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• W2002071785 abstract "Pax5/B cell lineage specific activator protein (BSAP) is a B lineage-specific regulator that controls the B lineage-specific gene expression program and immunoglobulin gene VH to DJH recombination. Despite extensive studies on its multiple functions, little is known about how the activity of Pax5 is regulated. Here, we show that co-expression of histone acetyltransferase E1A binding protein p300 dramatically enhances Pax5-mediated transcriptional activation. The p300-mediated enhancement is dependent on its intrinsic histone acetyltransferase activity. Moreover, p300 interacts with the C terminus of Pax5 and acetylates multiple lysine residues within the paired box DNA binding domain of Pax5. Mutations of lysine residues 67 and 87/89 to alanine within Pax5 abolish p300-mediated enhancement of Pax5-induced Luc-CD19 reporter expression in HEK293 cells and prevent Pax5 to activate endogenous Cd19 and Blnk expression in Pax5−/− murine pro B cells. These results uncover a novel level of regulation of Pax5 function by p300-mediated acetylation. Pax5/B cell lineage specific activator protein (BSAP) is a B lineage-specific regulator that controls the B lineage-specific gene expression program and immunoglobulin gene VH to DJH recombination. Despite extensive studies on its multiple functions, little is known about how the activity of Pax5 is regulated. Here, we show that co-expression of histone acetyltransferase E1A binding protein p300 dramatically enhances Pax5-mediated transcriptional activation. The p300-mediated enhancement is dependent on its intrinsic histone acetyltransferase activity. Moreover, p300 interacts with the C terminus of Pax5 and acetylates multiple lysine residues within the paired box DNA binding domain of Pax5. Mutations of lysine residues 67 and 87/89 to alanine within Pax5 abolish p300-mediated enhancement of Pax5-induced Luc-CD19 reporter expression in HEK293 cells and prevent Pax5 to activate endogenous Cd19 and Blnk expression in Pax5−/− murine pro B cells. These results uncover a novel level of regulation of Pax5 function by p300-mediated acetylation. Pax5/BSAP is an important regulator for B lineage cell development (1Cobaleda C. Schebesta A. Delogu A. Busslinger M. Nat. Immunol. 2007; 8: 463-470Crossref PubMed Scopus (422) Google Scholar, 2Schebesta M. Heavey B. Busslinger M. Curr. Opin. Immunol. 2002; 14: 216-223Crossref PubMed Scopus (125) Google Scholar). It belongs to the PAX family of transcription factors with a highly conserved paired box DNA binding domain (PRD) 2The abbreviations used are: PRD, paired box DNA binding domain; Luc, luciferase; HAT, histone acetyltransferase; CBP, CREB-binding protein. (3Czerny T. Schaffner G. Busslinger M. Genes Dev. 1993; 7: 2048-2061Crossref PubMed Scopus (349) Google Scholar, 4Czerny T. Busslinger M. Mol. Cell. Biol. 1995; 15: 2858-2871Crossref PubMed Scopus (260) Google Scholar). The expression of Pax5 is initiated in the common lymphoid progenitor cells, continued through all stages of B lineage cells, and turned off in terminally differentiated plasma cells (1Cobaleda C. Schebesta A. Delogu A. Busslinger M. Nat. Immunol. 2007; 8: 463-470Crossref PubMed Scopus (422) Google Scholar, 2Schebesta M. Heavey B. Busslinger M. Curr. Opin. Immunol. 2002; 14: 216-223Crossref PubMed Scopus (125) Google Scholar). Pax5 controls the B lineage cell developmental progress at multiple steps. First, Pax5 activates the transcription of many B lineage-specific genes, such as Cd19, Mb1, Blnk, Ebf1, and Aid (1Cobaleda C. Schebesta A. Delogu A. Busslinger M. Nat. Immunol. 2007; 8: 463-470Crossref PubMed Scopus (422) Google Scholar, 2Schebesta M. Heavey B. Busslinger M. Curr. Opin. Immunol. 2002; 14: 216-223Crossref PubMed Scopus (125) Google Scholar, 5Nutt S.L. Morrison A.M. Dörfler P. Rolink A. Busslinger M. EMBO J. 1998; 17: 2319-2333Crossref PubMed Scopus (258) Google Scholar). In Pax5−/− mice, B cell development is blocked at the pro B cell stage (6Urbánek P. Wang Z.Q. Fetka I. Wagner E.F. Busslinger M. Cell. 1994; 79: 901-912Abstract Full Text PDF PubMed Scopus (668) Google Scholar, 7Nutt S.L. Urbánek P. Rolink A. Busslinger M. Genes Dev. 1997; 11: 476-491Crossref PubMed Scopus (337) Google Scholar). Recent studies showed that Pax5 is responsible for the induction of 170 target genes that encode proteins responsible for multiple functions in B lineage cells (8Schebesta A. McManus S. Salvagiotto G. Delogu A. Busslinger G.A. Busslinger M. Immunity. 2007; 27: 49-63Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 9Pridans C. Holmes M.L. Polli M. Wettenhall J.M. Dakic A. Corcoran L.M. Smyth G.K. Nutt S.L. J. Immunol. 2008; 180: 1719-1728Crossref PubMed Scopus (95) Google Scholar, 10Fuxa M. Busslinger M. J. Immunol. 2007; 178: 8222-8228PubMed Google Scholar). Second, Pax5 specifies the B lineage developmental program through repressing the expression of lineage-inappropriate genes, such as Notch1, M-CSFR/c-fms, and Ccl3 (1Cobaleda C. Schebesta A. Delogu A. Busslinger M. Nat. Immunol. 2007; 8: 463-470Crossref PubMed Scopus (422) Google Scholar, 11Nutt S.L. Heavey B. Rolink A.G. Busslinger M. Nature. 1999; 401: 556-562Crossref PubMed Scopus (1) Google Scholar, 12Cobaleda C. Busslinger M. Curr. Opin. Immunol. 2008; 20: 139-148Crossref PubMed Scopus (43) Google Scholar). Without these restrictions, Pax5−/− pro B cells are able to differentiate into Natural killer (NK) cells, dendritic cells, macrophages, osteoclasts, granulocytes, or T lineage cells under appropriate conditions (11Nutt S.L. Heavey B. Rolink A.G. Busslinger M. Nature. 1999; 401: 556-562Crossref PubMed Scopus (1) Google Scholar, 13Rolink A.G. Nutt S.L. Melchers F. Busslinger M. Nature. 1999; 401: 603-606Crossref PubMed Scopus (307) Google Scholar). Conditionally deleting Pax5 in mature B cells can lead to de-differentiation to uncommitted progenitors back into the bone marrow and then redevelop into T lineage cells (12Cobaleda C. Busslinger M. Curr. Opin. Immunol. 2008; 20: 139-148Crossref PubMed Scopus (43) Google Scholar, 14Nutt S.L. N. Engl. J. Med. 2008; 358: 82-83Crossref PubMed Scopus (6) Google Scholar). Third, Pax5 controls the B lineage differentiation progression through repressing the expression of differentiation stage-inappropriate genes, such as Flt3, Sca-1, Igj, Blimp1, and Cd28 (15Delogu A. Schebesta A. Sun Q. Aschenbrenner K. Perlot T. Busslinger M. Immunity. 2006; 24: 269-281Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 16Nera K.P. Kohonen P. Narvi E. Peippo A. Mustonen L. Terho P. Koskela K. Buerstedde J.M. Lassila O. Immunity. 2006; 24: 283-293Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Loss of Pax5 in avian or murine mature B cells also leads to early expression of plasma cell-specific genes (15Delogu A. Schebesta A. Sun Q. Aschenbrenner K. Perlot T. Busslinger M. Immunity. 2006; 24: 269-281Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 16Nera K.P. Kohonen P. Narvi E. Peippo A. Mustonen L. Terho P. Koskela K. Buerstedde J.M. Lassila O. Immunity. 2006; 24: 283-293Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Last, Pax5 plays an important role in regulation of the B lineage-specific immunoglobulin heavy chain gene (IgH) VH to DJH recombination. In Pax5−/− pro B cells, although the DH to JH recombination occurs normally, rearrangement of most of the upstream VH7183 and VHJ558 genes is severely compromised, except for a few DH-proximal VH7183 genes (7Nutt S.L. Urbánek P. Rolink A. Busslinger M. Genes Dev. 1997; 11: 476-491Crossref PubMed Scopus (337) Google Scholar, 17Hesslein D.G. Pflugh D.L. Chowdhury D. Bothwell A.L. Sen R. Schatz D.G. Genes Dev. 2003; 17: 37-42Crossref PubMed Scopus (131) Google Scholar). Conversely, forced expression of Pax5 transgenes in early T lineage cells can induce the recombination of a few DH-proximal VH7183 genes to the DJH locus (18Fuxa M. Skok J. Souabni A. Salvagiotto G. Roldan E. Busslinger M. Genes Dev. 2004; 18: 411-422Crossref PubMed Scopus (308) Google Scholar, 19Hsu L.Y. Liang H.E. Johnson K. Kang C. Schlissel M.S. J. Exp. Med. 2004; 199: 825-830Crossref PubMed Scopus (27) Google Scholar, 20Roldán E. Fuxa M. Chong W. Martinez D. Novatchkova M. Busslinger M. Skok J.A. Nat. Immunol. 2005; 6: 31-41Crossref PubMed Scopus (203) Google Scholar). It has also been shown that Pax5 is required for histone hypomethylation across the IgH locus (21Johnson K. Pflugh D.L. Yu D. Hesslein D.G. Lin K.I. Bothwell A.L. Thomas-Tikhonenko A. Schatz D.G. Calame K. Nat. Immunol. 2004; 5: 853-861Crossref PubMed Scopus (93) Google Scholar) and for contraction of the IgH locus prior to V(D)J recombination (18Fuxa M. Skok J. Souabni A. Salvagiotto G. Roldan E. Busslinger M. Genes Dev. 2004; 18: 411-422Crossref PubMed Scopus (308) Google Scholar, 20Roldán E. Fuxa M. Chong W. Martinez D. Novatchkova M. Busslinger M. Skok J.A. Nat. Immunol. 2005; 6: 31-41Crossref PubMed Scopus (203) Google Scholar), a mechanism that might facilitate the recombination of upstream VHJ558 genes by looping them closer to the DJH locus. Moreover, Pax5 has been found to associate with multiple VH gene coding regions and to interact with the recombination activating gene product (RAG)1/RAG2 protein complexes (22Zhang Z. Espinoza C.R. Yu Z. Stephan R. He T. Williams G.S. Burrows P.D. Hagman J. Feeney A.J. Cooper M.D. Nat. Immunol. 2006; 7: 616-624Crossref PubMed Scopus (58) Google Scholar). Through these interactions, Pax5 transactivates RAG-mediated VH to DJH recombination (22Zhang Z. Espinoza C.R. Yu Z. Stephan R. He T. Williams G.S. Burrows P.D. Hagman J. Feeney A.J. Cooper M.D. Nat. Immunol. 2006; 7: 616-624Crossref PubMed Scopus (58) Google Scholar). Pax5 interacts with many cellular factors to exert its multiple biological functions. For example, Pax5 forms a ternary complex with Ets-1 and binds to a complex Pax5/Ets binding site on the Mb-1 promoter to fully activate the Mb-1 gene transcription (23Fitzsimmons D. Hodsdon W. Wheat W. Maira S.M. Wasylyk B. Hagman J. Genes Dev. 1996; 10: 2198-2211Crossref PubMed Scopus (204) Google Scholar, 24Fitzsimmons D. Lutz R. Wheat W. Chamberlin H.M. Hagman J. Nucleic Acids Res. 2001; 29: 4154-4165Crossref PubMed Scopus (31) Google Scholar, 25Garvie C.W. Hagman J. Wolberger C. Mol. Cell. 2001; 8: 1267-1276Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 26Sigvardsson M. Clark D.R. Fitzsimmons D. Doyle M. Akerblad P. Breslin T. Bilke S. Li R. Yeamans C. Zhang G. Hagman J. Mol. Cell. Biol. 2002; 22: 8539-8551Crossref PubMed Scopus (86) Google Scholar). On the other hand, Pax5 interacts with Groucho protein family members of transcriptional co-repressors to repress transcription of target genes (27Eberhard D. Busslinger M. Cancer Res. 1999; 59: 1716s-1724sPubMed Google Scholar, 28Linderson Y. Eberhard D. Malin S. Johansson A. Busslinger M. Pettersson S. EMBO Rep. 2004; 5: 291-296Crossref PubMed Scopus (53) Google Scholar). The interaction between DAXX and Pax5 also modulates Pax5-mediated functions in different cell types (29Emelyanov A.V. Kovac C.R. Sepulveda M.A. Birshtein B.K. J. Biol. Chem. 2002; 277: 11156-11164Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). It has been well documented that acetylation of lysine residues within histone tails provides an important regulatory mechanism for transcription along DNA templates packed into the chromatin structure (30Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2356) Google Scholar, 31Yang X.J. Seto E. Mol. Cell. 2008; 31: 449-461Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). A group of histone acetyltransferases (HATs), including general control of amino acid synthesis 5 (GCN5/KAT2A), E1A binding protein p300 or Ep300 (p300), CREB-binding protein (CBP), p300/CBP-associating factor, and TATA Box Binding protein associated factor (TAFII250), can transfer acetyl groups to the ϵ-NH2 of lysine residues at the N terminus of histones (30Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2356) Google Scholar, 31Yang X.J. Seto E. Mol. Cell. 2008; 31: 449-461Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). Besides histones, HATs can acetylate many other cellular proteins, including transcription factors, to modulate their functions (31Yang X.J. Seto E. Mol. Cell. 2008; 31: 449-461Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). For example, acetylation of p53, GATA-1, NFκB p50, STAT6, and Runx1 enhances their binding to cognate DNA binding elements and augments their transcriptional activities (32Boyes J. Byfield P. Nakatani Y. Ogryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (628) Google Scholar, 33Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2144) Google Scholar, 34Hayakawa F. Abe A. Kitabayashi I. Pandolfi P.P. Naoe T. J. Biol. Chem. 2008; 283: 24420-24425Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 35Gingras S. Simard J. Groner B. Pfitzner E. Nucleic Acids Res. 1999; 27: 2722-2729Crossref PubMed Scopus (106) Google Scholar, 36Sheppard K.A. Rose D.W. Haque Z.K. Kurokawa R. McInerney E. Westin S. Thanos D. Rosenfeld M.G. Glass C.K. Collins T. Mol. Cell. Biol. 1999; 19: 6367-6378Crossref PubMed Google Scholar). Conversely, acetylation of Bcl-6 diminishes its DNA binding activity and attenuates Bcl-6-mediated transcriptional repression (37Chen L.F. Williams S.A. Mu Y. Nakano H. Duerr J.M. Buckbinder L. Greene W.C. Mol. Cell. Biol. 2005; 25: 7966-7975Crossref PubMed Scopus (359) Google Scholar). Acetylation of E2A enhances its transcriptional activation through increasing its nuclear retention (38Bradney C. Hjelmeland M. Komatsu Y. Yoshida M. Yao T.P. Zhuang Y. J. Biol. Chem. 2003; 278: 2370-2376Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Acetylation of p53 excludes polyubiquitination on the same lysine residue and thus stabilizes p53 proteins by preventing them from proteasome-dependent degradation (39Nakamura S. Roth J.A. Mukhopadhyay T. Mol. Cell. Biol. 2000; 20: 9391-9398Crossref PubMed Scopus (160) Google Scholar). In searching for potential regulators for Pax5-mediated function, several lines of evidence led us to explore the functional interaction between Pax5 and p300. It has been shown that CBP and p300 are involved in the regulation of numerous transcription factors in B lineage cells, including Pu.1, E47, EBF, and NF-κB (40Busslinger M. Annu. Rev. Immunol. 2004; 22: 55-79Crossref PubMed Scopus (374) Google Scholar). In particular, Pax5 interacts with GcN5 and CBP through an adaptor protein, Ada2, and thus recruits GcN5 and CBP as transcriptional co-activators (41Barlev N.A. Emelyanov A.V. Castagnino P. Zegerman P. Bannister A.J. Sepulveda M.A. Robert F. Tora L. Kouzarides T. Birshtein B.K. Berger S.L. Mol. Cell. Biol. 2003; 23: 6944-6957Crossref PubMed Scopus (52) Google Scholar). The requirement of p300 during early B lineage cell commitment and development had been demonstrated in conditional p300 knockout mice (p300flox/flox;Mx-Cre mice) (42Xu W. Fukuyama T. Ney P.A. Wang D. Rehg J. Boyd K. van Deursen J.M. Brindle P.K. Blood. 2006; 107: 4407-4416Crossref PubMed Scopus (50) Google Scholar) as well as in mice carrying KIX domain-deleted p300 proteins (p300KIX/KIX mice) (43Kasper L.H. Boussouar F. Ney P.A. Jackson C.W. Rehg J. van Deursen J.M. Brindle P.K. Nature. 2002; 419: 738-743Crossref PubMed Scopus (154) Google Scholar). In this study, we focused on the functional interaction between Pax5 and p300. We found that histone acetyltransferase p300 interacts with and acetylates Pax5 and thus acts as a potent co-activator for Pax5. HEK293 or ΦNX cells were maintained in DMEM (Cellgro, Herndon, VA) supplemented with 10% heat-inactive fetal bovine serum, 25 mm HEPES, 1 mm sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin. Human B lineage EU12 cells, Abelson retroviral transformed mouse pre B 2A cells, and Pax5−/− G5 pro B cells were maintained in RPMI medium (Cellgro) supplemented with 10% heat-inactive fetal bovine serum, 12.5 μm β-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin. The Luc-CD19 reporter construct and expression vectors for human Pax5, ΔHD, and ΔPRD constructs were kindly provided by the laboratory of Dr. Meinrad Busslinger. The wild-type p300 expression vector was obtained from the laboratory of Dr. Gary Nabel. The p300ΔHAT expression vector was obtained from the laboratory of Dr. Tso-Peng Yao. The Pax5 lysine-to-alanine mutant constructs were generated from the FLAG-Pax5 expression vector by a PCR-based mutagenesis approach using specific primers listed in the supplemental list of oligonucleotide sequences. All the mutant sites were confirmed by DNA sequencing. The coding regions of wild-type Pax5 and different Pax5 lysine-to-alanine mutants were subcloned into the pMI retroviral vectors in front of the Internal Ribosome Entry Site-GFP expression cassette to generate retroviral vectors for recombinant retrovirus production. The Luc-CD19 reporter constructs (0.5 μg) were transfected into HEK293 cells together with different combinations of expression vectors using the Fugene 6 (Roche) reagents following the manufacturer's instructions. A pCMV-Renilla luciferase expression vector (0.01 μg) was always included in each transient transfection to monitor the transfection efficiency. One day after transfection, cells were lysed, and luciferase activities were measured using the dual luciferase assay kit (Promega) on a luminometer (Turner Design or BMG). Nuclear extracts were prepared from EU12, 2A, or G5 cells, or HEK293 cells transiently transfected with different Pax5 constructs as described elsewhere (22Zhang Z. Espinoza C.R. Yu Z. Stephan R. He T. Williams G.S. Burrows P.D. Hagman J. Feeney A.J. Cooper M.D. Nat. Immunol. 2006; 7: 616-624Crossref PubMed Scopus (58) Google Scholar). After preclearance with non-relevant goat or rabbit IgG and protein A/G-agarose beads, immunoprecipitation was performed in HEGN150 buffer (10 mm Hepes, 0.25 mm EDTA, 10% glycerol, 150 mm NaCl, 0.1 mm DTT, 0.1 mm PMSF) using antibodies specific for Pax5 (N-19 and C-20, Santa Cruz Biotechnology) or p300 (C-20, Santa Cruz biotechnology) at 4 °C overnight with slow rotation. EtBr (10 μg/ml) was always included in the immunoprecipitation reaction to avoid nonspecific DNA binding. Immunoprecipitates were collected by additional incubation with protein G-agarose (50 μl) for 30 min. After washing four times with HEGN150 buffer, samples were eluted by boiling with 1× SDS gel loading buffer and analyzed by Western blotting. For detection of the full-length Pax5, monoclonal anti-Pax5 (A11, Santa Cruz Biotechnology) was used. For detection of different Pax5 truncation products, goat polyclonal anti-Pax5 antibodies (Santa Cruz Biotechnology), either the C-20 antibody recognizing the C terminus of Pax5 or the N-19 antibody recognizing the N terminus of Pax5, were used. Anti-GST antibody (Sigma) was used to detect the GST fusion Pax5. For Western blotting, the primary antibodies were diluted in Tris-Buffered Saline Tween-20 (TBST) with 5% nonfat dry milk, except for the anti-acetyl lysine antibodies (4G12, Upstate, Lake Placid, NY), which were diluted in TBST (1:200). Bound antibodies were detected using HRP-conjugated secondary antibodies, followed by standard ECL detection procedures. For co-immunoprecipitation assays, Trueblot secondary antibodies (eBioscience, San Diego, CA) were used in co-immunoprecipitation experiments to avoid detection of the immunoprecipitation antibodies. GST fusion full-length Pax5 protein or different Pax5 truncation constructs were expressed and purified from HEK293 cells using glutathione-agarose beads. After extensive washing with HEGN600 buffer (10 mm HEPES, 0.25 mm EDTA, 10% glycerol, 600 mm NaCl, 0.1 mm DTT, 0.1 mm PMSF), different GST-Pax5 constructs or GST protein retained on the beads was incubated with purified recombinant p300 (200 ng) (Active Motif, Carlsbad, CA) in 30 μl of HEGN150 buffer for 2 h at 4 °C. DNase (0.1 units/ml) and ethidium bromide (10 μg/ml) were included in the binding reaction to reduce nonspecific DNA binding. After washing four times with 1 ml HEGN150 buffer, bound proteins were eluted by boiling in 1× SDS gel loading buffer and analyzed by Western blotting. Tandem mass spectral analyses were performed at the University of Alabama at Birmingham Mass Spectroscopy Core and at the University of Nebraska Medical Center Mass Spectrometry Core Facility. Briefly, GST-Pax5 protein was purified from about 100 × 106 HEK293 cells co-transfected with GST-Pax5 and p300 expression vectors. Purified GST-Pax5 proteins (1–5 μg) were separated on premade SDS-PAGE gels (Bio-Rad) and stained with Coomassie Blue. A single band that correlates with the size of GST-Pax5 protein was excised and subjected to tryptic digestion at 37 °C for 16 h. The resulting peptides were purified and analyzed with the Q-TOF2 mass spectrometer (Micromass, Manchester, UK) at the University of Alabama at Birmingham or the LTQ Orbitrap XL ETD (Thermo Fisher Scientific) at the University of Nebraska Medical Center. The obtained results at the University of Alabama at Birmingham were analyzed manually to identify the acetylated peptides derived from the Pax5 protein. The results obtained at the University of Nebraska Medical Center were analyzed by Mascot via automated database searching of Mascot generic format files against the Human IPI protein database version 3.52. For in vitro acetylation assays, purified GST fusion Pax5 (1 μg) from HEK293 cells was incubated with full-length recombinant p300 (Active Motif) in the presence of 5 mm Tris HCl (pH8.0), 1% glycerol, 0.1 mm DTT, 0.1 mm PMSF, 1 mm sodium butyrate, and 12 mm acetyl CoA at room temperature for 1 h with gentle shaking. Reaction samples were separated by SDS-PAGE, and acetylated proteins were detected by monoclonal anti-acetyl-lysine antibodies (4G12, Upstate). Recombinant retroviruses were produced by co-transfection of the retroviral expression vectors together with the pGP and pEco booster vectors into semi-confluent ΦNX packaging cells using the Fugene 6 reagent (Roche). Culture supernatant containing recombinant retroviruses was collected 48 h after transfection and filtered through a 0.45-μm filter. The resulting live viruses were used to transduce Pax5−/− mouse pro B cells with the help of Polybrene (4 μg/ml). The culture medium was changed to Polybrene-free RPMI growth medium the following day. Transduction efficiency was monitored by FACS analysis evaluating the frequency of GFP-positive cells. All Pax5 constructs are expressed together with GFP from a bicistronic RNA transcript. The expression of Pax5 constructs can be tracked by analyzing GFP+ cells. After washing with FACS buffer (PBS supplied with 2% fetal bovine serum), cells (106) were stained with phycoerythin (PE) conjugated anti-mouse CD19 antibodies (BD Biosciences) on ice for 30 min. Cells were washed twice with 1 ml FACS buffer and resuspended in 500 μl FACS buffer containing propidium iodide (1 μg/ml). Samples were analyzed on a FACScalibur (BD Biosciences), and FACS data were analyzed using the Winmid 2.8 software. Pax5−/− pro B cells (G5) were reconstituted with retroviruses expressing wild-type or different mutant Pax5 constructs. The reconstituted (GFP+) cells were purified on a MoFlow FACS sorter (BD Biosciences) and directly collected in TRIzol reagent (Invitrogen). Total RNA was purified following the manufacturer's protocol. First-strand cDNA was synthesized using the Superscript III kit (Invitrogen). The cDNA samples were subjected to real-time PCR analysis on an ABI Prism 7900 sequence detection system (Applied Biosystems) using the SYBRGreen PCR master mix (SuperArray). Primers used are listed in the supplemental list of oligonucleotide sequences. The BSAP binding probe, derived from the Pax5 binding site within the Cd19 promoter, was labeled with Alexa Fluor 488 at the 5′ end. Nuclear extracts were prepared from HEK293 cells transiently transfected with expression vectors for the wild-type Pax5 or different lysine-to-alanine mutants without or with co-transfection of p300 expression vectors. Binding reactions were carried out in 20 μl of buffer containing 1 μg/μl poly-dI/dC, 10 mm HEPES (pH 7.9), 50 mm KCl, 10% glycerol, 0.2 mg/ml BSA, 0.1 mm DTT, and 0.1 mm PMSF at room temperature for 20 min. Samples were separated on 5% native polyacrylamide gel at 4 °C with Tris borate-EDTA buffer (8.9 mm Tris, 8.9 mm boric acid, 0.2 mm EDTA). Wet gels were directly analyzed with a FluorChemQ Multi Imager III (Alpha Innotech). Pax5/BSAP is a B lineage-specific transcription factor that activates many B lineage-specific genes (40Busslinger M. Annu. Rev. Immunol. 2004; 22: 55-79Crossref PubMed Scopus (374) Google Scholar). On the other hand, it has been shown that histone acetyltransferase p300 plays an important role during early B lineage cell commitment and development (42Xu W. Fukuyama T. Ney P.A. Wang D. Rehg J. Boyd K. van Deursen J.M. Brindle P.K. Blood. 2006; 107: 4407-4416Crossref PubMed Scopus (50) Google Scholar, 43Kasper L.H. Boussouar F. Ney P.A. Jackson C.W. Rehg J. van Deursen J.M. Brindle P.K. Nature. 2002; 419: 738-743Crossref PubMed Scopus (154) Google Scholar). To explore the potential functional interaction between p300 and Pax5, we performed luciferase reporter assays in HEK293 cells using the Pax5-responsive Luc-CD19 reporter construct with different combinations of the Pax5 and p300 expression vectors. Co-expression of Pax5 with the Luc-CD19 reporter construct in HEK293 cells resulted in a 7–10-fold induction of luciferase activities, which was dramatically enhanced by co-expression of p300 together with Pax5 (60-fold higher than that of the basal level) but not by expression of p300 alone (Fig. 1A). The p300-mediated enhancement is dependent on the level of p300 expression, as shown in a series of transient transfection assays using increasing amounts of p300 expression vectors (Fig. 1B and supplemental Fig. 1A). Overexpression of p300 did not affect Pax5 protein expression levels in these transient transfection assays (supplemental Fig. 1A). It has been well characterized that p300 possesses intrinsic HAT activity, which is required for its co-activator function (30Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2356) Google Scholar). Therefore, we compared the effects of the wild-type p300 and p300ΔHAT constructs on Pax5-mediated function. In contrast to the wild-type p300, the p300ΔHAT construct failed to enhance Pax5-mediated induction of the Luc-CD19 reporter expression (Fig. 1C), indicating that the p300 HAT activity is required for enhancing Pax5 function. In addition, the p300-mediated enhancement of Pax5 transcriptional activity was further augmented by blocking histone deacetylase activity with trichostatin A treatment (Fig. 1D). Conversely, treatment of transfected cells with anacardic acid to inhibit HAT activity attenuated p300-mediated enhancement (Fig. 1E). The p300-mediated enhancement is dependent on the DNA binding domain and transcriptional activation domain of Pax5 (supplemental Fig. 1B). Taken together, these results show that histone acetyltransferase p300 acts as a transcriptional co-activator for Pax5 and that the intrinsic HAT activity of p300 is essential for enhancing the transcriptional activity of Pax5. It has been shown that Pax5 can recruit histone acetyltransferase CBP and Gcn5 as co-activators for transactional regulation (41Barlev N.A. Emelyanov A.V. Castagnino P. Zegerman P. Bannister A.J. Sepulveda M.A. Robert F. Tora L. Kouzarides T. Birshtein B.K. Berger S.L. Mol. Cell. Biol. 2003; 23: 6944-6957Crossref PubMed Scopus (52) Google Scholar). Next, we performed co-immunoprecipitation assays to determine whether Pax5 interacts with p300. Using anti-p300 antibodies but not the control rabbit IgG, we were able to immunoprecipitate Pax5 from crude nuclear extracts prepared from human B lineage EU12 cells containing endogenous Pax5 and p300 proteins (Fig. 2A). Using anti-Pax5 antibodies, we were also able to immunoprecipitate p300 from HEK293 cells transiently transfected with the Pax5 and p300 expression vectors (Fig. 2B). To further dissect the domains of Pax5 responsible for interaction with p300, we performed co-immunoprecipitation assays using a series of Pax5 truncation constructs transiently expressed in HEK293 cells. Using anti-p300 antibodies, we were able to immunoprecipitate Pax5 and the ΔHD proteins and, to a lesser extent, the ΔB construct. Under the same experimental conditions, we could not detect the ΔC and PRD constructs in immunoprecipitated samples (Fig. 2C). To confirm the physical interaction between Pax5 and p300, we performed GST fusion protein pull-down assays. Different GST fusion Pax5 constructs were expressed and purified from HEK293 cells and used to interact with purified full-length p300 proteins. The p300 proteins can be efficiently pulled down by GST-Pax5, ΔHD, and ΔB constructs but not by the GST-PRD construct (Fig. 2D). These results are consistent with our co-immunoprecipitation results and demonstrate that the C terminus of Pax5 is required for interaction with p300 (Fig. 2E). Originally identified as a histone acetyltransferase, p300 can also acetylate many non-histone proteins, such as transcription factors, to modulate their functions. Next, we analyzed if overexpression of p300 enhances Pax5 acetylation. In our transient transfection, overexpression of p300 resulted in global acetylation of man" @default.
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• W2002071785 date "2011-04-01" @default.
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• W2002071785 title "Histone Acetyltransferase p300 Acetylates Pax5 and Strongly Enhances Pax5-mediated Transcriptional Activity" @default.
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