PCNA connects DNA replication to epigenetic inheritance in yeast

Article metrics

Abstract

Formation of a heterochromatin-like structure results in transcriptional silencing at the HM mating-type loci and telomeres in Saccharomyces cerevisiae1,2,3. Once formed, such epigenetically determined structures are inherited for many mitotic divisions4. Here we show that mutations in the proliferating cell nuclear antigen (PCNA), an essential component at the DNA replication fork5, reduced repression of genes near a telomere and at the silent mating-type locus, HMR. The pol30-8 mutant displayed coexistence of both repressed (pink) and de-repressed (white) cells within a single colony when assayed with the ADE2 gene inserted at HMR. Unlike pol30-8, the pol30-6 and pol30-79 mutants partially reduced gene silencing at telomeres and the HMR and synergistically decreased silencing in cells lacking chromatin assembly factor 1 (CAF-1). All silencing defective mutants showed reduced binding to CAF-1 in vitro and altered chromatin association of the CAF-1 large subunit in vivo. Thus, PCNA participates in inheritance of both DNA and epigenetic chromatin structures during the S phase of the cell cycle, the latter by at least two mechanisms.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The effect of PCNA mutations on telomeric silencing.
Figure 2: PCNA mutants affected HMR repression.
Figure 3: Interactions between PCNA and CAF-1.
Figure 4: Chromatin association of Cac1 in wild-type PCNA or mutant strains.
Figure 5: Molecular surface of the PCNA trimer.

References

  1. 1

    Loo, S. & Rine, J. Silencing and heritable domains of gene expression. Annu. Rev. Cell Dev. Biol. 11, 519–548 (1995).

  2. 2

    Grunstein, M. Yeast heterochromatin: regulation of its assembly and inheritance by histones. Cell 93, 325–328 (1998).

  3. 3

    Lustig, A. J. Mechanisms of silencing in Saccharomyces cerevisiae. Curr. Opin. Genet. Dev. 8, 233–239 (1998 ); erratum Curr. Opin. Genet. Dev. 8, 721 (1998).

  4. 4

    Pillus, L. & Rine, J. Epigenetic inheritance of transcriptional states in S. cerevisiae. Cell 59, 637–647 (1989).

  5. 5

    Waga, S. & Stillman, B. The DNA replication fork in eukaryotic cells. Annu. Rev. Biochem. 67, 721– 751 (1998).

  6. 6

    Verreault, A. De novo nucleosome assembly: new pieces in an old puzzle. Genes Dev. 14, 1430–1438 ( 2000).

  7. 7

    Shibahara, K. & Stillman, B. Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell 96, 575–585 ( 1999).

  8. 8

    Moggs, J. G. et al. A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage. Mol. Cell Biol. 20, 1206–1218 (2000).

  9. 9

    Kaufman, P. D., Kobayashi, R. & Stillman, B. Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I. Genes Dev. 11, 345– 357 (1997).

  10. 10

    Enomoto, S. & Berman, J. Chromatin assembly factor I contributes to the maintenance, but not the re-establishment, of silencing at the yeast silent mating loci. Genes Dev. 12, 219– 232 (1998).

  11. 11

    Henderson, D. S., Banga, S. S., Grigliatti, T. A. & Boyd, J. B. Mutagen sensitivity and suppression of position-effect variegation result from mutations in mus209, the Drosophila gene encoding PCNA. EMBO J. 13, 1450–1459 ( 1994).

  12. 12

    Gottschling, D. E., Aparicio, O. M., Billington, B. L. & Zakian, V. A. Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63, 751– 762 (1990).

  13. 13

    Sussel, L., Vannier, D. & Shore, D. Epigenetic switching of transcriptional states: cis- and trans-acting factors affecting establishment of silencing at the HMR locus in Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 3919–3928 (1993).

  14. 14

    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. 5, 605– 615 (1991).

  15. 15

    Liang, C. & Stillman, B. Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants. Genes Dev. 11, 3375–3386 (1997).

  16. 16

    Kaufman, P. D., Kobayashi, R., Kessler, N. & Stillman, B. The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication. Cell 81, 1105–1114 (1995).

  17. 17

    Krishna, T. S., Kong, X. P., Gary, S., Burgers, P. M. & Kuriyan, J. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell 79, 1233– 1243 (1994).

  18. 18

    Ayyagari, R., Impellizzeri, K. J., Yoder, B. L., Gary, S. L. & Burgers, P. M. A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair. Mol. Cell. Biol. 15, 4420 –4429 (1995).

  19. 19

    Eissenberg, J. C., Ayyagari, R., Gomes, X. V. & Burgers, P. M. Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon. Mol. Cell. Biol. 17, 6367–6378 (1997).

  20. 20

    Miller, A. M. & Nasmyth, K. A. Role of DNA replication in the repression of silent mating type loci in yeast. Nature 312, 247–251 (1984).

  21. 21

    Triolo, T. & Sternglanz, R. Role of interactions between the origin recognition complex and SIR1 in transcriptional silencing. Nature 381, 251–253 ( 1996).

  22. 22

    Chien, C. T., Buck, S., Sternglanz, R. & Shore, D. Targeting of SIR1 protein establishes transcriptional silencing at HM loci and telomeres in yeast. Cell 75, 531–541 (1993).

  23. 23

    Fox, C. A., Ehrenhofer-Murray, A. E., Loo, S. & Rine, J. The origin recognition complex, SIR1, and the S phase requirement for silencing. Science 276, 1547–1551 (1997).

  24. 24

    Murzina, N., Verreault, A., Laue, E. & Stillman, B. Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins. Mol. Cell 4, 529– 540 (1999).

  25. 25

    Le, S., Davis, C., Konopka, J. B. & Sternglanz, R. Two new S-phase-specific genes from Saccharomyces cerevisiae. Yeast 13, 1029–1042 ( 1997).

  26. 26

    Tyler, J. K. et al. The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402, 555– 560 (1999).

  27. 27

    Verreault, A., Kaufman, P. D., Kobayashi, R. & Stillman, B. Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4. Cell 87, 95–104 ( 1996).

  28. 28

    Ehrenhofer-Murray, A. E., Kamakaka, R. T. & Rine, J. A role for the replication proteins PCNA, RF-C, polymerase epsilon and Cdc45 in transcriptional silencing in Saccharomyces cerevisiae. Genetics 153, 1171–1182 (1999).

  29. 29

    Laman, H., Balderes, D. & Shore, D. Disturbance of normal cell cycle progression enhances the establishment of transcriptional silencing in Saccharomyces cerevisiae . Mol. Cell. Biol. 15, 3608– 3617 (1995).

  30. 30

    Scott, M. P. Development: the natural history of genes. Cell 100 , 27–40 (2000).

Download references

Acknowledgements

We thank P. Burgers, P. Kaufman, R. Sternglanz and D. Shore for plasmids and yeast strains used in this study. We thank A. Stenlund for critical reading of the manuscript, T. Tully for statistical analysis of the data presented in Table 1, and members of the Stillman laboratory, especially L. Zou, for helpful discussions. This work is supported by a grant from the National Institutes of Health (to B. S.). Z. Z. is supported by a postdoctoral fellowship from the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation. K. S. is a Leukemia Society of America Special Fellow.

Author information

Additional information

Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, New York 11724, USA

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.