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. 1998 Feb 16;17(4):1096–1106. doi: 10.1093/emboj/17.4.1096

The NOT proteins are part of the CCR4 transcriptional complex and affect gene expression both positively and negatively.

H Y Liu 1, V Badarinarayana 1, D C Audino 1, J Rappsilber 1, M Mann 1, C L Denis 1
PMCID: PMC1170458  PMID: 9463387

Abstract

The CCR4 transcriptional regulatory complex consisting of CCR4, CAF1, DBF2 and other unidentified factors is one of several groups of proteins that affect gene expression. Using mass spectrometry, we have identified the 195, 185 and 116 kDa species which are part of the CCR4 complex. The 195 and 185 kDa proteins were found to be NOT1 and the 116 kDa species was identical to NOT3. NOT1, 2, 3 and 4 proteins are part of a regulatory complex that negatively affects transcription. All four NOT proteins were found to co-immunoprecipitate with CCR4 and CAF1, and NOT1 co-purified with CCR4 and CAF1 through three chromatographic steps in a complex estimated to be 1.2x10(6) Da in size. Mutations in the NOT genes affected many of the same genes and processes that are affected by defects in the CCR4 complex components, including reduction in ADH2 derepression, defective cell wall integrity and increased sensitivity to monoand divalent ions. Similarly, ccr4, caf1 and dbf2 alleles negatively regulated FUS1-lacZ expression, as do defects in the NOT genes. These results indicate that the NOT proteins are physically and functionally part of the CCR4 complex which forms a unique and novel complex that affects transcription both positively and negatively.

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Selected References

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  1. Breeden L., Nasmyth K. Cell cycle control of the yeast HO gene: cis- and trans-acting regulators. Cell. 1987 Feb 13;48(3):389–397. doi: 10.1016/0092-8674(87)90190-5. [DOI] [PubMed] [Google Scholar]
  2. Cade R. M., Errede B. MOT2 encodes a negative regulator of gene expression that affects basal expression of pheromone-responsive genes in Saccharomyces cerevisiae. Mol Cell Biol. 1994 May;14(5):3139–3149. doi: 10.1128/mcb.14.5.3139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Collart M. A., Struhl K. CDC39, an essential nuclear protein that negatively regulates transcription and differentially affects the constitutive and inducible HIS3 promoters. EMBO J. 1993 Jan;12(1):177–186. doi: 10.1002/j.1460-2075.1993.tb05643.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Collart M. A., Struhl K. NOT1(CDC39), NOT2(CDC36), NOT3, and NOT4 encode a global-negative regulator of transcription that differentially affects TATA-element utilization. Genes Dev. 1994 Mar 1;8(5):525–537. doi: 10.1101/gad.8.5.525. [DOI] [PubMed] [Google Scholar]
  5. Collart M. A. The NOT, SPT3, and MOT1 genes functionally interact to regulate transcription at core promoters. Mol Cell Biol. 1996 Dec;16(12):6668–6676. doi: 10.1128/mcb.16.12.6668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cook W. J., Chase D., Audino D. C., Denis C. L. Dissection of the ADR1 protein reveals multiple, functionally redundant activation domains interspersed with inhibitory regions: evidence for a repressor binding to the ADR1c region. Mol Cell Biol. 1994 Jan;14(1):629–640. doi: 10.1128/mcb.14.1.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Denis C. L., Draper M. P., Liu H. Y., Malvar T., Vallari R. C., Cook W. J. The yeast CCR4 protein is neither regulated by nor associated with the SPT6 and SPT10 proteins and forms a functionally distinct complex from that of the SNF/SWI transcription factors. Genetics. 1994 Dec;138(4):1005–1013. doi: 10.1093/genetics/138.4.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Denis C. L. Identification of new genes involved in the regulation of yeast alcohol dehydrogenase II. Genetics. 1984 Dec;108(4):833–844. doi: 10.1093/genetics/108.4.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Denis C. L., Malvar T. The CCR4 gene from Saccharomyces cerevisiae is required for both nonfermentative and spt-mediated gene expression. Genetics. 1990 Feb;124(2):283–291. doi: 10.1093/genetics/124.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Draper M. P., Liu H. Y., Nelsbach A. H., Mosley S. P., Denis C. L. CCR4 is a glucose-regulated transcription factor whose leucine-rich repeat binds several proteins important for placing CCR4 in its proper promoter context. Mol Cell Biol. 1994 Jul;14(7):4522–4531. doi: 10.1128/mcb.14.7.4522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grant P. A., Duggan L., Côté J., Roberts S. M., Brownell J. E., Candau R., Ohba R., Owen-Hughes T., Allis C. D., Winston F. Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. Genes Dev. 1997 Jul 1;11(13):1640–1650. doi: 10.1101/gad.11.13.1640. [DOI] [PubMed] [Google Scholar]
  12. Guarente L., Mason T. Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site. Cell. 1983 Apr;32(4):1279–1286. doi: 10.1016/0092-8674(83)90309-4. [DOI] [PubMed] [Google Scholar]
  13. Igual J. C., Johnson A. L., Johnston L. H. Coordinated regulation of gene expression by the cell cycle transcription factor Swi4 and the protein kinase C MAP kinase pathway for yeast cell integrity. EMBO J. 1996 Sep 16;15(18):5001–5013. [PMC free article] [PubMed] [Google Scholar]
  14. Irie K., Yamaguchi K., Kawase K., Matsumoto K. The yeast MOT2 gene encodes a putative zinc finger protein that serves as a global negative regulator affecting expression of several categories of genes, including mating-pheromone-responsive genes. Mol Cell Biol. 1994 May;14(5):3150–3157. doi: 10.1128/mcb.14.5.3150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jensen O. N., Podtelejnikov A., Mann M. Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps. Rapid Commun Mass Spectrom. 1996;10(11):1371–1378. doi: 10.1002/(SICI)1097-0231(199608)10:11<1371::AID-RCM682>3.0.CO;2-5. [DOI] [PubMed] [Google Scholar]
  16. Lamond A. I., Mann M. Cell biology and the genome projects a concerted strategy for characterizing multiprotein complexes by using mass spectrometry. Trends Cell Biol. 1997 Apr;7(4):139–142. doi: 10.1016/S0962-8924(97)01031-3. [DOI] [PubMed] [Google Scholar]
  17. Levin D. E., Bartlett-Heubusch E. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J Cell Biol. 1992 Mar;116(5):1221–1229. doi: 10.1083/jcb.116.5.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liu H. Y., Toyn J. H., Chiang Y. C., Draper M. P., Johnston L. H., Denis C. L. DBF2, a cell cycle-regulated protein kinase, is physically and functionally associated with the CCR4 transcriptional regulatory complex. EMBO J. 1997 Sep 1;16(17):5289–5298. doi: 10.1093/emboj/16.17.5289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mann M., Wilm M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal Chem. 1994 Dec 15;66(24):4390–4399. doi: 10.1021/ac00096a002. [DOI] [PubMed] [Google Scholar]
  20. McKenzie E. A., Kent N. A., Dowell S. J., Moreno F., Bird L. E., Mellor J. The centromere and promoter factor, 1, CPF1, of Saccharomyces cerevisiae modulates gene activity through a family of factors including SPT21, RPD1 (SIN3), RPD3 and CCR4. Mol Gen Genet. 1993 Sep;240(3):374–386. doi: 10.1007/BF00280389. [DOI] [PubMed] [Google Scholar]
  21. Natsoulis G., Dollard C., Winston F., Boeke J. D. The products of the SPT10 and SPT21 genes of Saccharomyces cerevisiae increase the amplitude of transcriptional regulation at a large number of unlinked loci. New Biol. 1991 Dec;3(12):1249–1259. [PubMed] [Google Scholar]
  22. Neubauer G., Gottschalk A., Fabrizio P., Séraphin B., Lührmann R., Mann M. Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry. Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):385–390. doi: 10.1073/pnas.94.2.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sakai A., Chibazakura T., Shimizu Y., Hishinuma F. Molecular analysis of POP2 gene, a gene required for glucose-derepression of gene expression in Saccharomyces cerevisiae. Nucleic Acids Res. 1992 Dec 11;20(23):6227–6233. doi: 10.1093/nar/20.23.6227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schild D. Suppression of a new allele of the yeast RAD52 gene by overexpression of RAD51, mutations in srs2 and ccr4, or mating-type heterozygosity. Genetics. 1995 May;140(1):115–127. doi: 10.1093/genetics/140.1.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shevchenko A., Jensen O. N., Podtelejnikov A. V., Sagliocco F., Wilm M., Vorm O., Mortensen P., Shevchenko A., Boucherie H., Mann M. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14440–14445. doi: 10.1073/pnas.93.25.14440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Toyn J. H., Araki H., Sugino A., Johnston L. H. The cell-cycle-regulated budding yeast gene DBF2, encoding a putative protein kinase, has a homologue that is not under cell-cycle control. Gene. 1991 Jul 31;104(1):63–70. doi: 10.1016/0378-1119(91)90465-n. [DOI] [PubMed] [Google Scholar]
  27. Verdone L., Cesari F., Denis C. L., Di Mauro E., Caserta M. Factors affecting Saccharomyces cerevisiae ADH2 chromatin remodeling and transcription. J Biol Chem. 1997 Dec 5;272(49):30828–30834. doi: 10.1074/jbc.272.49.30828. [DOI] [PubMed] [Google Scholar]
  28. Wade P. A., Werel W., Fentzke R. C., Thompson N. E., Leykam J. F., Burgess R. R., Jaehning J. A., Burton Z. F. A novel collection of accessory factors associated with yeast RNA polymerase II. Protein Expr Purif. 1996 Aug;8(1):85–90. doi: 10.1006/prep.1996.0077. [DOI] [PubMed] [Google Scholar]
  29. Wilm M., Mann M. Analytical properties of the nanoelectrospray ion source. Anal Chem. 1996 Jan 1;68(1):1–8. doi: 10.1021/ac9509519. [DOI] [PubMed] [Google Scholar]
  30. Wilm M., Shevchenko A., Houthaeve T., Breit S., Schweigerer L., Fotsis T., Mann M. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature. 1996 Feb 1;379(6564):466–469. doi: 10.1038/379466a0. [DOI] [PubMed] [Google Scholar]
  31. Wilson C. J., Chao D. M., Imbalzano A. N., Schnitzler G. R., Kingston R. E., Young R. A. RNA polymerase II holoenzyme contains SWI/SNF regulators involved in chromatin remodeling. Cell. 1996 Jan 26;84(2):235–244. doi: 10.1016/s0092-8674(00)80978-2. [DOI] [PubMed] [Google Scholar]

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