Histone H3 Thr 45 phosphorylation is a replication-associated post-translational modification in S. cerevisiae

Abstract

Post-translational histone modifications are crucial for the regulation of numerous DNA-templated processes, and are thought to mediate both alteration of chromatin dynamics and recruitment of effector proteins to specific regions of the genome. In particular, histone Ser/Thr phosphorylation regulates multiple nuclear functions in the budding yeast Saccharomyces cerevisiae, including transcription, DNA damage repair, mitosis, apoptosis and sporulation. Although modifications to chromatin during replication remain poorly understood, a number of recent studies have described acetylation of the histone H3 N-terminal alpha-helix (alphaN helix) at Lys 56 as a modification that is important for maintenance of genomic integrity during DNA replication and repair. Here, we report phosphorylation of H3 Thr 45 (H3-T45), a histone modification also located within the H3 alphaN helix in S. cerevisiae. Thr 45 phosphorylation peaks during DNA replication, and is mediated by the S phase kinase Cdc7-Dbf4 as part of a multiprotein complex identified in this study. Furthermore, loss of phosphorylated H3-T45 causes phenotypes consistent with replicative defects, and prolonged replication stress results in H3-T45 phosphorylation accumulation over time. Notably, the phenotypes described here are independent of Lys 56 acetylation status, and combinatorial mutations to both Thr 45 and Lys 56 of H3 cause synthetic growth defects. Together, these data identify and characterize H3-T45 phosphorylation as a replication-associated histone modification in budding yeast.

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, the peak of histone kinase activity from a. Proteins identified by mass spectrometry and/or confirmed by western blot are labelled; unlabelled bands await confirmation. (c) Histone kinase assays using proteins immunoprecipitated by either anti-Cdc7 antibody or pre-immune sera from extracts that were bound to and eluted from Ni²⁺-NTA agarose resin. (d) Kinase assays using histone octamers and the purified native Cdc7–TAP complex as indicated.

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 derivatives are indicated by (Pr) and (OMe), respectively. Underlined mass values indicate b and y ions identified. (b) Western blots of H3 kinase assays using recombinant Cdc7 and Dbf4 either co-expressed or expressed independently. (c) Dot blot showing specificity of anti-phospho H3-T45 (H3-T45 phosp) antibody. (d) T45A mutation or CDC7 deletion reduces H3-T45 phosphorylation signal in western blots.

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, and/or Tel1 were either left untreated or treated with HU (200 mM) for 3 h, extracted with trichloroacetic acid, and assessed by western blotting for histone H3 T45 phosphorylation and total H3. (c) Western blots of wild-type yeast released from M phase arrest were probed with the indicated antibodies.

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 of the wild-type (WT) plasmid. (b) Growth curve analysis of H3-T45A mutation and CDC7 deletion relative to parental strains. (c) Serial dilution to assess hypersensitivities of H3-T45A and cdc7Δ deleted yeast to methyl methanesulphonate (MMS), hydroxyurea (HU), or camptothecin (CPT). (d) Treatment with HU or CPT increases H3-T45 phosphorylation, as detected by western blotting. Results in panels b are shown as the mean ± s.e.m. (n = 3). Uncropped images of blots are shown in Supplementary Information, Fig. S6.

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 to parental strain. OD; optical density. (c) Western blots showing Thr 45 phosphorylation and Lys 56 acetylation are unaffected by mutation of the opposite residue. (d) Flow cytometry profiles of asynchronous yeast cultures expressing WT, K56R, or T45A histone H3. Fractions of cell populations in G1, S, and G2/M phases and estimated temporal lengths of each phase are indicated. Results in panels b are shown as the mean ± s.e.m. (n = 3). Uncropped images of blots are shown in Supplementary Information, Fig. S6.