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Transcriptional Repression of the Cystic Fibrosis Transmembrane Conductance Regulator Gene, Mediated by CCAAT Displacement Protein/cut Homolog, Is Associated with Histone Deacetylation

1999; Elsevier BV; Volume: 274; Issue: 12 Linguagem: Inglês

10.1074/jbc.274.12.7803

1083-351X

SiDe Li, Libia Moy, Nanci Pittman, Gongliang Shue, Barbara Aufiero, Ellis J. Neufeld, Neal S. LeLeiko, Martin J. Walsh,

Epigenetics and DNA Methylation

Human cystic fibrosis transmembrane conductance regulator gene (CFTR) transcription is tightly regulated by nucleotide sequences upstream of the initiator sequences. Our studies of human CFTR transcription focus on identifying transcription factors bound to an inverted CCAAT consensus or "Y-box element." The human homeodomain CCAAT displacement protein/cut homolog (CDP/cut) can bind to the Y-box element through a cut repeat and homeobox. Analysis of stably transfected cell lines with wild-type and mutant humanCFTR-directed reporter genes demonstrates that human histone acetyltransferase GCN5 and transcription factor ATF-1 can potentiate CFTR transcription through the Y-box element. We have found 1) that human CDP/cut acts as a repressor ofCFTR transcription through the Y-box element by competing for the sites of transactivators hGCN5 and ATF-1; 2) that the ability of CDP/cut to repress activities of hGCN5 and ATF-1 activity is contingent on the amount of CDP/cut expression; 3) that histone acetylation may have a role in the regulation of gene transcription by altering the accessibility of the CFTRY-box for sequence-specific transcription factors; 4) that trichostatin A, an inhibitor of histone deacetylase activity, activates transcription of CFTR through the Y-box element; 5) that the inhibition of histone deacetylase activity leads to an alteration of local chromatin structure requiring an intact Y-box sequence inCFTR; 6) that immunocomplexes of CDP/cutpossess an associated histone deacetylase activity; 7) that the carboxyl region of CDP/cut, responsible for the transcriptional repressor function, interacts with the histone deacetylase, HDAC1. We propose that CFTR transcription may be regulated through interactions with factors directing the modification of chromatin and requires the conservation of the inverted CCAAT (Y-box) element of the CFTR promoter. Human cystic fibrosis transmembrane conductance regulator gene (CFTR) transcription is tightly regulated by nucleotide sequences upstream of the initiator sequences. Our studies of human CFTR transcription focus on identifying transcription factors bound to an inverted CCAAT consensus or "Y-box element." The human homeodomain CCAAT displacement protein/cut homolog (CDP/cut) can bind to the Y-box element through a cut repeat and homeobox. Analysis of stably transfected cell lines with wild-type and mutant humanCFTR-directed reporter genes demonstrates that human histone acetyltransferase GCN5 and transcription factor ATF-1 can potentiate CFTR transcription through the Y-box element. We have found 1) that human CDP/cut acts as a repressor ofCFTR transcription through the Y-box element by competing for the sites of transactivators hGCN5 and ATF-1; 2) that the ability of CDP/cut to repress activities of hGCN5 and ATF-1 activity is contingent on the amount of CDP/cut expression; 3) that histone acetylation may have a role in the regulation of gene transcription by altering the accessibility of the CFTRY-box for sequence-specific transcription factors; 4) that trichostatin A, an inhibitor of histone deacetylase activity, activates transcription of CFTR through the Y-box element; 5) that the inhibition of histone deacetylase activity leads to an alteration of local chromatin structure requiring an intact Y-box sequence inCFTR; 6) that immunocomplexes of CDP/cutpossess an associated histone deacetylase activity; 7) that the carboxyl region of CDP/cut, responsible for the transcriptional repressor function, interacts with the histone deacetylase, HDAC1. We propose that CFTR transcription may be regulated through interactions with factors directing the modification of chromatin and requires the conservation of the inverted CCAAT (Y-box) element of the CFTR promoter. cystic fibrosis transmembrane conductance regulator cyclic AMP-responsive element p300/CBP-associated factor CRE-binding protein CREB-binding protein/p300 wild-type histone deacetylase 1/2 base pair(s) CCAAT-binding factor/nuclear factor Y CCAAT displacement protein/cut homolog activating transcription factor 1 human growth hormone cytomegalovirus thymidine kinase glutathioneS-transferase polymerase chain reaction bromo cyclic AMP trichostatin lysis washing electrophoretic mobility shift assay CAATenhancer-binding protein The gene responsible for cystic fibrosis encodes thecystic fibrosis transmembrane conductance regulator (CFTR)1 protein (1Rommens J.M. Iannuzzi M.C. Kerem B.-S. Drumm M.L. Melmer G. Dean M. Rozmahel R. Cole J. Kennedy D. Hidaka N. Zsiga M. Buchwald Riordan J.R. Tsui L.-C. Collins F.S. Science. 1989; 245: 1059-1065Crossref PubMed Scopus (2493) Google Scholar, 2Riordan J.R. Rommens J.M. Kerem B.-S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.-L. Drumm M.-L. Iannuzzi M.C. Collins F.S. Tsui L.-C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5854) Google Scholar). The expression of CFTRis confined primarily to specific epithelial cell types and is ordinarily expressed in low levels. The low levels and cell type-specific expression ofCFTR appear to be dictated primarily by sequences upstream of the transcriptional start sites of CFTR which correspond to functional promoter sequences (3Yoshimura K. Nakamura H. Trapnell B.C. Dalemans W. Pavirani A. Lecocq J.-P. Crystal R.G. J. Biol. Chem. 1991; 266: 9140-9144Abstract Full Text PDF PubMed Google Scholar, 4Chou J.-L. Rozmahel R. Tsui L.-C. J. Biol. Chem. 1991; 266: 24471-24476Abstract Full Text PDF PubMed Google Scholar). The elements required for active, cell type-specific CFTR transcription lie in a narrow band of nucleotide sequences, proximal to the multiple transcript initiation (5Koh J. Sferra T.J. Collins F.S. J. Biol. Chem. 1993; 268: 15912-15921Abstract Full Text PDF PubMed Google Scholar). The levels of CFTR transcripts in individual cell types appear to correspond to the ability of specific epithelia to modulate expression of CFTR variably in response to 1) levels of cAMP (6Breuer W. Kartner N. Riordan J.R. Cabantchik Z.I. J. Biol. Chem. 1992; 267: 10465-10469Abstract Full Text PDF PubMed Google Scholar), 2) the stimulation of protein kinase A and C activities (7Bargon J. Trapnell B.C. Chu C.-S. Rosenthal E.R. Yoshimura K. Guggino W.B. Dalemans W. Pavirani A. Lecocq J.P. Crystal R.G. Mol. Cell. Biol. 1992; 12: 1872-1878Crossref PubMed Scopus (26) Google Scholar, 8Bargon J. Trapnell B.C. Yoshimura K. Dalemans W. Pavirani A. Lecocq J.-P. Crystal R.G. J. Biol. Chem. 1992; 267: 16056-16060Abstract Full Text PDF PubMed Google Scholar, 9Breuer W. Glickstein H. Kartner N. Riordan J.R. Ausiello D.A. Cabantchik I.Z. J. Biol. Chem. 1993; 268: 13935-13939Abstract Full Text PDF PubMed Google Scholar), and 3) phorbol esters (10Trapnell B.C. Zeitlin P.L. Chu C.S. Yoshimura K. Nakamura H. Guggino W.B. Bargon J. Banks T.C. Dalemans W. Pavirani A. Lecocq J.-P. Crystal R.G. J. Biol. Chem. 1991; 266: 10319-10323Abstract Full Text PDF PubMed Google Scholar) likely through signaling pathways or mechanisms that converge ultimately on gene transcription (for review, see Ref. 11Hunter T. Karin M. Cell. 1992; 70: 375-387Abstract Full Text PDF PubMed Scopus (1116) Google Scholar).The tight transcriptional control of CFTR requires a conserved inverted CCAAT (Y-box) element (12Pittman N. Shue G. LeLeiko N.S. Walsh M.J. J. Biol. Chem. 1995; 270: 28848-28857Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Despite the absence of a TATA signal, the human CFTR gene can initiate RNA transcription through multiple, discrete start sites requiring conserved residues within the Y-box. Thus, an intact CCAAT consensus on the opposing strand of the human CFTR appears to be a requirement for accurate transcript initiation (12Pittman N. Shue G. LeLeiko N.S. Walsh M.J. J. Biol. Chem. 1995; 270: 28848-28857Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Both basal and cAMP-mediated regulation of CFTR transcription have implicated the Y-box (12Pittman N. Shue G. LeLeiko N.S. Walsh M.J. J. Biol. Chem. 1995; 270: 28848-28857Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) in tandem with a weak CRE nucleotide consensus downstream (13Matthews R.P. McKnight G.S. J. Biol. Chem. 1996; 271: 31869-31877Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). CFTR transcription appears to be influenced by additional cis-acting elements, including a site located in the first intron of the human CFTR (14Smith A. Barth M.L. McDowell T.L. Moulin D.S. Nuthall H.N. Hollingsworth M.A. Harris A. J. Biol. Chem. 1996; 271: 9947-9954Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The activation of CFTR transcription by cAMP correlates with increasing sensitivity to DNase I overlapping the Y-box element (12Pittman N. Shue G. LeLeiko N.S. Walsh M.J. J. Biol. Chem. 1995; 270: 28848-28857Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). This may reflect the role of trans-acting factors in modifying local chromatin structure. Although it has been shown previously that individual members of bZIP families of transcription factors have the capacity to bind and activate CFTRtranscription (12Pittman N. Shue G. LeLeiko N.S. Walsh M.J. J. Biol. Chem. 1995; 270: 28848-28857Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 13Matthews R.P. McKnight G.S. J. Biol. Chem. 1996; 271: 31869-31877Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), little is known about the parameters and proteins involved in directing transcription of the CFTR in vivo.Histone modification plays an important role in regulating gene transcription (15Brownell 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 (1277) Google Scholar, 16Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (463) Google Scholar). The level of acetylation of conserved residues on the core histones H3 and H4 appears to plays a critical role in the regulation of gene transcription (17Wolffe A.P. Science. 1996; 272: 371-372Crossref PubMed Scopus (271) Google Scholar), and the presence of histone acetyltransferase activity correlates with the activation of other genes (18Wade P.A. Wolffe A.P. Curr. Biol. 1997; 7: 82-84Abstract Full Text Full Text PDF PubMed Google Scholar, 19Wade P.A. Pruss D. Wolffe A.P. Trends Biochem. Sci. 1997; 22: 128-132Abstract Full Text PDF PubMed Scopus (403) Google Scholar). Conversely, it is believed that gene repression is associated with the increased activity of histone deacetylases (17Wolffe A.P. Science. 1996; 272: 371-372Crossref PubMed Scopus (271) Google Scholar,20Wolffe A.P. Pruss D. Cell. 1996; 84: 817-819Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Although it is accepted that histone acetyltransferases and deacetylases are tightly associated with transcriptional machinery through several well characterized transcriptional cofactors or adaptor proteins (21Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1523) Google Scholar, 22Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2246) Google Scholar, 23Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1524) Google Scholar), little is known about how such activities are directed to a specific gene. Several genes encoding histone acetyltransferase and deacetylase activities have been identified in human. Interestingly, many of these genes encode both transcriptional coactivators and corepressors, such as hGCN5, P/CAF, p300/CBP, TAF250, and HDAC1/2 (for review see Ref. 18Wade P.A. Wolffe A.P. Curr. Biol. 1997; 7: 82-84Abstract Full Text Full Text PDF PubMed Google Scholar). Recent observations have suggested that transcriptional cofactors such as p300/CBP and HDAC1/2 encode proteins for the enzymatic histone acetyltransferase and deacetylase activities, respectively (22Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2246) Google Scholar, 24Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (516) Google Scholar). These proteins can form complexes with sequence-specific nuclear hormone receptors and transcription factors (25Nagy L. Kao H.Y. Chakravarti D. Lin R.J. Hassig C.A. Ayer D.E. Schreiber S.L. Evans R.M. Cell. 1997; 89: 373-380Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar, 26Minucci S. Horn V. Bhattacharyya N. Russanova V. Ogryzko V.V. Gabriele L. Howard B.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11295-11300Crossref PubMed Scopus (103) Google Scholar, 27Currie R.A. J. Biol. Chem. 1998; 273: 1430-1434Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 28Jin S. Scotto K.W. Mol. Cell. Biol. 1998; 18: 4377-4384Crossref PubMed Google Scholar). Although these cofactors do not possess sequence-specific DNA binding activities, a transcriptional cofactor can target a specific gene when tethered to a gene promoter through a heterologous DNA binding domain as shown previously (29Wang H. Stillman D.J. Mol. Cell. Biol. 1993; 13: 1805-1814Crossref PubMed Scopus (102) Google Scholar). Therefore, transcriptional cofactors may regulate the level of histone acetylation of a specific gene through interactions with sequence-specific transcription factors.Our present objective was to identify proteins bound to the humanCFTR promoter through the Y-box nucleotide consensus of theCFTR. We have now identified two such proteins: CDP/cut, a homeodomain repressor, and the transcription factor complex CBF·NF-Y. CBF·NF-Y is a highly conserved complex of heterotrimeric subunits shown to interact with CCAAT sequence elements common to many eukaryotic promoters (30Maity S.N. Golumbek P.T. Karsenty G. de Crombrugghe B. Science. 1988; 241: 582-585Crossref PubMed Scopus (150) Google Scholar, 31Maity S.N. Sinha S. Ruteshouser E.C. de Crombrugghe B. J. Biol. Chem. 1992; 267: 16574-16580Abstract Full Text PDF PubMed Google Scholar). Conserved residues within subunits of the CBF·NF-Y complex, which include a region homologous to the histone-fold motif found in histones H2A and H2B, are necessary for subunit interaction to form the heterotrimeric complex (32Sinha S. Kim I.-S. Sohn K.-Y. de Crombrugghe B. Maity S.N. Mol. Cell. Biol. 1996; 16: 328-337Crossref PubMed Scopus (144) Google Scholar). Domains of CBF·NF-Y, located in two of the subunits (CBF-c and CBF-b) are necessary for the transactivation by the CBF·NF-Y complex (33Coustry F. Maity S.N. Sinha S. de Crombrugghe B. J. Biol. Chem. 1996; 271: 14485-14491Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The CBF·NF-Y-bound complex can form tight interactions with known histone acetyltransferases hGCN5 and P/CAF to transactivate transcription (27Currie R.A. J. Biol. Chem. 1998; 273: 1430-1434Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Mammalian CCAAT displacementprotein/cut (CDP/cut) repressor is a 180–190-kDa polypeptide (34Neufeld E.J. Skalnik D.G. Lievens P.M. Orkin S.H. Nat. Genet. 1992; 1: 50-55Crossref PubMed Scopus (186) Google Scholar, 35Aufiero B. Neufeld E.J. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7757-7761Crossref PubMed Scopus (105) Google Scholar, 36Andres V. Nadal-Ginard B. Mahdavi V. Development. 1992; 116: 321-334PubMed Google Scholar) closely related to the cutprotein of Drosophila (37Blochlinger K. Bodmer R. Jack J. Jan L.Y. Jan Y.N. Nature. 1988; 333: 629-635Crossref PubMed Scopus (222) Google Scholar), which determines cell fate inDrosophila (38Jack J. Dorsett D. Delotto Y. Liu S. Development. 1991; 113: 735-747Crossref PubMed Google Scholar, 39Blochlinger K. Jan L.Y. Jan Y.N. Development. 1993; 117: 441-450PubMed Google Scholar). CDP was first described as a CCAAT-box-binding protein in the sea urchin sperm histone promoter (40Barberis A. Superti-Furga G. Busslinger M. Cell. 1987; 50: 347-359Abstract Full Text PDF PubMed Scopus (261) Google Scholar). It was defined subsequently as a gene-specific transcriptional repressor associated with the differential regulation of the gp91phox gene in myeloid cell differentiation (41Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar). Human CDP/cut also binds to promoters of the c-myc, N-cam, thymidine kinase, c-mos, and histone genes in a variety of cell types (42Dufort D. Nepveu A. Mol. Cell. Biol. 1994; 14: 4251-4257Crossref PubMed Scopus (84) Google Scholar, 43Valarche I. Tissier-Seta J.P. Hirsh M.R. Martinez S. Goridis C. Brunet J.F. Development. 1993; 119: 881-896Crossref PubMed Google Scholar, 44Kim E.C. Lau J.S. Rawlings S. Lee A.S. Cell Growth Differ. 1997; 8: 1329-1338PubMed Google Scholar, 45Higgy N.A. Tarnasky H.A. Valarche I. Nepveu A. van der Hoorn F.A. Biochim. Biophys. Acta. 1997; 1351: 313-324Crossref PubMed Scopus (28) Google Scholar, 46el-Hodiri H.M. Perry M. Mol. Cell. Biol. 1995; 15: 3587-3596Crossref PubMed Scopus (49) Google Scholar) and is implicated in the regulation of gene transcription through sequence elements associated with nuclear matrix attachment (47Banan M. Rojas I.C. Lee W.-H. King H.L. Harriss J.V. Kobayashi R. Webb C. Gottlieb P.D. J. Biol. Chem. 1997; 272: 18440-18452Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The competing activities between CBF·NF-Y and CDP/cut have been postulated as a mechanism to regulate several genes through the CCAAT motif (44Kim E.C. Lau J.S. Rawlings S. Lee A.S. Cell Growth Differ. 1997; 8: 1329-1338PubMed Google Scholar, 48Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 18203-18210Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 49Mantovani R. Nucleic Acids Res. 1998; 26: 1135-1143Crossref PubMed Scopus (443) Google Scholar).The objective of this study was to determine further whether transcription of CFTR is regulated by nuclear factors associated with histone acetylation. Because the regulationCFTR transcription is potentiated through cAMP and the CREB/ATF activators (12Pittman N. Shue G. LeLeiko N.S. Walsh M.J. J. Biol. Chem. 1995; 270: 28848-28857Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 13Matthews R.P. McKnight G.S. J. Biol. Chem. 1996; 271: 31869-31877Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), we postulate that mechanisms directing the recruitment of histone acetyltransferase coactivators could, conversely, direct a histone deacetylase complex through a separate set of CCAAT-binding factors within the context of the humanCFTR promoter Y-box element. In this report, we present evidence that the Y-box element of the CFTR promoter is a site that intersects histone acetylation and deacetylation processes with mechanisms to modulate transcription of CFTR.DISCUSSIONThe architecture of chromatin plays a fundamental role in the regulation of genes. The underlying behavior of gene transcriptionin vivo may be determined more by the overall structure of chromatin as a result of the cumulative function transcription factors have in regulating local chromatin structure. The relationship between the remodeling of chromatin by the modification of histones and gene-specific transcription factors recently has become more apparent (15Brownell 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 (1277) Google Scholar, 20Wolffe A.P. Pruss D. Cell. 1996; 84: 817-819Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Since the observation of transcriptional regulatory proteins encoding intrinsic activity for the acetylation or deacetylation of histones, this mechanism for altering chromatin structure has gained prominence in the study of gene regulation (19Wade P.A. Pruss D. Wolffe A.P. Trends Biochem. Sci. 1997; 22: 128-132Abstract Full Text PDF PubMed Scopus (403) Google Scholar). Although it is believed that acetylation of histones may neutralize charged lysine residues to allow for the movement or displacement of nucleosomes along a DNA template, making local chromatin more accessible and active for gene transcription, a precise mechanism for this model has yet to be elucidated.In this report, we provide evidence that the CFTR gene Y-box element is a potential target of the CBF·NF-Y activation complex. This was demonstrated by competitive EMSA and supershift analysis (Fig.1). CBF·NF-Y has been implicated previously in the assembly and remodeling of chromatin structure surrounding its cognate sequence (63Linhoff M.W. Wright K.L. Ting J.P.-Y. Mol. Cell. Biol. 1997; 17: 4589-4596Crossref PubMed Scopus (51) Google Scholar). The trimeric subunit complex of CBF·NF-Y has been shown previously to possess histone acetyltransferase activity through the tethering of hGCN5 and/or P/CAF to CBF·NF-Y (27Currie R.A. J. Biol. Chem. 1998; 273: 1430-1434Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 28Jin S. Scotto K.W. Mol. Cell. Biol. 1998; 18: 4377-4384Crossref PubMed Google Scholar). Previously, we have also demonstrated the binding of the CFTR inverted CCAAT (Y-box) sequence by the transcription factor C/EBP (12Pittman N. Shue G. LeLeiko N.S. Walsh M.J. J. Biol. Chem. 1995; 270: 28848-28857Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Although our study here has not included the role of C/EBP on CFTRtranscription, it is important to note that C/EBP can interact with CBP/p300 in vivo (64Mink S. Haenig B. Klempnauer K.H. Mol. Cell. Biol. 1997; 17: 6609-6617Crossref PubMed Google Scholar) and is the focus of other studies. We believe that C/EBP may play a particularly intriguing role inCFTR transcription. The absence of C/EBP in cell lines derived from transgenic "knockout" mice, when compared with wild-type littermates, show the lack of p300-containing complexes associated with the CFTR Y-box. However, this observation is in contrast to results showing CBP associated with the same Y-box element in both knockout and wild-type cell lines. 4M. J. Walsh, S.-D. Li, and G. Shue, manuscript in preparation. Interestingly, the rate of CFTR transcription in vivo in these same "knockout" cell lines is precisely half of that compared with the wild-type cell lines. It is plausible to consider that the composition of individual coactivator complexes could reflect a preferential stoichiometric arrangement or dosage between CBP and p300 (65Yao T.P. Oh S.P. Fuchs M. Zhou N.D. Ch'ng L.-E. Newsome D. Bronson R.T. Li E. Livingstone D.M. Eckner R. Cell. 1998; 93: 361-372Abstract Full Text Full Text PDF PubMed Scopus (813) Google Scholar) and may demonstrate some distinction and/or differential regulation between the two coactivators.We have demonstrated that transcription of CFTR is mediated by the transcription factor ATF-1 and the human histone acetyltransferase hGCN5 (Fig. 2). Transfection studies have demonstrated the activating potential of the transcription factor ATF-1 and the human histone acetyltransferase hGCN5 to stimulateCFTR transcription. Evidence that hGCN5 potentiates transcription of CFTR indicates a role for histone acetylation in the regulation of CFTR transcription. We also show that CDP/cut has a functional role in repressing transcription of CFTR. This result suggested to us that CDP/cut effectively competes with transcriptional activators hGCN5 and ATF-1 to antagonize directly the activation ofCFTR transcription (Figs. 3 and 4). The binding of CDP/cut (shown in Fig. 5, A and B) overlaps the Y-box of CFTR; therefore, we speculate that transcription factors bound to the Y-box may, differentially, alter the pattern of histone acetylation in chromatin proximal to transcript initiation to regulate transcription. In our studies, we have used the CDP/cut protein, from nuclear extracts, transfected COS-7 cells, and from protein expressed in bacteria to show binding to theCFTR Y-box (Fig. 5 B). How does the Y-box element function to regulate CFTR transcription in vivo? Our studies demonstrate that inhibition of histone deacetylase activity with TSA corresponds to the stimulation of CFTRtranscription mediated through an inverted and intact CCAAT sequence. Evidence in this report implies that activities competing for the association with the Y-box element regulate the level of histone acetylation in chromatin surrounding the transcript start sites, leading to either activation or repression of gene transcription. We have shown that inhibition of histone deacetylase activity by TSA potentiates the transcription of a CFTR-directed transgene through the Y-box element. Thus, the activation of CFTRtranscription could be linked to histone acetyltransferase activity through the inverted CCAAT nucleotide consensus (Y-box) sequence. Examination of the CFTR promoter in vivo using aBspMI endonuclease sensitivity assay suggests that TSA will promote cleavage of nucleotide sequences proximal to theCFTR Y-box element and CRE (Fig. 7). Furthermore, DNase I hypersensitivity assays shown in Fig. 8 demonstrate that an intact 5′-ATTGG-3′ sequence of human CFTR is required for DNase I hypersensitivity of nuclei. DNase I hypersensitivity of twoCFTR-directed transgenic constructs reveals that, in fact, the ATTGG consensus is an essential nucleotide requirement for the nuclease hypersensitivity of the CFTR promoter in chromatin as a result of the inhibition of histone deacetylase activity in vivo. This has been demonstrated in several randomly selected stably transfected clones (data not shown). Despite the inhibition of histone deacetylase activity by TSA to stimulate CFTRtranscription through the putative alteration of chromatin structure, overlapping the Y-box element, there is no direct evidence to show the modification of histones within the context of the CFTRpromoter to undergo changes in acetylation patterns as a result of TSA treatment. However, our observations suggest that the specialized sensitivity of local chromatin to nuclease hydrolysis is linked to the inhibition of histone deacetylase activity. We have yet to establish whether trans-acting factors bound to the CFTRY-box element (such as CDP/cut, C/EBP, or CBF·NF-Y) can target the enzymatic modification of local histones in vivo(66Kadosh D. Struhl K. Mol. Cell. Biol. 1998; 18: 5121-5127Crossref PubMed Scopus (267) Google Scholar). Although the correlation between histone acetylation and gene activation is established, further studies to determine the extent of local chromatin to undergo histone modification will have to be performed.Our observations suggest the association of CDP/cut with histone deacetylase activity. We propose that transcriptional repression by CDP/cut may recruit histone deacetylases toCFTR and that certain factors tethered to the DNA-binding proteins (such as CDP/cut) can target the deacetylation (or acetylation) of specific lysine residues of core histones such as those established through similar models (67Yang W.M. Inouye C. Zeng Y. Bearss D. Seto E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12845-12850Crossref PubMed Scopus (482) Google Scholar, 68Ayer D.E. Kretzner L. Eisenman R.N. Cell. 1993; 72: 211-222Abstract Full Text PDF PubMed Scopus (621) Google Scholar). Because CFTRtranscription is potentiated through TSA we tested whether CDP/cut may function to interact with any known histone deacetylase. Immunoprecipitation of CDP/cut demonstrates that along with CDP/cut, histone deacetylase activity is coimmunoprecipitated and is inhibited by TSA. Furthermore, studies using fusion proteins of CDP/cut indicate that CDP/cut may recruit interactions with histone deacetylase, HDAC1, through the COOH-terminal domain known to contain the repression domain of CDP/cut (57Mailly F. Bérubé G. Harada R. Mao P.-L. Phillips S. Nepveu A. Mol. Cell. Biol. 1996; 16: 5346-5357Crossref PubMed Scopus (105) Google Scholar). It is only implied here that CDP/cut may repress gene transcription of CFTRthrough a mechanism incorporating the histone deacetylation of chromatin in proximity to the CFTR promoter. Although the molecular basis for the repression mediated by CDP/cut in vivo is not known, it could conceptually involve competition between enzymes involved in histone acetylation associated with sequence-specific transcription factors. Although this simple model could explain the role CDP/cut may have in transcriptional repression of CFTR, still little is known about the precise mechanism directing specific alterations in local chromatin domainsin vivo.The acetylation state of local chromatin may represent a stoichiometric relationship between competing acetylation and deacetylation processes. Thus, the requirement for the Y-box in hGCN5-mediated transactivation of CFTR suggests that the Y-box element likely binds proteins necessary for the conformational changes in chromatin structure. There is evidence to suggest that certain transcription factors have higher affinity for their binding sites when the nucleotide sequences are embedded in chromatin assembled from hyperacetylated histones (69Vettese-Dadey M. Grant P.A. Hebbes T.R. Crane-Robinson C. Allis C.D. Workman J.L. EMBO J. 1996; 15: 2508-2518Crossref PubMed Scopus (374) Google Scholar). It is plausible that competing activities directed by CDP/cut or CBF·NF-Y, as an example, recruit activities favorable for either the repression or activation ofCFTR transcription, respectively. Although we suggest that CDP/cut recruits histone deacetylase activity, it is unclear precisely how such a ternary complex of CDP/cut is assembled in the context of the CFTR promoter Y-box element and whether such events do take place in viv