Journal of Molecular Biology
The Fission Yeast spSet1p is a Histone H3-K4 Methyltransferase that Functions in Telomere Maintenance and DNA Repair in an ATM Kinase Rad3-dependent Pathway
Introduction
The SET domain is a characteristic motif found in several chromatin regulators involved in epigenetic control of transcription. The SET domain currently characterizes more than 200 proteins ranging from yeast to man.1 The function of the SET domain was unknown until it was demonstrated that human SUV39H1 and its Schizosaccharomyces pombe homolog Clr4 have a SET-domain dependent histone H3 methyltranferase (HMTase) activity that methylates H3 on lysine 9 (K9).2 An HMTase activity with different specificities has been now found in other SET domain proteins.3 Importantly, H3 methylated at K9 by either SUV39H1 or Clr4 has the ability to recruit the heterochromatic protein HP1 through its chromodomain. HP1 in turn binds to SUV39H1, which could methylate H3 at K9 on the adjacent nucleosome, thereby facilitating the spreading of a heterochromatic structure.4., 5., 6. Consistent with these observations, loss of Suv39h impairs heterochromatin and genome stability in mammals.7 Finally, the idea that K9 methylation could be associated with transcriptional repression was reinforced by the demonstration that H3-K9 methylation localizes to a 20 kb silent heterochromatin region at the fission yeast mat locus.8
Whereas H3-K9 appears to be associated with transcriptional repression, methylation of H3 at lysine 4 (K4) may result in transcriptional activation.8., 9., 10., 11., 12., 13. However, H3 methylation at specific positions may have different effects, depending on the chromosomal location and on the complex interplay between covalent modifications of histone tails.3 For instance, inactivation of the SET domain of budding yeast Set1p, which is required for H3-K4 methylation,14., 15., 16. results in the alleviation of telomeric silencing17., 18., 19. and of rDNA silencing.20 It emerged from these studies that both gene silencing and activation may depend on multiple histone modifications, which generate binding sites for proteins responsible for chromatin-based regulation of genes.21
Budding yeast Set1p belongs to a complex of eight proteins, most of which are required to efficiently catalyze methylation of K4 on H3 in vivo.14., 16., 22. The molecular function associated with each member of the complex remains to be elucidated. The SET domain of Set1p interacts with the C-terminal region of the DNA damage checkpoint protein Mec3p.18 With Rad9p, Rad17p, Rad24p and Ddc1p, Mec3p belongs to the group of DNA damage sensor proteins of the checkpoint machinery. These proteins are thought to recognize DNA damage and generate a signal relayed by the central transducer Mec1p to the protein kinases Rad53p and Chk1p.23 In turn, this signal leads to cell-cycle delay and to transcriptional induction of DNA repair genes, thereby increasing the DNA repair capacities of the cell.24 We have observed that deletion of SET1 increases viability after DNA damage of all the checkpoint sensor mutants as well as of the transducer mec1 checkpoint mutant. This recovery depends on the presence of the Rad53p kinase, which induces hyperphosphorylation of the middle subunit of Replication Protein A (Rfa2p) leading to the transcriptional activation of DNA repair genes.25
In S. pombe, both DNA damage and replication checkpoints involve six checkpoint Rad proteins, Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1 which are homologous to the budding yeast proteins Rad17, Mec1, Ddc1, Rad24, Ddc2 and Mec3, respectively.26., 27. Central to both DNA damage and replication checkpoint is the fission yeast ATM-like Rad3 kinase which, in association with Rad26, activates the downstream effectors Chk1 and Cds1.28., 29., 30. While Cds1 kinase is the effector of the replication checkpoint through its interaction with the Mrc1 protein and is required for recovery under replicational stress,27., 31. the Chk1 kinase mediates mitotic arrest when DNA is damaged either in the late S or the G2 phase of the cell-cycle. Chk1 activation specifically requires the BRCT-containing domain protein Rhp9/Crb2.32., 33., 34., 35., 36. Both Chk1 and Cds1 delay cell-cycle progression by maintaining the inhibitory tyrosine 15 phosphorylation of the mitotic kinase Cdc2.37., 38. Besides being necessary for Chk1 activation, the Crb2 protein is required for recovery of cells treated with the replication inhibitor hydroxyurea (HU) and for the recovery of cells that express the thermosensitive DNA polymerase δ catalytic subunit mutated at R1064 (polδts3 allele).39 Both of these requirements are independent from the checkpoint function of Crb2.33., 36. Recently, it has been proposed that checkpoint-dependent hyperphosphorylation of Crb2 is required for efficient Top3 activity and thus for regulation of Rqh1 helicase when cells experience double-strand breaks in the G2 phase of the cell-cycle.40 Furthermore, the Crb2 protein and most checkpoint Rad proteins positively regulate telomere length in fission yeast.41., 42., 43. In addition to Rad3, S. pombe has an ATM homologue called Tel1 that is involved in telomere maintenance.44 While a tel1 mutant has telomeres of wild-type size, cells lacking both ATM kinases lose functional telomeres after successive cell divisions and maintain their genome by circularization of chromosomes.44 Telomere maintenance by Rad3 and Tel1 apparently occurs through two distinct pathways that do not involve the Rad3-activated kinases Cds1 and Chk1.41
In this study, we have characterized the S. pombe homolog of Set1p, which we named spSet1p. We found that spSet1p is required to methylate H3 at K4 in vivo and that recombinant spSet1 is able to methylate H3 tail at K4 in vitro. Our analysis of the role of spSet1p in both centromeric and telomeric silencing, telomere length regulation and survival of different checkpoint mutants to genotoxic treatments unveils similarities and differences between budding and fission yeast Set1p. In S. pombe, SpSet1p appears to function in a pathway that involves the ATM kinase Rad3. Our results bring new insights into the links between chromatin structure, telomeric functions and cellular response to DNA damage.
Section snippets
spSet1p, the S. pombe homolog of budding yeast Set1p
Several alignments pointing out the evolutionary conservation of the SET domain have been published.3., 45. Database searching identified candidate orthologs of Set1p in S. pombe, Caenorhabditis elegans, Drosophila and human.14 The S. pombe ortholog of Saccharomyces cerevisiae Set1p, which we have called spSet1, is a protein of 920 amino acid residues that is highly homologous throughout the whole Set1p sequence. A significant homology with budding yeast Set1p starts at position 155. Both
Discussion
In this study, we have identified and characterized the S. pombe homolog of the budding yeast Set1p. Like the budding yeast Set1p, spSet1p has H3 methyl transferase activity specific for K4. The fact that H3 K4 methylation is abolished in S. pombe Δspset1 cells indicates that spSet1p accounts for most, if not all of the H3-K4 methylation. The two proteins share a high degree of identity in their SET domains and exhibit 44% similarity throughout their whole sequence. Nevertheless, both proteins
S. pombe physiological and genetic methods
Strains used in this study are listed in Table 1. Double and triple mutants were obtained by standard genetic techniques. The genomic DNA fragment containing the spset1+ gene and extending from position −238 to nucleotide to +2856 (with respect to the ATG codon of spset1) was amplified by PCR from wild-type genomic DNA and cloned in the vector pCRscript (Stratagene). The fragment from −238 to +2116 was sub-cloned in pCRscript plasmid and the Kan MX6 cassette (from PFA6a-KanMX6) was inserted
Acknowledgements
We thank Assen Roguev and Francis Stewart for the swd2 and spp1 mutants, and for communication of unpublished data, Akash Gunjan and Alain Verreault for communication of unpublished results, Robin Allshire for the FY1862, Tony Carr for the rqh1 mutant, Paul Russel for the mus81 mutant, strains and advice, and Patrick Hughes for critical reading of the manuscript. The work in the Laboratory of V.G. was supported by “l'Association pour la Recherche sur le Cancer” and by “la Fondation pour la
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Cited by (0)
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J.K. and S.F. contributed equally to this work.