Title: NF-Y Is Associated with the Histone Acetyltransferases GCN5 and P/CAF
Abstract: The ubiquitous transcription factor, NF-Y, plays a pivotal role in the cell cycle regulation of the mammalian cyclin A,cdc25C, and cdc2 genes, in the S-phase activation of the ribonucleotide reductase R2 gene, in addition to its critical role as a key proximal promoter factor in the transcriptional regulation of the albumin, collagen, lipoprotein lipase, major histocompatibility complex class II, and a variety of other eukaryotic and viral genes. In this report, the NF-Y complex has been shown to possess histone acetyltransferase activity through physical association with the related histone acetyltransferase enzymes, human GCN5 and P/CAF in vivo. The assembled NF-YA:B:C complex, and the NF-YB:YC, NF-YB:YC (DNA binding-subunit interaction domain), and NF-YC:YB (DNA binding-subunit interaction domain) heterodimers were sufficient to support stable interaction with human GCN5 in vitro, suggesting that these histone acetyltransferases interact with a unique surface in the ancient YB:YC histone-fold motif. Deletion of either N- or C-terminal regions in human GCN5 disrupted interaction with NF-Y in vitro. In addition, human GCN5 was observed to activate NF-Y in transient transfections in vivo using a natural α2(I) collagen promoter. These results suggest that these associated histone acetyltransferases may serve to modulate NF-Y transactivation potential by aiding disruption of local chromatin structure thereby facilitating NF-Y access to its CCAAT box DNA binding sites. The ubiquitous transcription factor, NF-Y, plays a pivotal role in the cell cycle regulation of the mammalian cyclin A,cdc25C, and cdc2 genes, in the S-phase activation of the ribonucleotide reductase R2 gene, in addition to its critical role as a key proximal promoter factor in the transcriptional regulation of the albumin, collagen, lipoprotein lipase, major histocompatibility complex class II, and a variety of other eukaryotic and viral genes. In this report, the NF-Y complex has been shown to possess histone acetyltransferase activity through physical association with the related histone acetyltransferase enzymes, human GCN5 and P/CAF in vivo. The assembled NF-YA:B:C complex, and the NF-YB:YC, NF-YB:YC (DNA binding-subunit interaction domain), and NF-YC:YB (DNA binding-subunit interaction domain) heterodimers were sufficient to support stable interaction with human GCN5 in vitro, suggesting that these histone acetyltransferases interact with a unique surface in the ancient YB:YC histone-fold motif. Deletion of either N- or C-terminal regions in human GCN5 disrupted interaction with NF-Y in vitro. In addition, human GCN5 was observed to activate NF-Y in transient transfections in vivo using a natural α2(I) collagen promoter. These results suggest that these associated histone acetyltransferases may serve to modulate NF-Y transactivation potential by aiding disruption of local chromatin structure thereby facilitating NF-Y access to its CCAAT box DNA binding sites. Chromatin structure plays a vital role in the control and regulation of eukaryotic gene transcription, as nucleosomes are now known to be remodeled during transcription in a dynamic process that involves a number of multicomponent complexes that participate in enzymatic modification of chromatin structures (1Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2400) Google Scholar). Recent characterization of several ATP-dependent remodeling activities (2Peterson C.L. Tamkun J.W. Trends Biochem. Sci. 1995; 20: 143-146Abstract Full Text PDF PubMed Scopus (342) Google Scholar, 3Tsukiyama T. Wu C. Cell. 1995; 83: 1011-1020Abstract Full Text PDF PubMed Scopus (516) Google Scholar, 4Cairns B.R. Lorch Y. Li Y. Zhang M. Lacomis L. Erdjument-Bromage H. Tempst P. Du J. Laurent B. Kornberg R.D. Cell. 1996; 87: 1249-1260Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar), and the enzymes that acetylate or deacetylate specific N-terminal lysines in the core histones proteins, provide convincing evidence that chromatin structure is significantly involved in transcriptional regulation (5Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar, 6Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1534) Google Scholar). In addition, a growing subset of known transcriptional cofactors have been shown to possess intrinsic histone acetyltransferase (HAT) 1The abbreviations used are: HAT, histone acetyltransferase; IP, immunoprecipitation; CBP, CREB-binding protein; CMV, cytomegalovirus; DBD, DNA binding-subunit interaction domain; DTT, dithiothreitol; GST, glutathione S-transferase; yeast GCN5, yGCN5; hGCN5, human GCN5; NF-Y, nuclear factor-Y; PAGE, polyacrylamide gel electrophoresis; P/CAF, p300/CBP-associated factor; PCR, polymerase chain reaction; PBS, phosphate-buffered saline. activity, as well as established activation domains, and physical links to known DNA binding transcription factors (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar, 8Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1533) Google Scholar, 9Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.M. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2402) Google Scholar). The overall importance of HAT activity in transcriptional control mechanisms has recently been underscored by the observation that the DNA binding activity of the tumor suppressor protein, p53, can be regulated through acetylation of specific C-terminal lysine residues by p300/CBP (10Gu W. Roeder R.G. Cell. 1997; 87: 595-606Abstract Full Text Full Text PDF Scopus (2177) Google Scholar). Nuclear Factor-Y (NF-Y) (11Dorn A. Bollekens J. Staub A. Benoist C. Mathis D. Cell. 1987; 50: 863-872Abstract Full Text PDF PubMed Scopus (473) Google Scholar), also known as the CCAAT-binding factor (12Maity S.N. Golumbek P.T. Karsenty G. de Crombrugghe B. Science. 1988; 241: 582-585Crossref PubMed Scopus (150) Google Scholar) together with its Saccharomyces cerevisiae homolog, HAP2/3/5 (13McNabb D.S. Xing Y. Guarente L. Genes Dev. 1995; 9: 47-58Crossref PubMed Scopus (234) Google Scholar), is the only known transcription factor whose DNA binding domain is created through the interaction of three heterologous subunits (13McNabb D.S. Xing Y. Guarente L. Genes Dev. 1995; 9: 47-58Crossref PubMed Scopus (234) Google Scholar, 14Maity S.N. Sinha S. Ruteshouser E.C. de Crombrugghe B. J. Biol. Chem. 1992; 267: 16574-16580Abstract Full Text PDF PubMed Google Scholar, 15Sinha S. Maity S.N. Lu J. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1624-1628Crossref PubMed Scopus (251) Google Scholar). Biochemical analyses of the NF-Y complex have demonstrated that the NF-YB:YC subunits associate through a subdomain in the DNA binding-subunit interaction domain (DBD) (16Sinha S. Kim I.-S. Sohn K.-Y. de Crombrugghe B. Maity S.N. Mol. Cell. Biol. 1996; 16: 328-337Crossref PubMed Scopus (145) Google Scholar) referred to as the histone-fold “handshake” motif (17Arents G. Buringame R.W. Wang B.-C. Love W.E. Moudrianakis E.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10148-10152Crossref PubMed Scopus (606) Google Scholar, 18Baxevanis A.D. Arents G. Moudrianakis E.N. Landsman D. Nucleic Acids Res. 1995; 23: 2685-2691Crossref PubMed Scopus (181) Google Scholar), which resembles an α-helical structure first identified in the core histone proteins as primarily responsible for dimerization of the H2A/H2B and H3/H4 histone pairs. The NF-YB:YC histone-fold is most related to histones H2B/H2A, respectively (18Baxevanis A.D. Arents G. Moudrianakis E.N. Landsman D. Nucleic Acids Res. 1995; 23: 2685-2691Crossref PubMed Scopus (181) Google Scholar), and similarly contains a number of hydrophobic amino acids which project along one face of an α-helix. Both NF-YB:YC and these core histone pairs require strong denaturants to effect their biochemical separation. The NF-YB:YC histone-fold plays a crucial role in creation of a functional NF-Y CCAAT box DNA binding complex as the NF-YA subunit associates only with the YB:YC heterodimer (15Sinha S. Maity S.N. Lu J. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1624-1628Crossref PubMed Scopus (251) Google Scholar). The yeast protein GCN5 (yGCN5) has long been known to collaborate with yeast GCN4 in the transcriptional regulation of a large number of genes involved in yeast amino acid biosynthesis and to be involved in maximally increasing the transcriptional activity of several respiratory genes which depend on the yeast HAP2/3/4/5 complex (13McNabb D.S. Xing Y. Guarente L. Genes Dev. 1995; 9: 47-58Crossref PubMed Scopus (234) Google Scholar, 19Georgakopoulos T. Thireos G. EMBO J. 1992; 11: 4145-4152Crossref PubMed Scopus (255) Google Scholar,20Georgakopoulos T. Gounalaki N. Thireos G. Mol. Gen. Genet. 1995; 246: 723-728Crossref PubMed Scopus (54) Google Scholar). yGCN5 is now known to possess intrinsic HAT activity (5Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar) and is thought to acetylate specific N-terminal histone lysine resides as a consequence of its association with additional adaptor proteins in two large multicomponent complexes, referred to as the SAGA complexes (21Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (882) Google Scholar). In these complexes, additional protein components are thought to modulate yGCN5 substrate specificity, and together these large adaptor structures serve to link upstream activators with the basic RNA polymerase II machinery. Recent cloning and characterization of the human equivalents of these yeast adaptor components (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar, 22Candau R. Moore P.A. Wang L. Barlev N. Ying C.Y. Rosen C.A. Berger S.L. Mol. Cell. Biol. 1996; 16: 593-602Crossref PubMed Scopus (159) Google Scholar, 23Wang L. Mizzen C. Ying C. Candau R. Barlev N. Brownell J. Allis C.D. Berger S.L. Mol. Cell. Biol. 1997; 17: 519-527Crossref PubMed Scopus (145) Google Scholar) has suggested that human GCN5 (hGCN5) is likewise associated with additional protein components and is highly related to another HAT enzyme, P/CAF (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar), which has been shown to physically associate with the general transcriptional coactivator, p300/CBP (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar), and the hormone receptor cofactor, ACTR (24Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar). In this report the NF-Y complex has been shown to possess HAT activityin vivo through physical association with the known HAT enzymes, hGCN5 and P/CAF. hGCN5 activates an NF-Y CCAAT box reporterin vivo and associates with the NF-YB:YC histone-fold motif,in vitro. This report further identifies the first transcription factor target for hGCN5, suggests that yGCN5 likewise is associated with the yeast HAP2/3/5 complex through the HAP3/5 histone-fold, and thereby suggests a direct functional role for yGCN5 in global yeast respiratory gene regulation. HeLa and 293 cells were maintained in 10-cm dishes in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (HyClone) and grown at 37 °C, 5% CO2. Full-length human GCN5 (provided by X.-J. Yang and Y. Nakatani) (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar) was cloned into pcDNA3 (Invitrogen) using PCR. pH6 (25Coustry F. Maity S.N. de Chrombrugghe B. J. Biol. Chem. 1995; 270: 468-475Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) was used to generate a site-directed mutation of the proximal CCAAT box site contained in the α2(I) collagen promoter. Both the wild-type and NF-Y mutant promoters were cloned into the pGL3 luciferase vector (Promega) to generate pH6 GL3 and pH6m GL3, respectively, and verified using standard procedures (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). HeLa cells were transfected using the calcium phosphate coprecipitation method and assayed for both luciferase and β-galactosidase activities using the Dual-Light assay system (Tropix). Human GCN5 was cloned into pRSETA (Invitrogen) and pGEX2TK (Pharmacia Biotech Inc.) vectors using PCR. The C-terminal deletion mutant of hGCN5 was prepared from GST-hGCN5 by restriction enzyme digestion and contains amino acids 1–332; the “bromodomain” (27Haynes S.R. Sollard C. Winston F. Beck S. Trowsdale J. Dawid I.B. Nucleic Acids Res. 1992; 80: 2603Crossref Scopus (321) Google Scholar) containing N-terminal deletion of hGCN5 was cloned into pGEX2TK using PCR and contains amino acids 337–476 (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar). His-hGCN5 was purified from the soluble fraction of Escherichia coli BL21(DE3) lysates following induction with 0.1 mmisopropyl-β-d-thiogalactopyranoside for 1 h at 37 °C using Ni2+ chelating resin (Novagen). GST fusion proteins were expressed and purified as described (28Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). YA (DBD), YB (DBD), and YC (DBD) were cloned into pGEX2TK using PCR and contain amino acids 234–303, 53–143, and 1–143, respectively (15Sinha S. Maity S.N. Lu J. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1624-1628Crossref PubMed Scopus (251) Google Scholar, 29van Huijsduijnen R.H. Li X.-Y. Black D. Matthes H. Benoist C. Mathis D. EMBO J. 1990; 9: 3119-3127Crossref PubMed Scopus (210) Google Scholar). The cloning expression and purification of full-length GST- and His-NF-YABC subunits has been described previously (30Nakshatri H. Bhat-Nakshatri P. Currie R.A. J. Biol. Chem. 1996; 271: 28784-28791Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). GST-HMG-Y (20–56) (provided by M. Wegner) (31Leger H. Sock E. Renner K. Grummt F. Wegner M. Mol. Cell. Biol. 1995; 15: 3738-3747Crossref PubMed Scopus (92) Google Scholar), GST-Dr1 (provided by D. Reinberg) (32Inostroza J.A. Mermelstein F.H. Ha I. Lane W.S. Reinberg D. Cell. 1992; 70: 477-489Abstract Full Text PDF PubMed Scopus (293) Google Scholar), and GST-PC4 (provided by R. Roeder) (33Ge H. Roeder R.G. Cell. 1994; 78: 513-523Abstract Full Text PDF PubMed Scopus (308) Google Scholar) have been described. HeLa cell nuclear extracts were prepared according to Dignam et al. (34Dignam J.D. Lebowitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1488Crossref PubMed Scopus (9160) Google Scholar). 293 cells were collected by scraping into 1 ml of ice-cold PBS and pelleted by gentle centrifugation. PBS was removed, and the cells were resuspended in 750 μl of lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm EDTA, 0.5% (v/v) Nonidet P-40, 0.1 mm phenylmethylsulfonyl fluoride). The lysis mixture was incubated on ice for 20 min, then cleared by centrifugation at 12,000 × g for 10 min at 4 °C. Antibodies were added to 100 μl of either HeLa nuclear extract or 293 whole cell extract at 4 °C for 2 h. Protein A-Sepharose:protein G-Sepharose (15 μl; 1:1) (Pharmacia) was added, and the mixture was rotated overnight at 4 °C. Immune complexes were pelleted by gentle centrifugation and washed six times at 4 °C with 1.5 ml of lysis buffer, followed by two washes with 1 × PBS (1 mmDTT), and two washes with 1 × HAT reaction buffer (50 mm Tris-HCl, pH 8.0, 10% (v/v) glycerol, 1 mmDTT, 1 mm phenylmethylsulfonyl fluoride, 0.1 mmEDTA) and assayed as described (34Dignam J.D. Lebowitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1488Crossref PubMed Scopus (9160) Google Scholar). Acetylation reactions were performed for 20 min at 30 °C, and products were resolved with 12% SDS-PAGE gels and fluorography (Amplify; Amersham Corp.). Affinity-purified polyclonal antibodies directed against the NF-YB and NF-YA subunits were prepared as described (36Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988: 511-552Google Scholar). Affinity-purified α-YB antibodies were chemically cross-linked to Affi-Gel 10 beads according to the manufacturer (Bio-Rad). In blocking experiments GST fusion proteins were incubated with α-YB antibodies at 4 °C for 2 h prior to the addition of HeLa nuclear extract; IP analyses were preformed as described above. Affinity-purified polyclonal antibodies directed against human GCN5 and P/CAF were kindly provided by X.-J. Yang and Y. Nakatani (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar). Antibodies directed against p300 (PharMingen) and E1A (Santa Cruz Biotechnology) were obtained commercially. Proteins bound to α-YB affinity beads following washing with IP lysis buffer were eluted using SDS-PAGE buffer with no reducing agents at room temperature. Immunoprecipitates in all cases were then heated to 95 °C under reducing conditions, electrophoresed through 12% SDS-PAGE gels, transferred to nitrocellulose membranes, and incubated with a 1:2000 dilution of primary affinity-purified antibodies overnight at 4 °C. Bound antibodies were detected using α-rabbit horseradish peroxidase-conjugated secondary antibodies (1:5000) and ECL (Amersham). Purified GST fusion proteins were incubated with glutathione-agarose beads (Sigma) (20 μl packed bead volume) with gentle mixing for 20 min at room temperature in 100 μl of 1 × PBS (1 mm DTT), then washed with 2 ml of PBS. In specific cases beads were further incubated with additional NF-Y subunits, then washed with PBS. Recombinant His-hGCN5 was added, and the incubation continued at 4 °C for 1 h with gentle mixing. Beads were washed and processed for HAT activity as described for immunoprecipitates above. YA (DBD) was cleaved from GST-YA (DBD) using thrombin and 32P-labeled using heart muscle creatine kinase (Sigma). GST fusion proteins were bound to glutatione-agarose beads then incubated with the assembled32P-NF-Y complex or the 32P-YA (DBD) subunit alone. Beads were washed as described above for immunoprecipitations, and retained 32P-YA (DBD) was eluted and analyzed using SDS-PAGE gels. During study of accessory protein cofactor interactions with the NF-Y complex (39Currie R.A. J. Biol. Chem. 1997; 272: 30880-30888Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), a CCAAT box DNA affinity-purified NF-Y fraction was observed to possess histone acetyltransferase activity. To address whether this HAT activity was copurifying or physically associated with NF-Y in vivo, affinity-purified α-NF-YB and α-NF-YA antibodies were used to immunoprecipitate NF-Y derived from HeLa nuclear and 293 whole cell extracts and tested using the IP-HAT assay (8Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1533) Google Scholar, 35Mizzen C.A. Yang X.-J. Kobubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 555-565Abstract Full Text Full Text PDF Scopus (623) Google Scholar) (Fig. 1, A andB). IP-HAT analysis of the NF-Y complex using either of these subunit directed antibodies resulted in acetylation of the core histone proteins, H3, H2B, H2A, and to a lesser extent, histone H4. Control α-p300 antibodies brought down HAT activity in both cell types in a manner similar to α-NF-Y antibodies, whereas α-E1A antibodies precipitated HAT activity only in 293 extracts and not HeLa extracts as predicted (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar, 8Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1533) Google Scholar, 35Mizzen C.A. Yang X.-J. Kobubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 555-565Abstract Full Text Full Text PDF Scopus (623) Google Scholar). Preincubation of affinity-purified α-YB antibodies with the purified immunogen, GST-YB, effectively blocked specific NF-Y-associated IP-HAT activity (Fig. 1 C). Recombinant human GCN5 (hGCN5) acetylated histone H3 predominantly in this liquid HAT assay as has been observed previously with both yeast and human GCN5 (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar, 35Mizzen C.A. Yang X.-J. Kobubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 555-565Abstract Full Text Full Text PDF Scopus (623) Google Scholar). Deletion of histone substrates or substitution with bovine serum albumin in the IP-HAT assay resulted in no acetylation, whereas both the α-YA and α-YB immunoprecipitates retained specific CCAAT box DNA binding activity, a property of the heterotrimeric NF-Y complex (data not shown). To identify the protein(s) responsible for the observed NF-Y-associated HAT activity, α-YB immunoprecipitates derived from HeLa extracts were analyzed for the presence of known HAT proteins using Western blot analysis (Fig. 1 D). Both hGCN5 and P/CAF HAT proteins were detected in α-YB affinity bead immunoprecipitates using α-hGCN5 affinity-purified antibodies, which were raised against full-length hGCN5 and cross-react with P/CAF (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar) (lane 2). P/CAF and NF-YB were also detected in HeLa α-YB immunoprecipitates using affinity-purified α-P/CAF antibodies, which were raised against the unique N-terminal region of P/CAF and do not cross-react with hGCN5 (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar) (lane 4) and affinity-purified α-YB antibodies (lane 6), respectively. These results suggested that hGCN5 and P/CAF were associated with NF-Y in vivo and responsible for the observed HAT activity. P/CAF has been shown to associate with the coactivator, p300/CBP (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar), which itself has intrinsic HAT activity, however p300/CBP was not detected in HeLa α-YB immunoprecipitates using Western analysis (data not shown). To examine the possible functional role of hGCN5 in modulating NF-Y transcriptional activityin vivo, HeLa cells were transfected with hGCN5 and an NF-Y CCAAT box containing promoter reporter derived from the murine collagen α2(I) gene (25Coustry F. Maity S.N. de Chrombrugghe B. J. Biol. Chem. 1995; 270: 468-475Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) (Fig. 2). The observed ∼4-fold activation of this reporter by hGCN5 was dependent on an intact proximal CCAAT box element, suggesting that hGCN5 functionally interacts and regulates NF-Y transactivation potential on a natural CCAAT box-containing promoter. To examine the possibility that the NF-Y complex possesses intrinsic HAT activity, individual and specific combinations of recombinant NF-Y subunits were tested for HAT activity using the liquid assay. Neither the complete functional NF-YA:B:C complex, the NF-YB:C heterodimer, nor any individual NF-Y subunit was observed to possess HAT activity (data not shown). An in vitro GST fusion protein “pull-down” assay was developed to determine the specific NF-Y subunit requirements for physical association with hGCN5 (Fig.3). Recombinant hGCN5 was incubated with the assembled NF-Y complex, the NF-YB:C heterodimer, and individual NF-Y subunits that were tethered to glutathione-agarose beads, then assayed for HAT activity. Using this approach hGCN5 was shown to stably associate with the complete NF-Y complex and the NF-YB:C heterodimer. hGCN5 did not associate with any individual NF-Y subunit, whereas hGCN5 was observed to associate with the full-length YB:YC complex (data not shown) and in heterodimers composed of a full-length subunit and its complementing YB (DBD) or YC (DBD) partner (Fig. 3, lanes 6and 8, respectively). Recombinant hGCN5 (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar, 35Mizzen C.A. Yang X.-J. Kobubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 555-565Abstract Full Text Full Text PDF Scopus (623) Google Scholar), P/CAF (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar), and yGCN5 (37Kuo M.-H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 383: 269-272Crossref PubMed Scopus (507) Google Scholar) are known to acetylate histone H3 predominantly in the liquid HAT assay when presented with the core histone proteins, whereas acetylation of any of the core histone proteins in nucleosomes by yeast GCN5 requires additional protein components assembed in the large molecular mass SAGA complexes (21Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (882) Google Scholar, 38Candau R. Zhou J. Allis C.D. Berger S.L. EMBO J. 1997; 16: 555-565Crossref PubMed Scopus (179) Google Scholar). Predominant acetylation of histone H3 by hGCN5 tethered to recombinant NF-Y or NF-YB:YC complexes (Fig. 3) suggests that the histone specificity of hGCN5, and possibly P/CAF, is altered when associated in the native NF-Y complex, since histones H2A and H2B were additionally acetylated by immunoprecipitated NF-Y in the IP-HAT assay (Fig. 1). In an attempt to map relevant functional domains in hGCN5 that are required for stable interaction with NF-Y, several GST-hGCN5 deletion mutants were tested using an in vitro GST pull-down assay (Fig. 4). NF-YA (DBD) has been used previously to assemble a functional heterotrimeric NF-Y complex and as an individual NF-YA subunit derivative to demonstrate that each stably interacts with a single AT-hook motif present in the non-histone chromosomal proteins, HMG-I(Y) (39Currie R.A. J. Biol. Chem. 1997; 272: 30880-30888Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) (Fig. 4, A and B, lane 6). Interaction of hGCN5 with NF-Y was dependent on an intact NF-Y complex (Fig. 4 A, lane 3), since no specific interaction with YA (DBD) alone was observed (Fig. 4 B, lane 3). Deletion of either the N- or C-terminal regions of hGCN5 severely inhibited its stable interaction with the assembled NF-Y complex (Fig. 4 A, lanes 4 and 5, respectively). The hGCN5 C-terminal bromodomain (5Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar, 27Haynes S.R. Sollard C. Winston F. Beck S. Trowsdale J. Dawid I.B. Nucleic Acids Res. 1992; 80: 2603Crossref Scopus (321) Google Scholar) contained in the N-terminal deletion mutant is not itself sufficient to support stable interaction between NF-Y and hGCN5 (Fig. 4 A, lane 5). These results suggest that interaction with NF-Y requires specific domains in both the N- and C-terminal regions or that the primary interaction site maps to a region close to the deletion site. Both single nucleosomes and higher order chromatin structure are generally thought to represent structural impediments to gene transcription (1Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2400) Google Scholar). Recent isolation of chromatin remodeling activities has provided new insights into the mechanisms responsible for altering chromatin structure during transcription and the means to begin approaching questions regarding the targeting of specific activities to specific promoters (2Peterson C.L. Tamkun J.W. Trends Biochem. Sci. 1995; 20: 143-146Abstract Full Text PDF PubMed Scopus (342) Google Scholar, 3Tsukiyama T. Wu C. Cell. 1995; 83: 1011-1020Abstract Full Text PDF PubMed Scopus (516) Google Scholar, 4Cairns B.R. Lorch Y. Li Y. Zhang M. Lacomis L. Erdjument-Bromage H. 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Together these observations now suggest that acetylation of the N-terminal histone lysines residues is involved in disrupting the structure between nucleosomes in local regions in chromatin thereby facilitating access of transcription factors to specific promoters. In addition, recent reports have demonstrated that p53 is a substrate for p300 (10Gu W. Roeder R.G. Cell. 1997; 87: 595-606Abstract Full Text Full Text PDF Scopus (2177) Google Scholar), and the HAT enzymes, p300, P/CAF, and TAFII250, are capable of acetylating components of the general RNA polymerase II machinery (i.e. TFIIEβ and TFIIF) (42Imhof A. Yang X.-J. Ogryzko V.V. Nakatani Y. Wolffe A.P. Ge H. Curr. Biol. 1997; 7: 689-692Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). These results suggest that HAT enzymes play additional roles in modulating the DNA binding activity of an important tumor suppressor protein, and the functional activity of general initiation factors, and further suggest important implications for their role as cofactors in modulating upstream transcription factor transactivation potential. This report identifies the NF-Y complex as the first mammalian transcription factor target for the human acetyltransferase, GCN5, the first DNA-binding transcription factor associated with P/CAF, in vivo, and maps the site of interaction of hGCN5 to the highly conserved DBD elements of the NF-YB:C heterodimer. Anti-YB immunoprecipitates, which contain the NF-Y complex, were observed to be associated with the two highly related HAT enzymes, GCN5 and P/CAF. The C-terminal region of P/CAF is 86% identical on the amino acid level to hGCN5 (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar), which includes both the region responsible for HAT activity and the bromodomain, a motif thought to be involved in additional protein-protein interactions (5Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar, 27Haynes S.R. Sollard C. Winston F. Beck S. Trowsdale J. Dawid I.B. Nucleic Acids Res. 1992; 80: 2603Crossref Scopus (321) Google Scholar). The N-terminal region of P/CAF is unique, and α-P/CAF antibodies (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar) that recognize both N- and C-terminal regions were observed to detect P/CAF in α-YB immunoprecipitates (Fig. 1 D). P/CAF has been shown to be associated with two cofactors, p/300/CBP (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar) and ACTR (24Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar); however, the site(s) of physical interaction in either case has not been identified. While P/CAF is known to interact with p300/CBP, hGCN5 does not (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar), and presently no mammalian transcription factor has been shown to interact with both hGCN5 and P/CAF. NF-Y is the first DNA-binding transcription factor shown to be associated with P/CAF and hGCN5.In vitro binding studies between NF-Y and hGCN5 strongly suggest that P/CAF interacts with NF-Y likewise through its C-terminal region, which is highly related to hGCN5. Presently it is not known if these NF-Y: HAT complexes exhibit differential activities with regard to specific NF-Y-responsive promoters in vivo, and if the composition of NF-Y:HAT complexes differ with regard to additional protein components. Comparison of immunoprecipitated NF-Y-associated HAT activity within vitro reconstituted NF-Y:hGCN5 HAT activity showed an altered histone substrate specificity. Both histone H2A and H2B were observed to be acetylated when presented to NF-Y immunoprecipitates in addition to the predominant product, H3, while in vitroreconstituted complexes acetylated histone H3 exclusively. These results suggest that native NF-Y may be associated with additional protein components in vivo, in a manner possibly analogous to the yeast SAGA complexes that contain yGCN5, in addition to the characterized components, Spt 3/7/20, ADA2, ADA3, and other unknown components (21Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (882) Google Scholar). hGCN5, yGCN5, and P/CAF are known to acetylate histone H3 predominantly when presented with the core histone proteins and to be unable to acetylate histones in isolated nucleosomes in the absence of additional protein components (7Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar, 37Kuo M.-H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 383: 269-272Crossref PubMed Scopus (507) Google Scholar). Recent isolation of hADA2, the human homolog to the yeast adaptor protein, yADA2 (22Candau R. Moore P.A. Wang L. Barlev N. Ying C.Y. Rosen C.A. Berger S.L. Mol. Cell. Biol. 1996; 16: 593-602Crossref PubMed Scopus (159) Google Scholar), suggests that some fraction of both hGCN5 and P/CAF may be associated in vivo with hADA2 and mammalian equivalents of the SAGA complexes. Further analysis of α-NF-Y immunoprecipitates will determine if hADA2 is present and the identity of additional protein components that may play a role in regulating both hGCN5 and P/CAF histone substrate specificity. Mutational analysis of the NF-YB subunit has established that the YC interaction domain and the region required for DNA binding activity largely overlap in the ∼90-amino acid YB (DBD) (16Sinha S. Kim I.-S. Sohn K.-Y. de Crombrugghe B. Maity S.N. Mol. Cell. Biol. 1996; 16: 328-337Crossref PubMed Scopus (145) Google Scholar). In contrast, the NF-YA (DBD) element is more clearly defined and contains separable subdomains used for DNA binding and interaction with the YB:YC heterodimer. The YB:YC histone-fold motifs appear to play crucial roles in subunit interactions through creation of unique surfaces for interaction with the YA (DBD) and with the HAT enzymes, hGCN5 and P/CAF. The YB:YC histone-fold may make additional nonspecific contacts with DNA sequences flanking the CCAAT box, in a manner analogous to the H2B:H2A histone pair, whereas the YA (DBD) may make the majority of sequence-specific DNA contact in the CCAAT box, in addition to providing binding surfaces for HMG-I(Y) and PC4/p15 (39Currie R.A. J. Biol. Chem. 1997; 272: 30880-30888Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Clearly, x-ray crystallographic analyses of the NF-Y (DBD) regions in association with CCAAT box DNA and these newly identified HAT enzymes in the future will further our understanding of how these proteins are structurally organized and provide additional insights into how they function in vivo. I am grateful to X.-J. Yang and Y. Nakatani for kindly providing their hGCN5 and P/CAF reagents and to Drs. D. McNabb, L. Guarante, X.-Y. Yang, and Y. Nakatani for their comments and suggestions.