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

Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Nature Cell Biology (Impact Factor: 19.68). 02/2010; 12(3):294-8. DOI: 10.1038/ncb2030
Source: PubMed


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.

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    • "It is important to note that Thr45 is located within the histone fold domain (Fig. S1). The phosphorylation of this site was described recently by three groups [Hurd et al., 2009; Baker et al., 2010; Villagrasa et al., 2012]. Having established that recombinant S6K2 has a much higher affinity towards histone H3 than its relative kinase homologue S6K1 in vitro, we then investigated if the phosphorylation site in H3 recognised by S6K2 in vitro is Thr45. "
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    ABSTRACT: The activity of S6 kinases (S6K) is highly induced in cancer cells highlighting an essential role in carcinogenesis. The S6K family has two members: S6K1 and S6K2 which bear common as well as distinct features. In an attempt to identify S6K2 unique sequence features compared to S6K1, we applied extensive bioinformatic analysis and motif search approaches. Interestingly, we identified 14 unique protein signatures which are present in proteins directly connected to chromatin and/or involved in transcription regulation. Using chromatin binding assay, we biochemically showed that S6K2 is bound to chromatin as well as nuclear matrix cellular fractions in HEK293 cells. The presence of S6K2 in chromatin fractions raised the possibility that it may be in close proximity to a number of chromatin substrates. For that, we then searched for S6K phosphorylation consensus sites RXRXXT/S in mammalian proteins using the SWISS-PROT database. Interestingly, we identified some potential phosphorylation sites in histone H3 (Thr45). Using in vitro kinase assays and siRNA-based knockdown strategy; we confirmed that S6K2 but not S6K1 or AKT is essential for histone H3-Thr45 phosphorylation in HEK293 cells. Furthermore, we show that the nuclear localisation sequence in the S6K2 C-terminus is essential for this modification. We have found that, H3-Thr45 phosphorylation correlates to S6K activation in response to mitogens and TPA-induced cell differentiation of leukaemic cell lines U937, HL60 and THP1. Overall, we demonstrate that S6K2 is a novel kinase that can phosphorylate histone H3 at position Thr45, which may play a role during cell proliferation and/or differentiation. J. Cell. Biochem. 115: 1048–1062, 2014. © 2013 Wiley Periodicals, Inc.
    Journal of Cellular Biochemistry 06/2014; 115(6). DOI:10.1002/jcb.24566 · 3.26 Impact Factor
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    • "In S. cerevisiae, histone H3 is phosphorylated at threonine 45 in a cell cycle-dependent manner by the Cdc7-Dbf4 kinase. Mutating this residue to alanine causes sensitivity to hydroxyurea and camptothecin, indicating its importance in proper DNA replication (Baker et al. 2010). Because all organisms except S. cerevisiae lack common nucleotide sequence motifs at origins, discovering a chromatin signature that promotes origin function is essential to understanding the location and activity of replication zones in higher eukaryotes. "
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    ABSTRACT: DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication.
    Genetics 07/2012; 192(2):371-84. DOI:10.1534/genetics.112.142349 · 5.96 Impact Factor
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    • "To determine whether the H4G94P mutant had normal cell cycle progression, we examined the DNA content of an asynchronous population of yeast cells by flow cytometry (Figure 2C). This method has been used in previous yeast histone mutant studies [31,32]. Compared to the WT H4 control, which shows defined 1 C and 2 C DNA content peaks, the cell cycle profile of the H4G94P mutant is highly disrupted and shifted rightward (Figure 2C). "
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