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Reb1p-dependent DNA Bending Effects Nucleosome Positioning and Constitutive Transcription at the Yeast Profilin Promoter

2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês

10.1074/jbc.m301806200

1083-351X

Michaela Angermayr, Ulrich Oechsner, Wolfhard Bandlow,

Fungal and yeast genetics research

The molecular basis of constitutive gene activation is largely unknown. The yeast profilin gene (PFY1), encoding a housekeeping component of the actin cytoskeleton, is constitutively transcribed at a moderate level. ThePFY1 promoter dispenses with classical transactivators and a consensus TATA box; however, it contains a canonic site for the abundant multifunctional nuclear factor rDNA enhancer-binding protein (Reb1p) combined with a dA·dT element. Reb1p binds specificallyin vitro. Mutation of this site reduces PFY1expression to about 35%. A nucleosome-free gap of about 190 bp is centered at the genomic Reb1p binding site in vivo and spans the presumptive core promoter and transcriptional initiation sites. Nucleosomes at the border of the gap are positioned. Mutation of the Reb1p motif in the genomic PFY1 promoter abolishes nucleosome positioning, fills the gap with a non-positioned nucleosome, and reduces transcription by a factor of 3. From permutation studies we conclude that Reb1p induces a strong bend into the DNA. Phasing analyses indicate that it is directed toward the major groove. The data suggest that Reb1p plays an architectural role on DNA and that Reb1p-dependent DNA bending leads to a DNA conformation that is incompatible with packaging into nucleosomes and concomitantly facilitates constitutive transcription. In the absence of other transcription activators, Reb1p excludes nucleosomes and moderately stimulates transcription by distorting DNA. The molecular basis of constitutive gene activation is largely unknown. The yeast profilin gene (PFY1), encoding a housekeeping component of the actin cytoskeleton, is constitutively transcribed at a moderate level. ThePFY1 promoter dispenses with classical transactivators and a consensus TATA box; however, it contains a canonic site for the abundant multifunctional nuclear factor rDNA enhancer-binding protein (Reb1p) combined with a dA·dT element. Reb1p binds specificallyin vitro. Mutation of this site reduces PFY1expression to about 35%. A nucleosome-free gap of about 190 bp is centered at the genomic Reb1p binding site in vivo and spans the presumptive core promoter and transcriptional initiation sites. Nucleosomes at the border of the gap are positioned. Mutation of the Reb1p motif in the genomic PFY1 promoter abolishes nucleosome positioning, fills the gap with a non-positioned nucleosome, and reduces transcription by a factor of 3. From permutation studies we conclude that Reb1p induces a strong bend into the DNA. Phasing analyses indicate that it is directed toward the major groove. The data suggest that Reb1p plays an architectural role on DNA and that Reb1p-dependent DNA bending leads to a DNA conformation that is incompatible with packaging into nucleosomes and concomitantly facilitates constitutive transcription. In the absence of other transcription activators, Reb1p excludes nucleosomes and moderately stimulates transcription by distorting DNA. yeast gene encoding actin yeast gene encoding profilin position(s) uracil TATA-binding protein The actin cytoskeleton is fascinating because of both the complexity of its functions and the dynamics of its structure. In yeast, it has been shown to be involved in the establishment of cell polarity and bud site selection, in intracellular transport, and in signal transduction as well as in cytokinesis. According to the plethora of functions, the organization of the microfilament system and intracellular distribution of actin is highly dynamic and closely linked to the progression of the cell cycle. Despite continuous cell cycle-controlled reorganization of the cytoskeleton, the single actin gene in yeast (ACT1)1 is expressed constitutively (1McLean M. Hubberstey A.V. Bouman D.J. Pece N. Mastrangelo P. Wildeman A.G. Mol. Microbiol. 1995; 18: 605-614Crossref PubMed Scopus (15) Google Scholar). The polymerization state and organization of actin is controlled posttranscriptionally by accessory proteins (2Way M. Weeds A. Nature. 1990; 344: 292-294Crossref PubMed Scopus (34) Google Scholar, 3Stossel T.P. Chaponnier C. Ezzell R.M. Hartwig J.H. Janmey P.A. Kwiatkowski D.J. Lind S.E. Smith D.B. Southwick F.S. Yin H.L. Zaner K.S. Annu. Rev. Cell Biol. 1985; 1: 353-402Crossref PubMed Scopus (382) Google Scholar, 4Weeds A. Nature. 1982; 296: 811-816Crossref PubMed Scopus (314) Google Scholar), and actin as well as most of the actin-binding proteins are expressed constitutively.One of the actin-binding proteins is profilin (3Stossel T.P. Chaponnier C. Ezzell R.M. Hartwig J.H. Janmey P.A. Kwiatkowski D.J. Lind S.E. Smith D.B. Southwick F.S. Yin H.L. Zaner K.S. Annu. Rev. Cell Biol. 1985; 1: 353-402Crossref PubMed Scopus (382) Google Scholar, 5Carlsson L. Nystrøm L.-E. Lindberg U. Kannan K.K. Cid-Dresdner H. Lovgren S. Jornvall H. J. Mol. Biol. 1976; 105: 353-366Crossref PubMed Scopus (110) Google Scholar, 6Pollard T.D. Cooper J.A. Annu. Rev. Biochem. 1986; 55: 987-1935Crossref PubMed Google Scholar, 7Reichstein E. Korn E.D. J. Biol. Chem. 1979; 254: 6174-6179Abstract Full Text PDF PubMed Google Scholar, 8Magdolen V. Oechsner U. Müller G. Bandlow W. Mol. Cell. Biol. 1988; 8: 5108-5115Crossref PubMed Scopus (80) Google Scholar), which has been thought previously to regulate actin filament formation exclusively by sequestering G-actin monomers and thereby to antagonize actin polymerization (3Stossel T.P. Chaponnier C. Ezzell R.M. Hartwig J.H. Janmey P.A. Kwiatkowski D.J. Lind S.E. Smith D.B. Southwick F.S. Yin H.L. Zaner K.S. Annu. Rev. Cell Biol. 1985; 1: 353-402Crossref PubMed Scopus (382) Google Scholar). However, recent results point to a more complex regulation (9Goldschmidt-Clermont P.J. Janmey P.A. Cell. 1991; 66: 419-421Abstract Full Text PDF PubMed Scopus (73) Google Scholar). It involves the binding of phosphoinositides to profilin and their controlled cleavage by phospholipase C, which could provide the basis for the regulation of profilin-actin interaction (10Lassing I. Lindberg U. Nature. 1985; 314: 472-474Crossref PubMed Scopus (634) Google Scholar,11Lassing I. Lindberg U. J. Cell. Biochem. 1988; 37: 255-267Crossref PubMed Scopus (119) Google Scholar) and, as a consequence, in signaling to actin (9Goldschmidt-Clermont P.J. Janmey P.A. Cell. 1991; 66: 419-421Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 12Hartwig J.H. Chambers K.A. Hopcia K.L. Kwiatkowski D.J. J. Cell Biol. 1989; 109: 1571-1579Crossref PubMed Scopus (71) Google Scholar, 13Hartwig J.H. Kwiatkowski J.D. Curr. Opin. Cell Biol. 1991; 3: 87-97Crossref PubMed Scopus (217) Google Scholar, 14Goldschmidt-Clermont P.J. Machesky L.M. Baldassare J.J. Pollard T.D. Science. 1990; 247: 1575-1578Crossref PubMed Scopus (364) Google Scholar). In fact, results of recent experiments on yeast profilin and CAP, a component of the yeast adenylyl cyclase complex, functionally link the growth signaling pathway to the control of the cytoskeleton (15Vojtek A. Haarer B. Field J. Gerst J. Pollard T.D. Brown S.S. Wigler M. Cell. 1991; 66: 497-505Abstract Full Text PDF PubMed Scopus (149) Google Scholar). Previously we have isolated and characterized the single structural gene for yeast profilin (PFY1) and shown that its deletion leads to a temperature-conditional phenotype. PFY1 is constitutively transcribed at a moderately high level (8Magdolen V. Oechsner U. Müller G. Bandlow W. Mol. Cell. Biol. 1988; 8: 5108-5115Crossref PubMed Scopus (80) Google Scholar, 16Haarer B.K. Lillie S.H. Adams A.E.M. Magdolen V. Bandlow W. Brown S.S. J. Cell Biol. 1990; 110: 105-114Crossref PubMed Scopus (190) Google Scholar, 17Magdolen V. Drubin D.G. Mages G. Bandlow W. FEBS Lett. 1993; 316: 41-47Crossref PubMed Scopus (60) Google Scholar).Amazingly little is known about the chromatin constellation at constitutive promoters and about transcription factors involved in transcription of housekeeping genes. It is supposed that constitutive transcription reflects a static situation in which the promoter is in a permanently activated state. Accordingly these promoters are presumed to be constitutively kept free of nucleosomes (18Scott E.W. Baker H.V. Mol. Cell. Biol. 1993; 13: 543-550Crossref PubMed Scopus (78) Google Scholar, 19Angermayr M. Oechsner U. Gregor K. Schroth G.P. Bandlow W. Nucleic Acids Res. 2002; 30: 4199-4207Crossref PubMed Scopus (20) Google Scholar), but the principles and mechanisms underlying nucleosome exclusion are far from clear. Moreover it is unknown whether constitutive transcription is basal transcription involving only the basal transcription apparatus or whether specific transactivators are required in addition. Sincecis-acting sites for any of the classical transactivators are absent from the 5′-flank of the PFY1 gene, we tested the hypothesis that constitutive transcription of this gene dispenses with classical transcription activators. We have dissected thePFY1 promoter and demonstrate that it harbors a binding site for the rDNA enhancer-binding protein (Reb1p; also known as Grf2p, factor Y, and factor Q).Reb1p is among the abundant so-called "general regulatory factors." It is multifunctional, as it is involved in transcriptional termination (20Lang W.H. Reeder R.H. Mol. Cell. Biol. 1993; 13: 649-658Crossref PubMed Scopus (87) Google Scholar, 21Lang W.H. Reeder R.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9781-9785Crossref PubMed Scopus (46) Google Scholar), binds to telomeres and centromeres (22Chasman D.I. Lue N.F. Buchman A.R. LaPointe J.W. Lorch Y. Kornberg R.D. Genes Dev. 1990; 4: 503-514Crossref PubMed Scopus (160) Google Scholar), and plays a role in transcriptional regulation of a plethora of functionally unrelated genes transcribed by either polymerase I or II (18Scott E.W. Baker H.V. Mol. Cell. Biol. 1993; 13: 543-550Crossref PubMed Scopus (78) Google Scholar, 22Chasman D.I. Lue N.F. Buchman A.R. LaPointe J.W. Lorch Y. Kornberg R.D. Genes Dev. 1990; 4: 503-514Crossref PubMed Scopus (160) Google Scholar, 23Morrow B.E. Johnson S.P. Warner J.R. J. Biol. Chem. 1989; 264: 9061-9068Abstract Full Text PDF PubMed Google Scholar, 24Graham I.R. Chambers A. Mol. Microbiol. 1994; 12: 931-940Crossref PubMed Scopus (21) Google Scholar, 25Angermayr M. Bandlow W. Mol. Gen. Genet. 1997; 256: 682-689Crossref PubMed Scopus (11) Google Scholar, 26Angermayr M. Bandlow W. J. Biol. Chem. 1997; 272: 31630-31635Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). However, the direct transcriptional activation potential of Reb1p is marginal compared with specific activator proteins, but combinations of Reb1p binding sites with cognate motifs for weak transcription activators or dA·dT elements cause considerable synergistic effects (22Chasman D.I. Lue N.F. Buchman A.R. LaPointe J.W. Lorch Y. Kornberg R.D. Genes Dev. 1990; 4: 503-514Crossref PubMed Scopus (160) Google Scholar, 27Struhl K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8419-8423Crossref PubMed Scopus (305) Google Scholar, 28Struhl K. Cell. 1987; 49: 295-297Abstract Full Text PDF PubMed Scopus (274) Google Scholar, 29Moreira J.M. Hörz W. Holmberg S. J. Biol. Chem. 2002; 277: 3202-3209Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Interactions between Reb1p and the basal transcription machinery are discussed as well (1McLean M. Hubberstey A.V. Bouman D.J. Pece N. Mastrangelo P. Wildeman A.G. Mol. Microbiol. 1995; 18: 605-614Crossref PubMed Scopus (15) Google Scholar, 22Chasman D.I. Lue N.F. Buchman A.R. LaPointe J.W. Lorch Y. Kornberg R.D. Genes Dev. 1990; 4: 503-514Crossref PubMed Scopus (160) Google Scholar, 26Angermayr M. Bandlow W. J. Biol. Chem. 1997; 272: 31630-31635Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 30Packham E.A. Graham I.R. Chambers A. Mol. Gen. Genet. 1996; 250: 348-356PubMed Google Scholar, 31Erkine A.M. Adams C.C. Diken T. Gross D.S. Mol. Cell. Biol. 1996; 16: 7004-7017Crossref PubMed Scopus (39) Google Scholar). Reb1p is encoded by an essential gene (32Ju Q. Morrow B.E. Warner J.R. Mol. Cell. Biol. 1990; 1: 5226-5234Crossref Google Scholar); however, the reason for its indispensability has not yet been established. The 125-kDa protein binds as a monomer to its site on DNA with the consensus YNNYYACCCG, and its DNA-binding domain, which bears some similarity to the vertebrate proto-oncogenemyb, is extraordinarily large (about 400 amino acids) (32Ju Q. Morrow B.E. Warner J.R. Mol. Cell. Biol. 1990; 1: 5226-5234Crossref Google Scholar, 33Morrow B.E. Ju Q. Warner J.R. Mol. Cell. Biol. 1993; 13: 1173-1182Crossref PubMed Scopus (50) Google Scholar). The analysis of the chromatin structure at theGAL1-GAL10 promoter, which contains a Reb1p site overlapping with a motif for binding of Gal4p, has revealed a nucleosome-free gap of 230 bp. Previous studies indicated that Reb1p binding is responsible for nucleosome exclusion from the GAL1-GAL10 promoter (34Fedor M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Crossref PubMed Scopus (183) Google Scholar). However, more recent results have demonstrated that the chromatin structure in this intergenic region is not influenced by Reb1p binding (35Reagan M.S. Majors J.E. Mol. Gen. Genet. 1998; 259: 142-149Crossref PubMed Scopus (17) Google Scholar). Thus, the importance of Reb1p binding for the arrangement of nucleosomes and the efficiency of transcriptional initiation is still obscure. Whether nucleosome exclusion is a general feature of Reb1p remains to be elucidated and is controversially discussed (22Chasman D.I. Lue N.F. Buchman A.R. LaPointe J.W. Lorch Y. Kornberg R.D. Genes Dev. 1990; 4: 503-514Crossref PubMed Scopus (160) Google Scholar, 29Moreira J.M. Hörz W. Holmberg S. J. Biol. Chem. 2002; 277: 3202-3209Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 31Erkine A.M. Adams C.C. Diken T. Gross D.S. Mol. Cell. Biol. 1996; 16: 7004-7017Crossref PubMed Scopus (39) Google Scholar). More importantly, if nucleosome exclusion is a general feature, it is yet unknown which property of Reb1p prevents assembly of nucleosomes in the flanks of its binding site extending over distances as long as about 100 bp to either side.We show that constitutive transcription at the PFY1 promoter dispenses with classical transactivators. We demonstrate that Reb1p has an architectural role, and its DNA binding is necessary and sufficient to keep nucleosomes off the DNA region spanning the core promoter and the transcription initiation sites of the PFY1 promoter. Destruction of the Reb1p binding motif in the genomic context by site-directed point mutation leads to occupation of the promoter by randomly arranged nucleosomes and reduction of mRNA synthesis by a factor of 3. Permutation studies imply that nucleosome exclusion could be related to the strong bending of the DNA structure induced by Reb1p binding in conjunction with a neighboring dA·dT element. We conclude that, at the PFY1 promoter, transcription may ensue spontaneously as long as the core promoter is prebent and accessible to polymerase II holoenzyme. The actin cytoskeleton is fascinating because of both the complexity of its functions and the dynamics of its structure. In yeast, it has been shown to be involved in the establishment of cell polarity and bud site selection, in intracellular transport, and in signal transduction as well as in cytokinesis. According to the plethora of functions, the organization of the microfilament system and intracellular distribution of actin is highly dynamic and closely linked to the progression of the cell cycle. Despite continuous cell cycle-controlled reorganization of the cytoskeleton, the single actin gene in yeast (ACT1)1 is expressed constitutively (1McLean M. Hubberstey A.V. Bouman D.J. Pece N. Mastrangelo P. Wildeman A.G. Mol. Microbiol. 1995; 18: 605-614Crossref PubMed Scopus (15) Google Scholar). The polymerization state and organization of actin is controlled posttranscriptionally by accessory proteins (2Way M. Weeds A. Nature. 1990; 344: 292-294Crossref PubMed Scopus (34) Google Scholar, 3Stossel T.P. Chaponnier C. Ezzell R.M. Hartwig J.H. Janmey P.A. Kwiatkowski D.J. Lind S.E. Smith D.B. Southwick F.S. Yin H.L. Zaner K.S. Annu. Rev. Cell Biol. 1985; 1: 353-402Crossref PubMed Scopus (382) Google Scholar, 4Weeds A. Nature. 1982; 296: 811-816Crossref PubMed Scopus (314) Google Scholar), and actin as well as most of the actin-binding proteins are expressed constitutively. One of the actin-binding proteins is profilin (3Stossel T.P. Chaponnier C. Ezzell R.M. Hartwig J.H. Janmey P.A. Kwiatkowski D.J. Lind S.E. Smith D.B. Southwick F.S. Yin H.L. Zaner K.S. Annu. Rev. Cell Biol. 1985; 1: 353-402Crossref PubMed Scopus (382) Google Scholar, 5Carlsson L. Nystrøm L.-E. Lindberg U. Kannan K.K. Cid-Dresdner H. Lovgren S. Jornvall H. J. Mol. Biol. 1976; 105: 353-366Crossref PubMed Scopus (110) Google Scholar, 6Pollard T.D. Cooper J.A. Annu. Rev. Biochem. 1986; 55: 987-1935Crossref PubMed Google Scholar, 7Reichstein E. Korn E.D. J. Biol. Chem. 1979; 254: 6174-6179Abstract Full Text PDF PubMed Google Scholar, 8Magdolen V. Oechsner U. Müller G. Bandlow W. Mol. Cell. Biol. 1988; 8: 5108-5115Crossref PubMed Scopus (80) Google Scholar), which has been thought previously to regulate actin filament formation exclusively by sequestering G-actin monomers and thereby to antagonize actin polymerization (3Stossel T.P. Chaponnier C. Ezzell R.M. Hartwig J.H. Janmey P.A. Kwiatkowski D.J. Lind S.E. Smith D.B. Southwick F.S. Yin H.L. Zaner K.S. Annu. Rev. Cell Biol. 1985; 1: 353-402Crossref PubMed Scopus (382) Google Scholar). However, recent results point to a more complex regulation (9Goldschmidt-Clermont P.J. Janmey P.A. Cell. 1991; 66: 419-421Abstract Full Text PDF PubMed Scopus (73) Google Scholar). It involves the binding of phosphoinositides to profilin and their controlled cleavage by phospholipase C, which could provide the basis for the regulation of profilin-actin interaction (10Lassing I. Lindberg U. Nature. 1985; 314: 472-474Crossref PubMed Scopus (634) Google Scholar,11Lassing I. Lindberg U. J. Cell. Biochem. 1988; 37: 255-267Crossref PubMed Scopus (119) Google Scholar) and, as a consequence, in signaling to actin (9Goldschmidt-Clermont P.J. Janmey P.A. Cell. 1991; 66: 419-421Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 12Hartwig J.H. Chambers K.A. Hopcia K.L. Kwiatkowski D.J. J. Cell Biol. 1989; 109: 1571-1579Crossref PubMed Scopus (71) Google Scholar, 13Hartwig J.H. Kwiatkowski J.D. Curr. Opin. Cell Biol. 1991; 3: 87-97Crossref PubMed Scopus (217) Google Scholar, 14Goldschmidt-Clermont P.J. Machesky L.M. Baldassare J.J. Pollard T.D. Science. 1990; 247: 1575-1578Crossref PubMed Scopus (364) Google Scholar). In fact, results of recent experiments on yeast profilin and CAP, a component of the yeast adenylyl cyclase complex, functionally link the growth signaling pathway to the control of the cytoskeleton (15Vojtek A. Haarer B. Field J. Gerst J. Pollard T.D. Brown S.S. Wigler M. Cell. 1991; 66: 497-505Abstract Full Text PDF PubMed Scopus (149) Google Scholar). Previously we have isolated and characterized the single structural gene for yeast profilin (PFY1) and shown that its deletion leads to a temperature-conditional phenotype. PFY1 is constitutively transcribed at a moderately high level (8Magdolen V. Oechsner U. Müller G. Bandlow W. Mol. Cell. Biol. 1988; 8: 5108-5115Crossref PubMed Scopus (80) Google Scholar, 16Haarer B.K. Lillie S.H. Adams A.E.M. Magdolen V. Bandlow W. Brown S.S. J. Cell Biol. 1990; 110: 105-114Crossref PubMed Scopus (190) Google Scholar, 17Magdolen V. Drubin D.G. Mages G. Bandlow W. FEBS Lett. 1993; 316: 41-47Crossref PubMed Scopus (60) Google Scholar). Amazingly little is known about the chromatin constellation at constitutive promoters and about transcription factors involved in transcription of housekeeping genes. It is supposed that constitutive transcription reflects a static situation in which the promoter is in a permanently activated state. Accordingly these promoters are presumed to be constitutively kept free of nucleosomes (18Scott E.W. Baker H.V. Mol. Cell. Biol. 1993; 13: 543-550Crossref PubMed Scopus (78) Google Scholar, 19Angermayr M. Oechsner U. Gregor K. Schroth G.P. Bandlow W. Nucleic Acids Res. 2002; 30: 4199-4207Crossref PubMed Scopus (20) Google Scholar), but the principles and mechanisms underlying nucleosome exclusion are far from clear. Moreover it is unknown whether constitutive transcription is basal transcription involving only the basal transcription apparatus or whether specific transactivators are required in addition. Sincecis-acting sites for any of the classical transactivators are absent from the 5′-flank of the PFY1 gene, we tested the hypothesis that constitutive transcription of this gene dispenses with classical transcription activators. We have dissected thePFY1 promoter and demonstrate that it harbors a binding site for the rDNA enhancer-binding protein (Reb1p; also known as Grf2p, factor Y, and factor Q). Reb1p is among the abundant so-called "general regulatory factors." It is multifunctional, as it is involved in transcriptional termination (20Lang W.H. Reeder R.H. Mol. Cell. Biol. 1993; 13: 649-658Crossref PubMed Scopus (87) Google Scholar, 21Lang W.H. Reeder R.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9781-9785Crossref PubMed Scopus (46) Google Scholar), binds to telomeres and centromeres (22Chasman D.I. Lue N.F. Buchman A.R. LaPointe J.W. Lorch Y. Kornberg R.D. Genes Dev. 1990; 4: 503-514Crossref PubMed Scopus (160) Google Scholar), and plays a role in transcriptional regulation of a plethora of functionally unrelated genes transcribed by either polymerase I or II (18Scott E.W. Baker H.V. Mol. Cell. Biol. 1993; 13: 543-550Crossref PubMed Scopus (78) Google Scholar, 22Chasman D.I. Lue N.F. Buchman A.R. LaPointe J.W. Lorch Y. Kornberg R.D. Genes Dev. 1990; 4: 503-514Crossref PubMed Scopus (160) Google Scholar, 23Morrow B.E. Johnson S.P. Warner J.R. J. Biol. Chem. 1989; 264: 9061-9068Abstract Full Text PDF PubMed Google Scholar, 24Graham I.R. Chambers A. Mol. Microbiol. 1994; 12: 931-940Crossref PubMed Scopus (21) Google Scholar, 25Angermayr M. Bandlow W. Mol. Gen. Genet. 1997; 256: 682-689Crossref PubMed Scopus (11) Google Scholar, 26Angermayr M. Bandlow W. J. Biol. Chem. 1997; 272: 31630-31635Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). However, the direct transcriptional activation potential of Reb1p is marginal compared with specific activator proteins, but combinations of Reb1p binding sites with cognate motifs for weak transcription activators or dA·dT elements cause considerable synergistic effects (22Chasman D.I. Lue N.F. Buchman A.R. LaPointe J.W. Lorch Y. Kornberg R.D. Genes Dev. 1990; 4: 503-514Crossref PubMed Scopus (160) Google Scholar, 27Struhl K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8419-8423Crossref PubMed Scopus (305) Google Scholar, 28Struhl K. Cell. 1987; 49: 295-297Abstract Full Text PDF PubMed Scopus (274) Google Scholar, 29Moreira J.M. Hörz W. Holmberg S. J. Biol. Chem. 2002; 277: 3202-3209Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Interactions between Reb1p and the basal transcription machinery are discussed as well (1McLean M. Hubberstey A.V. Bouman D.J. Pece N. Mastrangelo P. Wildeman A.G. Mol. Microbiol. 1995; 18: 605-614Crossref PubMed Scopus (15) Google Scholar, 22Chasman D.I. Lue N.F. Buchman A.R. LaPointe J.W. Lorch Y. Kornberg R.D. Genes Dev. 1990; 4: 503-514Crossref PubMed Scopus (160) Google Scholar, 26Angermayr M. Bandlow W. J. Biol. Chem. 1997; 272: 31630-31635Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 30Packham E.A. Graham I.R. Chambers A. Mol. Gen. Genet. 1996; 250: 348-356PubMed Google Scholar, 31Erkine A.M. Adams C.C. Diken T. Gross D.S. Mol. Cell. Biol. 1996; 16: 7004-7017Crossref PubMed Scopus (39) Google Scholar). Reb1p is encoded by an essential gene (32Ju Q. Morrow B.E. Warner J.R. Mol. Cell. Biol. 1990; 1: 5226-5234Crossref Google Scholar); however, the reason for its indispensability has not yet been established. The 125-kDa protein binds as a monomer to its site on DNA with the consensus YNNYYACCCG, and its DNA-binding domain, which bears some similarity to the vertebrate proto-oncogenemyb, is extraordinarily large (about 400 amino acids) (32Ju Q. Morrow B.E. Warner J.R. Mol. Cell. Biol. 1990; 1: 5226-5234Crossref Google Scholar, 33Morrow B.E. Ju Q. Warner J.R. Mol. Cell. Biol. 1993; 13: 1173-1182Crossref PubMed Scopus (50) Google Scholar). The analysis of the chromatin structure at theGAL1-GAL10 promoter, which contains a Reb1p site overlapping with a motif for binding of Gal4p, has revealed a nucleosome-free gap of 230 bp. Previous studies indicated that Reb1p binding is responsible for nucleosome exclusion from the GAL1-GAL10 promoter (34Fedor M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Crossref PubMed Scopus (183) Google Scholar). However, more recent results have demonstrated that the chromatin structure in this intergenic region is not influenced by Reb1p binding (35Reagan M.S. Majors J.E. Mol. Gen. Genet. 1998; 259: 142-149Crossref PubMed Scopus (17) Google Scholar). Thus, the importance of Reb1p binding for the arrangement of nucleosomes and the efficiency of transcriptional initiation is still obscure. Whether nucleosome exclusion is a general feature of Reb1p remains to be elucidated and is controversially discussed (22Chasman D.I. Lue N.F. Buchman A.R. LaPointe J.W. Lorch Y. Kornberg R.D. Genes Dev. 1990; 4: 503-514Crossref PubMed Scopus (160) Google Scholar, 29Moreira J.M. Hörz W. Holmberg S. J. Biol. Chem. 2002; 277: 3202-3209Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 31Erkine A.M. Adams C.C. Diken T. Gross D.S. Mol. Cell. Biol. 1996; 16: 7004-7017Crossref PubMed Scopus (39) Google Scholar). More importantly, if nucleosome exclusion is a general feature, it is yet unknown which property of Reb1p prevents assembly of nucleosomes in the flanks of its binding site extending over distances as long as about 100 bp to either side. We show that constitutive transcription at the PFY1 promoter dispenses with classical transactivators. We demonstrate that Reb1p has an architectural role, and its DNA binding is necessary and sufficient to keep nucleosomes off the DNA region spanning the core promoter and the transcription initiation sites of the PFY1 promoter. Destruction of the Reb1p binding motif in the genomic context by site-directed point mutation leads to occupation of the promoter by randomly arranged nucleosomes and reduction of mRNA synthesis by a factor of 3. Permutation studies imply that nucleosome exclusion could be related to the strong bending of the DNA structure induced by Reb1p binding in conjunction with a neighboring dA·dT element. We conclude that, at the PFY1 promoter, transcription may ensue spontaneously as long as the core promoter is prebent and accessible to polymerase II holoenzyme. The skillful technical assistance of G. Strobel is gratefully acknowledged. E. coli transformant strains either expressing recombinant Reb1p or harboring the cloning vector pET11a were donated to us by J. Warner (Bronx, NY). Anti-profilin and anti-actin antisera were kindly provided by S. Brown (Ann Arbor, MI) and A. Adams (Tucson, AZ), respectively.