We set out to identify histone kinases in S. cerevisiae
through extensive chromatographic separation of candidate activities. A number of histone-modifying enzymes, including kinases, have been successfully purified from yeast extracts using Ni2+
-NTA agarose resin in the absence of any affinity tags5,6
. In this study, yeast whole-cell extracts were bound to Ni2+
-NTA agarose and fractionated through multiple rounds of chromatography (Supplementary Information, Fig. S1a
). Resultant fractions containing distinct histone kinase activities from the final separation were silver-stained and analysed by tandem mass spectrometry (). This study revealed a multiprotein complex containing the S phase-regulating kinase Cdc7–Dbf4. Accompanying protein bands from this silver-stained complex were identified, but await validation. As Cdc7 has not previously been reported as a core histone kinase, it was subsequently immunoprecipitated from extracts to verify this activity (). Our results suggest that the native Cdc7–Dbf4 complex is capable of phosphorylating core histones.
Figure 1 Identification of the Cdc7–Dbf4 histone kinase complex. (a) Histone kinase assays using purified chromatographic fractions from yeast whole-cell extracts. Fractions from the final Mini Q column are shown. (b) Silver stain of Mini Q fraction 20, (more ...)
The Ser/Thr kinase Cdc7 is a key regulator of cell-cycle progression into S phase7
. Enzymatic activity of Cdc7 is regulated by association with the unstable regulatory protein Dbf4 (ref. 8
), whose expression fluctuates throughout the cell cycle, peaking late in G1 before onset of S phase9
. Cdc7 has been linked directly to chromatin and to numerous nuclear functions requiring access to DNA10
, and thus histones represent a likely target for its enzymatic activity. Specifically, Cdc7–Dbf4 binds to chromatin at replication origins11
, where it has been implicated in the phosphorylation of the Mcm2–7 helicase complex12,13
. To further investigate the histone kinase activity of Cdc7, we generated a yeast strain bearing a TAP tag located downstream of the endogenous CDC7
locus, allowing purification of native Cdc7 and its associated proteins by calmodulin affinity. The complex was partially purified by conventional chromatography, then bound to calmodulin–sepharose beads and eluted using EGTA (Supplementary Information, Fig. S1b
). Gel filtration chromatography indicates that Cdc7 exists in a multiprotein complex with a relative molecular mass exceeding 1,000K, consistent with the number of protein bands observed in our original silver stain (Supplementary Information, Fig. S1c
). The final calmodulin elution was subsequently assessed for kinase activity using histone core octamers to identify the specific histones most likely to be targets in vivo
. Our results indicate that histone H3 is a major in vitro
target of native Cdc7 when in the context of the histone octamer ().
To identify specific sites of histone phosphorylation, kinase assays were repeated using recombinant histone H3, and the products were analysed by tandem mass spectrometry. Trypsin digestion and MS/MS analysis of H3 phosphorylated in vitro
revealed a phospho-peptide corresponding to residues 41–49, with the sequence YRPGpTVALR (). To verify this result, we subsequently performed kinase assays with peptides containing Thr 45 or several previously described H3 phosphorylation sites, either pre-phosphorylated or unmodified. Only the peptide containing unmodified Thr 45 gave counts significantly greater than the auto-phosphorylation observed in the control experiment (no peptide) when incubated with the Cdc7–TAP complex (Supplementary Information, Fig. S2a
). Additionally, Cdc7 and Dbf4 co-expressed recombinantly and purified from Escherichia coli
were able to phosphorylate H3-T45 in vitro
(; Supplementary Information, Fig. S2b
). Taken together, these data indicate that H3-T45 is a specific in vitro
substrate of Cdc7.
Figure 2 Mapping of Cdc7-dependent histone phosphorylation. (a) Cdc7–TAP histone kinase assay reaction products were analysed by mass spectrometry. Tandem MS spectrum of the precursor [M + 2H]2+ ion at 591.8054 m/z is shown. Propionyl and methyl ester (more ...)
Thr 45 lies within the H3 αN helix, a remarkably conserved region that makes critical contacts with DNA when assembled into the nucleosome core particle (Supplementary Information, Fig. S3a, b
. In particular, Thr 45 is located precisely at the points of entry and exit of DNA on the nucleosome, and the Thr side chain of this residue interacts directly with the DNA entry gyre16
. Thus, phosphorylation of this residue may heavily influence DNA–histone interactions within the core particle. Interestingly, a number of recent papers have described acetylation of H3 Lys 56 (H3-K56), a residue that is also located within the αN helix17
. This modification is associated with proper cell-cycle progression, recovery from replicative stress, and promotion of genomic stability3,4,18
To verify that phosphorylation of Thr 45 occurs in vivo
, we generated an antibody specific for this modification. Peptide dot blots indicate that this antibody recognizes phosphorylated Thr 45, and does not cross-react with several known H3 phosphorylation sites (). Yeast cells expressing either wild-type or T45A
-mutated histone H3 were grown to mid-log phase, and whole-cell extracts were analysed by western blotting. As expected, H3-T45 phosporylation signal was observed in the wild-type extracts but absent from the T45A
mutant, indicating that Thr 45 is indeed phosphorylated in vivo
(). To investigate the dependence of this modification on Cdc7, we used bob1
and bob1 cdc7
Δ yeast strains. Mutation of the MCM5
gene, termed bob1
, allows for deletion of the otherwise essential CDC7
. When we performed the same western blot experiment in these yeast strains, we found that H3-T45 phosphorylation signal was markedly decreased in the absence of Cdc7 (), supporting the findings of our initial in vitro
assays. Thus, Cdc7 mediates phosphorylation of H3-T45 both in vitro
and in vivo
We next sought to determine whether this modification is enriched during replication. Yeast cultures were grown to mid-log phase and treated with the ribonucleotide reductase inhibitor hydroxyurea to cause S phase arrest. After 2 h of treatment, the level of Thr 45 phosphorylation was increased over that observed in asynchronous cells (; Supplementary Information, Fig. S4a
). Notably, Mec1 and Tel1, the yeast homologues of the ATM/ATR checkpoint kinases, are not required for induction of Thr 45 phosphorylation (). We also synchronized cells with nocodazole and collected histone samples at 15 min intervals subsequent to release (). Western blot analysis showed that H3-T45 phosphorylation fluctuates throughout the cell cycle and peaks before expression of the G2/M cyclin Clb2 and in coordination with H3-K56 acetylation, which is enriched during S phase3
. These findings link the timing of Thr 45 phosphorylation to replication.
Figure 3 Histone H3 T45 phosphorylation is linked to replication in yeast. (a) Yeast expressing His-tagged histone H3 were arrested with hydroxyurea (HU, 200 mM) and assessed for H3-T45 phosphorylation or total H3 (His). (b) Yeast strains lacking Sml1 (WT), Mec1, (more ...)
As Thr 45 phosphorylation occurs during S phase, we reasoned that loss of this modification may cause replication defects. In addition to the T45A mutant described above, we generated a yeast strain mutated at H3-T45 to glutamic acid, to investigate the effects in vivo of either inhibited or constitutive phosphorylation. After 5 days, growth of T45A mutants was slow, compared with wild-type yeast, whereas T45E mutants seemed non-viable, indicating that genome-wide constitutive incorporation of a negative charge at this residue is very poorly tolerated (). The slow growth of T45A was further investigated by growth curve analysis in culture, which shows that T45A mutants progress at a much slower rate than wild-type yeast, comparable to yeast lacking CDC7 ().
Figure 4 Mutation of T45 causes sensitivity to replication stress. (a) Viabilities of yeast expressing wild-type (WT), T45A, or T45E histone H3 plated on complete supplement mixture (CSM)-Leu for selection of the mutant plasmid, and 5′-FOA for counter-selection (more ...)
We also sought to determine whether loss of H3-T45 phosphorylation would lead to a heightened sensitivity to replication stress and/or DNA damage. Tenfold serial dilutions indicate that both T45A mutation and deletion of CDC7 cause sensitivity to hydroxyurea and the topoisomerase I inhibitor camptothecin (CPT), indicating that loss of Thr 45 phosphorylation causes replication defects (). Importantly, neither mutation of Thr 45 nor loss of Cdc7 affected Lys 56 acetylation levels (); thus, the phenotypes observed in the T45A and bob1 cdc7Δ mutants do not simply occur because of alterations in the proximal Lys 56 modification state.
We further reasoned that if phosphorylation of H3-T45 is important for resistance to hydroxurea and CPT, then treatment of yeast cultures with these agents may lead to accumulation of this modification. We found that replication stress does indeed increase Thr 45 phosphorylation levels after 1–2 h of treatment in culture (). Importantly, treatment of yeast with the DNA alkylating agent methyl methanesulphonate (MMS) was not found to induce Thr 45 phosphorylation. This result agrees with our finding that T45A yeast are highly sensitive to replication stress, but has no apparent sensitivity to MMS by serial dilution ().
Finally, we compared T45A
with both K56R
yeast to further verify that the two modifications function independently. The slow growth phenotype observed in T45A
seemed much more severe than that of K56R
, and was heightened when the two mutations were present in combination, as assessed by streaking on medium plates, or by growth in culture (). Furthermore, we found that neither mutation alone had a significant effect on the post-translational modification status of the other residue (). Finally, we analysed asynchronous cultures of the mutants by flow cytometry to assess their DNA content profiles (). Compared with yeast expressing wild-type H3, H3-K56R
yeast showed a higher proportion of cells in the G2-M stage of the cell cycle, consistent with the previously described profile of yeast lacking the H3-K56 acetyltransferase Rtt109 (ref. 18
). By contrast, T45A
yeast showed a delay at all cell-cycle stages, with a significantly greater amount of time spent in G1/S, compared with wild-type yeast. Our results show that the Thr 45 and Lys 56 mutants behave distinctly, and thus the post-translational modifications of the two residues function independently.
Figure 5 H3-T45 phosphorylation is distinct from H3-K56 acetylation. (a) Viability of yeast expressing wild-type (WT) H3, or mutations of H3-T45 and H3-K56, alone or in combination. (b) Growth curve analysis of H3-K56R, H3-T45A, and H3-T45A-K56R mutations relative (more ...)
Taken together, our findings describe H3-T45 phosphorylation, a post-translational modification in a crucial region of the nucleosome core particle. Additionally, we have identified a histone kinase responsible for generating this modification, the S phase regulatory kinase Cdc7–Dbf4, which exists partially in its native state as a component of a much larger multiprotein complex. Phosphorylated Thr 45 is present in greatest abundance during S phase, and loss of this modification results in slow growth and drug sensitivities, which indicates replication defects, consistent with the phenotypes caused by a loss of Cdc7.
Given that Thr 45 phosphorylation is not essential for cell survival, we find it likely that this modification is not required for entry into S phase. Although Cdc7 has been implicated in the firing of replication origins, it is also thought to function in preserving the integrity of actively progressing replication forks in the event of replication stress and fork stalling20
. A recent study showed that Xenopus laevis
Cdc7 regulates DNA replication reinitiation during the S phase checkpoint recovery in extracts that had been treated with etoposide21
. Furthermore, constitutive genomewide mimicking of Thr 45 phosphorylation via T45E
mutation is very poorly tolerated. This is not surprising given the close proximity of the negatively charged DNA-phosphate backbone to this residue, and may indicate that localized placement of a phosphate molecule on this Thr serves to disrupt DNA–histone contacts at the onset of replication, or adjacent to sites of stalled replication forks. Indeed, mutation of Thr 45 has recently been shown to affect nucleosome dynamics in vitro
with respect to the wrapping of DNA around the nucleosome core particle16
. Given the direct contact between the Thr 45 side chain and the DNA entry gyre, it seems likely that transient phosphorylation of this residue could have a dramatic effect on nucleosome stability and DNA accessibility.
Furthermore, recent studies have also identified Thr 45 phosphorylation in human neutrophils, where it functions in apoptosis22
. Notably, it is not uncommon for phosphorylation of a single histone residue to function in multiple pathways, as has been well documented for phosphorylation of histone H3 Ser 10 (ref. 23
). To investigate whether H3-T45 phosphorylation similarly functions in yeast apoptosis, we monitored the modification upon treatment of yeast cultures with H2
(1 mM). We found no discernable increase in Thr 45 phosphorylation levels after 2 h of treatment (Supplementary Information, Fig. S4b
), indicating that H3-T45 probably does not function in this apoptotic pathway in yeast. Nevertheless, the findings that this residue may function in different pathways across multiple organisms provide further evidence that its position in the histone octamer is critical to nucleosome function.
Interestingly, the phenotypes of the T45A
mutant in response to replication stress seemed more severe than those observed in the bob1 cdc7
Δ strain. It is possible therefore that Thr 45 is phosphorylated by another enzyme(s) that contributes to genomic stability. Furthermore, our studies showed that T45A
does not result in sensitivity to MMS, and Thr 45 phosphorylation is not induced by prolonged treatment with MMS in culture. Given the extreme sensitivity of rtt109
Δ and H3-K56
mutants to MMS reported in previous studies3,4,18
, along with our findings that Lys 56 acetylation and Thr 45 phosphorylation seem to have no interdependency, we propose that the phenotypes described here are independent of H3-K56 acetylation, and thus are indicative of a distinct replicative function. Our findings reveal a mechanism of Cdc7–Dbf4 function during S phase, which ultimately may provide insight into their possible roles in the development of cancer.