Abstract
It has been proposed that eukaryotic repressors of transcription can act by organizing chromatin, thereby preventing the accessibility of nearby DNA to activator proteins required for transcription initiation. In this study, we test this idea for the yeast alpha 2 repressor using a simple, artificial promoter that contains a single binding site for the activator protein Gal4 and a single binding site for the repressor alpha 2. When both the repressor and the activator are expressed in the same cell, the artificial promoter is efficiently repressed. In vivo footprinting experiments demonstrate that Gal4 can occupy its binding site even when the promoter is repressed. This result indicates that alpha 2-directed repression must result from interference with some stage in transcription initiation other than activator binding to DNA.
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- Axelrod J. D., Majors J. An improved method for photofootprinting yeast genes in vivo using Taq polymerase. Nucleic Acids Res. 1989 Jan 11;17(1):171–183. doi: 10.1093/nar/17.1.171. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bender A., Sprague G. F., Jr MAT alpha 1 protein, a yeast transcription activator, binds synergistically with a second protein to a set of cell-type-specific genes. Cell. 1987 Aug 28;50(5):681–691. doi: 10.1016/0092-8674(87)90326-6. [DOI] [PubMed] [Google Scholar]
- Bernardi F., Zatchej M., Thoma F. Species specific protein--DNA interactions may determine the chromatin units of genes in S.cerevisiae and in S.pombe. EMBO J. 1992 Mar;11(3):1177–1185. doi: 10.1002/j.1460-2075.1992.tb05158.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brewer A. C., Marsh P. J., Patient R. K. A simplified method for in vivo footprinting using DMS. Nucleic Acids Res. 1990 Sep 25;18(18):5574–5574. doi: 10.1093/nar/18.18.5574. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chatterjee S., Struhl K. Connecting a promoter-bound protein to TBP bypasses the need for a transcriptional activation domain. Nature. 1995 Apr 27;374(6525):820–822. doi: 10.1038/374820a0. [DOI] [PubMed] [Google Scholar]
- Chen J., Ding M., Pederson D. S. Binding of TFIID to the CYC1 TATA boxes in yeast occurs independently of upstream activating sequences. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11909–11913. doi: 10.1073/pnas.91.25.11909. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dolan J. W., Fields S. Cell-type-specific transcription in yeast. Biochim Biophys Acta. 1991 Feb 16;1088(2):155–169. doi: 10.1016/0167-4781(91)90051-m. [DOI] [PubMed] [Google Scholar]
- Ganter B., Tan S., Richmond T. J. Genomic footprinting of the promoter regions of STE2 and STE3 genes in the yeast Saccharomyces cerevisiae. J Mol Biol. 1993 Dec 20;234(4):975–987. doi: 10.1006/jmbi.1993.1652. [DOI] [PubMed] [Google Scholar]
- Giniger E., Ptashne M. Cooperative DNA binding of the yeast transcriptional activator GAL4. Proc Natl Acad Sci U S A. 1988 Jan;85(2):382–386. doi: 10.1073/pnas.85.2.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Giniger E., Varnum S. M., Ptashne M. Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell. 1985 Apr;40(4):767–774. doi: 10.1016/0092-8674(85)90336-8. [DOI] [PubMed] [Google Scholar]
- Goutte C., Johnson A. D. a1 protein alters the DNA binding specificity of alpha 2 repressor. Cell. 1988 Mar 25;52(6):875–882. doi: 10.1016/0092-8674(88)90429-1. [DOI] [PubMed] [Google Scholar]
- Herschbach B. M., Arnaud M. B., Johnson A. D. Transcriptional repression directed by the yeast alpha 2 protein in vitro. Nature. 1994 Jul 28;370(6487):309–311. doi: 10.1038/370309a0. [DOI] [PubMed] [Google Scholar]
- Herschbach B. M., Johnson A. D. Transcriptional repression in eukaryotes. Annu Rev Cell Biol. 1993;9:479–509. doi: 10.1146/annurev.cb.09.110193.002403. [DOI] [PubMed] [Google Scholar]
- Jackson M. E. Negative regulation of eukaryotic transcription. J Cell Sci. 1991 Sep;100(Pt 1):1–7. doi: 10.1242/jcs.100.1.1. [DOI] [PubMed] [Google Scholar]
- Johnson A. D., Herskowitz I. A repressor (MAT alpha 2 Product) and its operator control expression of a set of cell type specific genes in yeast. Cell. 1985 Aug;42(1):237–247. doi: 10.1016/s0092-8674(85)80119-7. [DOI] [PubMed] [Google Scholar]
- Johnson A. D. The price of repression. Cell. 1995 Jun 2;81(5):655–658. doi: 10.1016/0092-8674(95)90524-3. [DOI] [PubMed] [Google Scholar]
- Keleher C. A., Goutte C., Johnson A. D. The yeast cell-type-specific repressor alpha 2 acts cooperatively with a non-cell-type-specific protein. Cell. 1988 Jun 17;53(6):927–936. doi: 10.1016/s0092-8674(88)90449-7. [DOI] [PubMed] [Google Scholar]
- Keleher C. A., Redd M. J., Schultz J., Carlson M., Johnson A. D. Ssn6-Tup1 is a general repressor of transcription in yeast. Cell. 1992 Feb 21;68(4):709–719. doi: 10.1016/0092-8674(92)90146-4. [DOI] [PubMed] [Google Scholar]
- Kent N. A., Bird L. E., Mellor J. Chromatin analysis in yeast using NP-40 permeabilised sphaeroplasts. Nucleic Acids Res. 1993 Sep 25;21(19):4653–4654. doi: 10.1093/nar/21.19.4653. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kladde M. P., Simpson R. T. Positioned nucleosomes inhibit Dam methylation in vivo. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1361–1365. doi: 10.1073/pnas.91.4.1361. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Komachi K., Redd M. J., Johnson A. D. The WD repeats of Tup1 interact with the homeo domain protein alpha 2. Genes Dev. 1994 Dec 1;8(23):2857–2867. doi: 10.1101/gad.8.23.2857. [DOI] [PubMed] [Google Scholar]
- Kuchin S., Yeghiayan P., Carlson M. Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):4006–4010. doi: 10.1073/pnas.92.9.4006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Levine M., Manley J. L. Transcriptional repression of eukaryotic promoters. Cell. 1989 Nov 3;59(3):405–408. doi: 10.1016/0092-8674(89)90024-x. [DOI] [PubMed] [Google Scholar]
- Liao S. M., Zhang J., Jeffery D. A., Koleske A. J., Thompson C. M., Chao D. M., Viljoen M., van Vuuren H. J., Young R. A. A kinase-cyclin pair in the RNA polymerase II holoenzyme. Nature. 1995 Mar 9;374(6518):193–196. doi: 10.1038/374193a0. [DOI] [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
- Morse R. H. Nucleosome disruption by transcription factor binding in yeast. Science. 1993 Dec 3;262(5139):1563–1566. doi: 10.1126/science.8248805. [DOI] [PubMed] [Google Scholar]
- Mueller P. R., Wold B. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science. 1989 Nov 10;246(4931):780–786. doi: 10.1126/science.2814500. [DOI] [PubMed] [Google Scholar]
- Paranjape S. M., Kamakaka R. T., Kadonaga J. T. Role of chromatin structure in the regulation of transcription by RNA polymerase II. Annu Rev Biochem. 1994;63:265–297. doi: 10.1146/annurev.bi.63.070194.001405. [DOI] [PubMed] [Google Scholar]
- Patterton H. G., Simpson R. T. Nucleosomal location of the STE6 TATA box and Mat alpha 2p-mediated repression. Mol Cell Biol. 1994 Jun;14(6):4002–4010. doi: 10.1128/mcb.14.6.4002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roth S. Y. Chromatin-mediated transcriptional repression in yeast. Curr Opin Genet Dev. 1995 Apr;5(2):168–173. doi: 10.1016/0959-437x(95)80004-2. [DOI] [PubMed] [Google Scholar]
- Roth S. Y., Dean A., Simpson R. T. Yeast alpha 2 repressor positions nucleosomes in TRP1/ARS1 chromatin. Mol Cell Biol. 1990 May;10(5):2247–2260. doi: 10.1128/mcb.10.5.2247. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shimizu M., Roth S. Y., Szent-Gyorgyi C., Simpson R. T. Nucleosomes are positioned with base pair precision adjacent to the alpha 2 operator in Saccharomyces cerevisiae. EMBO J. 1991 Oct;10(10):3033–3041. doi: 10.1002/j.1460-2075.1991.tb07854.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Siliciano P. G., Tatchell K. Transcription and regulatory signals at the mating type locus in yeast. Cell. 1984 Jul;37(3):969–978. doi: 10.1016/0092-8674(84)90431-8. [DOI] [PubMed] [Google Scholar]
- Singh J., Klar A. J. Active genes in budding yeast display enhanced in vivo accessibility to foreign DNA methylases: a novel in vivo probe for chromatin structure of yeast. Genes Dev. 1992 Feb;6(2):186–196. doi: 10.1101/gad.6.2.186. [DOI] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Tatchell K., Nasmyth K. A., Hall B. D., Astell C., Smith M. In vitro mutation analysis of the mating-type locus in yeast. Cell. 1981 Nov;27(1 Pt 2):25–35. doi: 10.1016/0092-8674(81)90357-3. [DOI] [PubMed] [Google Scholar]
- Thoma F., Bergman L. W., Simpson R. T. Nuclease digestion of circular TRP1ARS1 chromatin reveals positioned nucleosomes separated by nuclease-sensitive regions. J Mol Biol. 1984 Aug 25;177(4):715–733. doi: 10.1016/0022-2836(84)90046-9. [DOI] [PubMed] [Google Scholar]
- Treitel M. A., Carlson M. Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3132–3136. doi: 10.1073/pnas.92.8.3132. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tzamarias D., Struhl K. Distinct TPR motifs of Cyc8 are involved in recruiting the Cyc8-Tup1 corepressor complex to differentially regulated promoters. Genes Dev. 1995 Apr 1;9(7):821–831. doi: 10.1101/gad.9.7.821. [DOI] [PubMed] [Google Scholar]
- Wahi M., Johnson A. D. Identification of genes required for alpha 2 repression in Saccharomyces cerevisiae. Genetics. 1995 May;140(1):79–90. doi: 10.1093/genetics/140.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Webster N., Jin J. R., Green S., Hollis M., Chambon P. The yeast UASG is a transcriptional enhancer in human HeLa cells in the presence of the GAL4 trans-activator. Cell. 1988 Jan 29;52(2):169–178. doi: 10.1016/0092-8674(88)90505-3. [DOI] [PubMed] [Google Scholar]
- Wolffe A. P. Transcription: in tune with the histones. Cell. 1994 Apr 8;77(1):13–16. doi: 10.1016/0092-8674(94)90229-1. [DOI] [PubMed] [Google Scholar]
- Workman J. L., Kingston R. E. Nucleosome core displacement in vitro via a metastable transcription factor-nucleosome complex. Science. 1992 Dec 11;258(5089):1780–1784. doi: 10.1126/science.1465613. [DOI] [PubMed] [Google Scholar]