs were mixed with 75 nm FLAG-hPARP-1 or 16% of in vitro translated and transcribed PARP-1 pieces in GST IP buffer (50 mm Tris-HCl, pH 7.4, 200 mm NaCl, 1 mm EDTA, 0.02% Nonidet P-40, 10% glycerol, 1 mm PMSF) and incubated with glutathione beads (Amersham Biosciences) for 1.5 h at 4 °C, then washed five times with 1 ml of GST IP buffer, then boiled in SDS-loading buffer, and proteins were separated by SDS-PAGE. Recombinant PARP-1 was detected using a 1:1000 dilution of anti-PARP-1 antibody directed against the DNA-binding region (3Kim M.Y. Mauro S. Gevry N. Lis J.T. Kraus W.L. Cell. 2004; 119: 803-814Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar), and HA was detected using mouse anti-HA.11 (Covance) 1:1000.PARP Enzyme Assays—PARP-1 activity assays to determine an IC50 with macroH2A family members were performed under the following conditions (1 μl of high specific activity human PARP-1 (Trevigen), 50 mm Tris-HCl, pH 8.0, 25 mm MgCl2, 10 mm β-mercaptoethanol, 400 μm NAD+, and [32P]NAD+ to a final specific activity of 0.5 μCi/nmol NAD+) in a final volume of 100 μl. Inhibition by Gst-macroH2A1.2-NHD-His deletion constructs was performed in 100-μl assays with 65 nm purified FLAG-hPARP-1, 50 mm Tris-HCl, pH 8.0, 25 mm MgCl2, 1 mm DTT, and 400 μm NAD+, and [32P]NAD+ to a final specific activity of 0.5 μCi/nmol NAD+. Kinetic analysis of GST-macroH2A1.2-NHD-His of PARP-1 was performed as above, except trichloroacetic acid-precipitatable counts were normalized for the specific activity of NAD+ for each reaction (μCi/nmol NAD+). Each reaction was incubated with either 10 μl of dilution buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10% glycerol) or the appropriate purified GST construct in dilution buffer. PARP-1 and GST constructs were preincubated for 30 min at 30 °C, then NAD+ was added for 10 min at 30 °C, and reactions were stopped with the addition an icecold solution of 5% trichloroacetic acid/1% inorganic phosphate. Reactions were precipitated on ice for 10 min, spun at 14,000 rpm in a microcentrifuge for 10 min, and washed twice more with ice cold 5% trichloroacetic acid/1% inorganic phosphate. Precipitatable counts were measured using a Beckman scintillation counter. Trichloroacetic acid-precipitatable PARP activity was in the linear range for reaction conditions (data not shown).Short Hairpin RNA Interference (shRNAi) and X Chromosome Reactivation Experiments—Plasmid for RNAi constructs carrying short hairpin RNA sequences expressed under the control of the U6 or the H1 promoter were made as previously described (27Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3942) Google Scholar, 28Sui G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google Scholar). The Parp-1 RNAi was obtained from G. M. Shah and subcloned into pSuper-RetroPuromycin (OligoEngine) (29Shah R.G. Ghodgaonkar M.M. Affar el B. Shah G.M. Biochem. Biophys. Res. Commun. 2005; 331: 167-174Crossref PubMed Scopus (20) Google Scholar). Mel-18 and macroH2A1 RNAi plasmid have been described previously (18Hernandez-Munoz I. Lund A.H. van der Stoop P. Boutsma E. Muijrers I. Verhoeven E. Nusinow D.A. Panning B. Marahrens Y. van Lohuizen M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7635-7640Crossref PubMed Scopus (245) Google Scholar). Reactivation of GFP in SV40 T antigen-transformed mouse embryonic fibroblasts with the X-inactivated GFP transgene was performed as described before (18Hernandez-Munoz I. Lund A.H. van der Stoop P. Boutsma E. Muijrers I. Verhoeven E. Nusinow D.A. Panning B. Marahrens Y. van Lohuizen M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7635-7640Crossref PubMed Scopus (245) Google Scholar). Briefly, Phoenix cells were transfected and used to generate retroviral stocks, and mouse embryonic fibroblasts cultures were transduced with the viral supernatants in the presence of polybrene (4 μg/ml). Puromycin selection was added to the cultures 24 h after viral transduction (4 μg/ml). After selection, the cells were exposed to 5-aza-2′-deoxycytidine (5-Aza-dC)(4 days at 300 nm) and to 500 nm trichostatin A (TSA) for the last 24h. The cells were subjected to fluorescence-activated cell sorter analysis counting a minimum of 100,000 cells per sample. Experiments were performed in duplicate at least three times.RESULTSMacroH2A1.2 Associates with PARP-1 via the NHD— MacroH2A1.2 contains a large C-terminal NHD of unknown function. To investigate function of the NHD, we identified proteins that associated with full-length macroH2A1.2 (mH2A1.2) but not canonical H2A or the histone domain of macroH2A1 (mH2A1-HD). HEK293 cell lines expressing mH2A1.2, H2A, or the mH2A1-HD fused to GFP were generated. All three GFP-tagged histones were detected in the nucleus and on mitotic chromosomes, demonstrating they were incorporated into chromatin (data not shown). Whole cell extracts from all three histone-GFP lines and HEK293 cells were immunoprecipitated with GFP antibodies. SYPRO Red staining of immunoprecipitated material showed co-precipitation of the core histones with the GFP-tagged histones (Fig. 1A), confirming that all three fusion proteins were incorporated into chromatin. A prominent band of ∼110 kDa present only in the mH2A1.2-GFP immunoprecipitates was identified as PARP-1 by mass spectrometry, a protein implicated in regulation of chromatin structure. Western blotting of GFP immunoprecipitates confirmed that mH2A1.2-GFP exhibited significant PARP-1 binding, while H2A-GFP and mH2A1-HD-GFP did not (Fig. 1B). In addition, we detected co-precipitation of endogenous PARP-1 with macroH2A1 from HEK293 cells, using polyclonal antiserum that recognizes both macroH2A1 splice variants (mH2A1.1 and mH2A1.2 (24Chu F. Nusinow D.A. Chalkley R.J. Plath K. Panning B. Burlingame A.L. Mol. Cell. Proteomics. 2006; 5: 194-203Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar); Fig. 1C). Together, these data show that mH2A1.2 interacts physically with PARP-1 and that the interaction requires the NHD.MacroH2A1.2 is enriched on the Xi relative to the active X chromosome and autosomes, and sequences sufficient for enrichment on the Xi lie within the mH2A1-HD (21Perche P.Y. Vourc'h C. Konecny L. Souchier C. Robert-Nicoud M. Dimitrov S. Khochbin S. Curr. Biol. 2000; 10: 1531-1534Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 30Chadwick B.P. Valley C.M. Willard H.F. Nucleic Acids Res. 2001; 29: 2699-2705Crossref PubMed Scopus (52) Google Scholar). We examined whether PARP-1 was also enriched on the Xi by immunostaining for PARP-1 in mitotic chromosome spreads from the macroH2A1.2-GFP, mH2A1-HD-GFP, H2A-GFP, and parental HEK293 cell lines. In mitotic spreads from HEK293 cells, PARP-1 was not appreciably detectable (data not shown). While the H2A-GFP line showed an overall increase in the amount of PARP-1 staining of all chromosomes, it did not show enrichment of PARP-1 on a particular chromosome. Both mH2A1.2-GFP and mH2A1.2-HD-GFP are enriched on the Xi (24Chu F. Nusinow D.A. Chalkley R.J. Plath K. Panning B. Burlingame A.L. Mol. Cell. Proteomics. 2006; 5: 194-203Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), which allows for easy identification of the Xi in spreads. In the mH2A1.2-GFP line, PARP-1 was enriched on the Xi in 79.3 ± 5.1% of spreads (Fig. 1D), while in the mH2A1-HD-GFP line PARP-1 was enriched on the Xi in 1 ± 0.8% of spreads (n = 400). These data indicate that enrichment of macroH2A1.2-GFP on the Xi promotes PARP-1 association in vivo, consistent with a role for the NHD in recruiting PARP-1.The PARP-1 Catalytic Domain Interacts with the MacroH2A1.2 NHD—To test whether the macroH2A1.2 NHD and PARP-1 interact directly, in the absence of additional proteins, as well as to map the domains of each protein required for association, we performed in vitro binding assays. The NHD consists of a basic region, a leucine zipper-like region, and an ADP-ribose phosphoesterase-like region (Fig. 2A). We expressed and purified GST and His6-tagged mH2A1.2-NHD, basic region (Basic), leucine zipper-like region (LZ), ADP-ribose phosphoesterase-like region (ADP), and the NHD without the leucine zipper-like region (NHDΔLZ). These were employed as bait in pulldown experiments, using recombinant, FLAG-tagged PARP-1 as the prey. PARP-1 associated strongly with the NHD, the leucine zipper-like region, the ADP-ribose phosphoesterase-like region, and the NHD without the leucine zipper-like region (Fig. 2B, lanes 3, 4, 6, and 7) but not with the basic region or GST alone (Fig. 2B, lanes 2 and 5). Thus, both the leucine zipper-like region and the ADP-ribose phosphoesterase-like region of the mH2A1.2-NHD can each independently interact with PARP-1 in vitro.FIGURE 2Mapping of the PARP-1 and macroH2A1.2 NHD interaction. A, diagram of the GST-His6 tagged macroH2A1.2 NHD (mH2A1.2-HD) constructs employed in pull down experiments. LZ indicates leucine-zipper-like region and ADP indicates ADP-ribose phosphoesterase-like region. B, GST pull downs of recombinant PARP-1 using tagged macroH2A1.2 NHD constructs. Left panels, Coomassie-stained SDS-PAGE gels showing the purified GST-His6-tagged NHD polypeptides and human FLAG-PARP-1 used in this study. Right panels, Western blot for PARP-1 (above). Ponceau S stain of the transferred GST-fusion proteins associated with the beads is shown (below). 10% of PARP-1 input is shown (In). C, diagram indicating regions of PARP-1 employed in co-immunoprecipitation experiments. ZnF indicates the zinc finger DNA binding region, BRCT indicates the region of the PARP-1 containing the BRCT automodification domain, and CAT indicates the catalytic domain. D, inputs (In) and pull downs (IP) of in vitro transcribed and translated HA-tagged PARP-1 pieces using GST (left) or GST-macroH2A1.2-NHD-His (right) as bait. Upper, Western blots showing the location of PARP-1-HA products. Lower, Ponceau S stain of the membranes, showing the bait in each pulldown sample. 10% of the PARP-1 input to the pulldown reaction is shown in the input lanes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, we examined which regions of PARP-1 associated with the macroH2A1.2 NHD. PARP-1 consists of a zinc finger (ZnF) DNA binding region, a BRCA1 C-terminal repeat region (BRCT) automodification domain, and a catalytic (CAT) domain (Fig. 2C). HA-tagged versions of these three regions of PARP-1 were transcribed and translated in vitro and used as prey in the presence of GST-His6 tagged mH2A1.2-NHD, which served as bait. Compared with GST-alone, the GST-mH2A1.2-NHD interacted with the zinc finger region (Fig. 2D, lane 8) and interacted even more strongly with the catalytic domain of PARP-1 (Fig. 2D, lane 12). The mH2A1.2-NHD did not interact with the BRCT automodification domain (Fig. 2D, lane 10). These data show that multiple domains of each protein facilitate direct interaction of macroH2A1.2 with PARP-1.The MacroH2A NHD Inhibits PARP-1 Enzymatic Activity— The interaction between the macroH2A1.2 NHD and the catalytic domain of PARP-1 prompted us to investigate whether the NHD had any affect on PARP-1 catalytic activity. Human PARP-1 and [32P]NAD+ were incubated with GST-mH2A1.2-NHD-His6, GST, or buffer alone and the products of the PARP-1 enzymatic reaction visualized by SDS-PAGE and autoradiography. The high molecular weight smear observed when PARP-1 and [32P]NAD+ were incubated with buffer or GST alone is indicative of the addition of poly(ADP-ribose) oligomers to PARP-1 or PARP-1 automodification (Fig. 3A, lanes 2–4). GST-mH2A1.2-NHD-His6 significantly inhibited PARP-1 automodification relative to buffer or GST alone (Fig. 3A, lanes 2–4). Quantitation of PARP-1 activity showed that GST-mH2A1.2-NHD-His6 inhibited PARP-1 enzymatic activity by ∼10-fold (Fig. 3B). To examine whether inhibition of PARP-1 was a common feature of the macroH2A family, we purified and tested the NHDs of other macroH2A variants: macroH2A1.1 and macroH2A2 (Fig. 2B, lanes 3 and 4). We found that GST fusions of these NHDs also inhibited PARP-1 activity ∼10-fold (Fig. 3B). In titration experiments, the NHDs of all three macroH2A isoforms exhibited a half-maximal inhibitory concentration (IC50) of ≈100 ± 20 nm (Fig. 3C), indicating that the ability to inhibit PARP-1 enzymatic activity is conserved amo

Referência(s)