Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Dec;17(12):7077–7087. doi: 10.1128/mcb.17.12.7077

DNA in transcriptionally silent chromatin assumes a distinct topology that is sensitive to cell cycle progression.

X Bi 1, J R Broach 1
PMCID: PMC232564  PMID: 9372939

Abstract

Transcriptionally silent regions of the Saccharomyces cerevisiae genome, the silent mating type loci and telomeres, represent the yeast equivalent of metazoan heterochromatin. To gain insight into the nature of silenced chromatin structure, we have examined the topology of DNA spanning the HML silent mating type locus by determining the superhelical density of mini-circles excised from HML (HML circles) by site-specific recombination. We observed that HML circles excised in a wild-type (SIR+) strain were more negatively supercoiled upon deproteinization than were the same circles excised in a sir- strain, in which silencing was abolished, even when HML alleles in which neither circle was transcriptionally competent were used. cis-acting sites flanking HML, called silencers, are required in the chromosome for establishment and inheritance of silencing. HML circles excised without silencers from cells arrested at any point in the cell cycle retained SIR-dependent differences in superhelical density. However, progression through the cell cycle converted SIR+ HML circles to a form resembling that of circles from sir- cells. This decay was not observed with circles carrying a silencer. These results establish that (i) DNA in transcriptionally silenced chromatin assumes a distinct topology reflecting a distinct organization of silenced versus active chromatin; (ii) the altered chromatin structure in silenced regions likely results from changes in packaging of individual nucleosomes, rather than changes in nucleosome density; and (iii) cell cycle progression disrupts the silenced chromatin structure, a process that is counteracted by silencers.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abraham J., Feldman J., Nasmyth K. A., Strathern J. N., Klar A. J., Broach J. R., Hicks J. B. Sites required for position-effect regulation of mating-type information in yeast. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):989–998. doi: 10.1101/sqb.1983.047.01.113. [DOI] [PubMed] [Google Scholar]
  2. Abraham J., Nasmyth K. A., Strathern J. N., Klar A. J., Hicks J. B. Regulation of mating-type information in yeast. Negative control requiring sequences both 5' and 3' to the regulated region. J Mol Biol. 1984 Jul 5;176(3):307–331. doi: 10.1016/0022-2836(84)90492-3. [DOI] [PubMed] [Google Scholar]
  3. Aparicio O. M., Billington B. L., Gottschling D. E. Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae. Cell. 1991 Sep 20;66(6):1279–1287. doi: 10.1016/0092-8674(91)90049-5. [DOI] [PubMed] [Google Scholar]
  4. Aparicio O. M., Gottschling D. E. Overcoming telomeric silencing: a trans-activator competes to establish gene expression in a cell cycle-dependent way. Genes Dev. 1994 May 15;8(10):1133–1146. doi: 10.1101/gad.8.10.1133. [DOI] [PubMed] [Google Scholar]
  5. Bell S. P., Kobayashi R., Stillman B. Yeast origin recognition complex functions in transcription silencing and DNA replication. Science. 1993 Dec 17;262(5141):1844–1849. doi: 10.1126/science.8266072. [DOI] [PubMed] [Google Scholar]
  6. Bi X., Liu L. F. recA-independent and recA-dependent intramolecular plasmid recombination. Differential homology requirement and distance effect. J Mol Biol. 1994 Jan 14;235(2):414–423. doi: 10.1006/jmbi.1994.1002. [DOI] [PubMed] [Google Scholar]
  7. Boscheron C., Maillet L., Marcand S., Tsai-Pflugfelder M., Gasser S. M., Gilson E. Cooperation at a distance between silencers and proto-silencers at the yeast HML locus. EMBO J. 1996 May 1;15(9):2184–2195. [PMC free article] [PubMed] [Google Scholar]
  8. Brand A. H., Breeden L., Abraham J., Sternglanz R., Nasmyth K. Characterization of a "silencer" in yeast: a DNA sequence with properties opposite to those of a transcriptional enhancer. Cell. 1985 May;41(1):41–48. doi: 10.1016/0092-8674(85)90059-5. [DOI] [PubMed] [Google Scholar]
  9. Braunstein M., Rose A. B., Holmes S. G., Allis C. D., Broach J. R. Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev. 1993 Apr;7(4):592–604. doi: 10.1101/gad.7.4.592. [DOI] [PubMed] [Google Scholar]
  10. Braunstein M., Sobel R. E., Allis C. D., Turner B. M., Broach J. R. Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern. Mol Cell Biol. 1996 Aug;16(8):4349–4356. doi: 10.1128/mcb.16.8.4349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bücking-Throm E., Duntze W., Hartwell L. H., Manney T. R. Reversible arrest of haploid yeast cells in the initiation of DNA synthesis by a diffusible sex factor. Exp Cell Res. 1973 Jan;76(1):99–110. doi: 10.1016/0014-4827(73)90424-2. [DOI] [PubMed] [Google Scholar]
  12. Chen-Cleland T. A., Smith M. M., Le S., Sternglanz R., Allfrey V. G. Nucleosome structural changes during derepression of silent mating-type loci in yeast. J Biol Chem. 1993 Jan 15;268(2):1118–1124. [PubMed] [Google Scholar]
  13. Diffley J. F., Cocker J. H. Protein-DNA interactions at a yeast replication origin. Nature. 1992 May 14;357(6374):169–172. doi: 10.1038/357169a0. [DOI] [PubMed] [Google Scholar]
  14. Dubey D. D., Davis L. R., Greenfeder S. A., Ong L. Y., Zhu J. G., Broach J. R., Newlon C. S., Huberman J. A. Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins. Mol Cell Biol. 1991 Oct;11(10):5346–5355. doi: 10.1128/mcb.11.10.5346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Feldman J. B., Hicks J. B., Broach J. R. Identification of sites required for repression of a silent mating type locus in yeast. J Mol Biol. 1984 Oct 5;178(4):815–834. doi: 10.1016/0022-2836(84)90313-9. [DOI] [PubMed] [Google Scholar]
  16. Ferguson B. M., Fangman W. L. A position effect on the time of replication origin activation in yeast. Cell. 1992 Jan 24;68(2):333–339. doi: 10.1016/0092-8674(92)90474-q. [DOI] [PubMed] [Google Scholar]
  17. Foss M., Rine J. Molecular definition of the PAS1-1 mutation which affects silencing in Saccharomyces cerevisiae. Genetics. 1993 Nov;135(3):931–935. doi: 10.1093/genetics/135.3.931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gartenberg M. R., Wang J. C. Identification of barriers to rotation of DNA segments in yeast from the topology of DNA rings excised by an inducible site-specific recombinase. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10514–10518. doi: 10.1073/pnas.90.22.10514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gartler S. M., Riggs A. D. Mammalian X-chromosome inactivation. Annu Rev Genet. 1983;17:155–190. doi: 10.1146/annurev.ge.17.120183.001103. [DOI] [PubMed] [Google Scholar]
  20. Germond J. E., Hirt B., Oudet P., Gross-Bellark M., Chambon P. Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc Natl Acad Sci U S A. 1975 May;72(5):1843–1847. doi: 10.1073/pnas.72.5.1843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gottschling D. E. Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo. Proc Natl Acad Sci U S A. 1992 May 1;89(9):4062–4065. doi: 10.1073/pnas.89.9.4062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Grant S. G., Chapman V. M. Mechanisms of X-chromosome regulation. Annu Rev Genet. 1988;22:199–233. doi: 10.1146/annurev.ge.22.120188.001215. [DOI] [PubMed] [Google Scholar]
  23. Guacci V., Hogan E., Koshland D. Chromosome condensation and sister chromatid pairing in budding yeast. J Cell Biol. 1994 May;125(3):517–530. doi: 10.1083/jcb.125.3.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hecht A., Laroche T., Strahl-Bolsinger S., Gasser S. M., Grunstein M. Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: a molecular model for the formation of heterochromatin in yeast. Cell. 1995 Feb 24;80(4):583–592. doi: 10.1016/0092-8674(95)90512-x. [DOI] [PubMed] [Google Scholar]
  25. Hecht A., Strahl-Bolsinger S., Grunstein M. Spreading of transcriptional repressor SIR3 from telomeric heterochromatin. Nature. 1996 Sep 5;383(6595):92–96. doi: 10.1038/383092a0. [DOI] [PubMed] [Google Scholar]
  26. Hereford L. M., Hartwell L. H. Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis. J Mol Biol. 1974 Apr 15;84(3):445–461. doi: 10.1016/0022-2836(74)90451-3. [DOI] [PubMed] [Google Scholar]
  27. Herskowitz I. A regulatory hierarchy for cell specialization in yeast. Nature. 1989 Dec 14;342(6251):749–757. doi: 10.1038/342749a0. [DOI] [PubMed] [Google Scholar]
  28. Holmes S. G., Broach J. R. Silencers are required for inheritance of the repressed state in yeast. Genes Dev. 1996 Apr 15;10(8):1021–1032. doi: 10.1101/gad.10.8.1021. [DOI] [PubMed] [Google Scholar]
  29. Holmes S. G., Rose A. B., Steuerle K., Saez E., Sayegh S., Lee Y. M., Broach J. R. Hyperactivation of the silencing proteins, Sir2p and Sir3p, causes chromosome loss. Genetics. 1997 Mar;145(3):605–614. doi: 10.1093/genetics/145.3.605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jacobs C. W., Adams A. E., Szaniszlo P. J., Pringle J. R. Functions of microtubules in the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1988 Oct;107(4):1409–1426. doi: 10.1083/jcb.107.4.1409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Johnson L. M., Kayne P. S., Kahn E. S., Grunstein M. Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6286–6290. doi: 10.1073/pnas.87.16.6286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kayne P. S., Kim U. J., Han M., Mullen J. R., Yoshizaki F., Grunstein M. Extremely conserved histone H4 N terminus is dispensable for growth but essential for repressing the silent mating loci in yeast. Cell. 1988 Oct 7;55(1):27–39. doi: 10.1016/0092-8674(88)90006-2. [DOI] [PubMed] [Google Scholar]
  33. Kimmerly W., Buchman A., Kornberg R., Rine J. Roles of two DNA-binding factors in replication, segregation and transcriptional repression mediated by a yeast silencer. EMBO J. 1988 Jul;7(7):2241–2253. doi: 10.1002/j.1460-2075.1988.tb03064.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kurtz S., Shore D. RAP1 protein activates and silences transcription of mating-type genes in yeast. Genes Dev. 1991 Apr;5(4):616–628. doi: 10.1101/gad.5.4.616. [DOI] [PubMed] [Google Scholar]
  35. Laurenson P., Rine J. Silencers, silencing, and heritable transcriptional states. Microbiol Rev. 1992 Dec;56(4):543–560. doi: 10.1128/mr.56.4.543-560.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Liu C., Mao X., Lustig A. J. Mutational analysis defines a C-terminal tail domain of RAP1 essential for Telomeric silencing in Saccharomyces cerevisiae. Genetics. 1994 Dec;138(4):1025–1040. doi: 10.1093/genetics/138.4.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Loo S., Fox C. A., Rine J., Kobayashi R., Stillman B., Bell S. The origin recognition complex in silencing, cell cycle progression, and DNA replication. Mol Biol Cell. 1995 Jun;6(6):741–756. doi: 10.1091/mbc.6.6.741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Loo S., Rine J. Silencers and domains of generalized repression. Science. 1994 Jun 17;264(5166):1768–1771. doi: 10.1126/science.8209257. [DOI] [PubMed] [Google Scholar]
  39. Lutter L. C. Thermal unwinding of simian virus 40 transcription complex DNA. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8712–8716. doi: 10.1073/pnas.86.22.8712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lynch A. S., Wang J. C. Use of an inducible site-specific recombinase to probe the structure of protein-DNA complexes involved in F plasmid partition in Escherichia coli. J Mol Biol. 1994 Feb 25;236(3):679–684. doi: 10.1006/jmbi.1994.1179. [DOI] [PubMed] [Google Scholar]
  41. Mahoney D. J., Broach J. R. The HML mating-type cassette of Saccharomyces cerevisiae is regulated by two separate but functionally equivalent silencers. Mol Cell Biol. 1989 Nov;9(11):4621–4630. doi: 10.1128/mcb.9.11.4621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Mahoney D. J., Marquardt R., Shei G. J., Rose A. B., Broach J. R. Mutations in the HML E silencer of Saccharomyces cerevisiae yield metastable inheritance of transcriptional repression. Genes Dev. 1991 Apr;5(4):605–615. doi: 10.1101/gad.5.4.605. [DOI] [PubMed] [Google Scholar]
  43. Monk M. Genomic imprinting. Genes Dev. 1988 Aug;2(8):921–925. doi: 10.1101/gad.2.8.921. [DOI] [PubMed] [Google Scholar]
  44. Moretti P., Freeman K., Coodly L., Shore D. Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1. Genes Dev. 1994 Oct 1;8(19):2257–2269. doi: 10.1101/gad.8.19.2257. [DOI] [PubMed] [Google Scholar]
  45. Nasmyth K. A. The regulation of yeast mating-type chromatin structure by SIR: an action at a distance affecting both transcription and transposition. Cell. 1982 Sep;30(2):567–578. doi: 10.1016/0092-8674(82)90253-7. [DOI] [PubMed] [Google Scholar]
  46. Nickels J. T., Broach J. R. A ceramide-activated protein phosphatase mediates ceramide-induced G1 arrest of Saccharomyces cerevisiae. Genes Dev. 1996 Feb 15;10(4):382–394. doi: 10.1101/gad.10.4.382. [DOI] [PubMed] [Google Scholar]
  47. Norton V. G., Imai B. S., Yau P., Bradbury E. M. Histone acetylation reduces nucleosome core particle linking number change. Cell. 1989 May 5;57(3):449–457. doi: 10.1016/0092-8674(89)90920-3. [DOI] [PubMed] [Google Scholar]
  48. Norton V. G., Marvin K. W., Yau P., Bradbury E. M. Nucleosome linking number change controlled by acetylation of histones H3 and H4. J Biol Chem. 1990 Nov 15;265(32):19848–19852. [PubMed] [Google Scholar]
  49. Osborne B. I., Guarente L. Transcription by RNA polymerase II induces changes of DNA topology in yeast. Genes Dev. 1988 Jun;2(6):766–772. doi: 10.1101/gad.2.6.766. [DOI] [PubMed] [Google Scholar]
  50. Park E. C., Szostak J. W. Point mutations in the yeast histone H4 gene prevent silencing of the silent mating type locus HML. Mol Cell Biol. 1990 Sep;10(9):4932–4934. doi: 10.1128/mcb.10.9.4932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Pillus L., Rine J. Epigenetic inheritance of transcriptional states in S. cerevisiae. Cell. 1989 Nov 17;59(4):637–647. doi: 10.1016/0092-8674(89)90009-3. [DOI] [PubMed] [Google Scholar]
  52. Reik W. Genomic imprinting and genetic disorders in man. Trends Genet. 1989 Oct;5(10):331–336. doi: 10.1016/0168-9525(89)90138-8. [DOI] [PubMed] [Google Scholar]
  53. Reynolds A. E., McCarroll R. M., Newlon C. S., Fangman W. L. Time of replication of ARS elements along yeast chromosome III. Mol Cell Biol. 1989 Oct;9(10):4488–4494. doi: 10.1128/mcb.9.10.4488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Shei G. J., Broach J. R. Yeast silencers can act as orientation-dependent gene inactivation centers that respond to environmental signals. Mol Cell Biol. 1995 Jul;15(7):3496–3506. doi: 10.1128/mcb.15.7.3496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Simpson R. T., Thoma F., Brubaker J. M. Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: a model system for study of higher order structure. Cell. 1985 Oct;42(3):799–808. doi: 10.1016/0092-8674(85)90276-4. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. Slater M. L. Effect of reversible inhibition of deoxyribonucleic acid synthesis on the yeast cell cycle. J Bacteriol. 1973 Jan;113(1):263–270. doi: 10.1128/jb.113.1.263-270.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Solter D. Differential imprinting and expression of maternal and paternal genomes. Annu Rev Genet. 1988;22:127–146. doi: 10.1146/annurev.ge.22.120188.001015. [DOI] [PubMed] [Google Scholar]
  59. Strahl-Bolsinger S., Hecht A., Luo K., Grunstein M. SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev. 1997 Jan 1;11(1):83–93. doi: 10.1101/gad.11.1.83. [DOI] [PubMed] [Google Scholar]
  60. Terleth C., van Sluis C. A., van de Putte P. Differential repair of UV damage in Saccharomyces cerevisiae. Nucleic Acids Res. 1989 Jun 26;17(12):4433–4439. doi: 10.1093/nar/17.12.4433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Thompson J. S., Johnson L. M., Grunstein M. Specific repression of the yeast silent mating locus HMR by an adjacent telomere. Mol Cell Biol. 1994 Jan;14(1):446–455. doi: 10.1128/mcb.14.1.446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Thompson J. S., Ling X., Grunstein M. Histone H3 amino terminus is required for telomeric and silent mating locus repression in yeast. Nature. 1994 May 19;369(6477):245–247. doi: 10.1038/369245a0. [DOI] [PubMed] [Google Scholar]
  63. Triolo T., Sternglanz R. Role of interactions between the origin recognition complex and SIR1 in transcriptional silencing. Nature. 1996 May 16;381(6579):251–253. doi: 10.1038/381251a0. [DOI] [PubMed] [Google Scholar]
  64. Volkert F. C., Broach J. R. Site-specific recombination promotes plasmid amplification in yeast. Cell. 1986 Aug 15;46(4):541–550. doi: 10.1016/0092-8674(86)90879-2. [DOI] [PubMed] [Google Scholar]
  65. Wang J. C. Appendix. I: An introduction to DNA supercoiling and DNA topoisomerase-catalyzed linking number changes of supercoiled DNA. Adv Pharmacol. 1994;29B:257–270. doi: 10.1016/s1054-3589(08)61142-4. [DOI] [PubMed] [Google Scholar]
  66. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  67. Wilson C., Bellen H. J., Gehring W. J. Position effects on eukaryotic gene expression. Annu Rev Cell Biol. 1990;6:679–714. doi: 10.1146/annurev.cb.06.110190.003335. [DOI] [PubMed] [Google Scholar]
  68. Wu H. Y., Shyy S. H., Wang J. C., Liu L. F. Transcription generates positively and negatively supercoiled domains in the template. Cell. 1988 May 6;53(3):433–440. doi: 10.1016/0092-8674(88)90163-8. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES