Terminating Histone Synthesis to Preserve Centromere Integrity

Histone protein synthesis is activated as cells enter S phase to allow packaging of the newly replicated DNA into chromatin. In this issue of Developmental Cell, Takayama and coworkers elucidate a mechanism for silencing histone expression at the end of S phase in S. pombe. Failure to shut off histone expression disrupts centromeric chromatin structure. Histone protein synthesis is activated as cells enter S phase to allow packaging of the newly replicated DNA into chromatin. In this issue of Developmental Cell, Takayama and coworkers elucidate a mechanism for silencing histone expression at the end of S phase in S. pombe. Failure to shut off histone expression disrupts centromeric chromatin structure. Replication of the genome in eukaryotes requires both replication of DNA and synthesis of large amounts of histones to package the newly replicated DNA into chromatin. In all eukaryotes, the levels of histone mRNAs are cell cycle regulated with their accumulation activated as cells approach entry into S phase and rapidly decreasing when DNA replication is completed. Recent findings clearly demonstrate that it is as important to shut off histone synthesis at the end of S phase as it is to initiate it at the start of S phase. The mRNAs that encode the bulk of the histone proteins, termed replication-dependent histone mRNAs, are tightly cell cycle regulated. In all organisms, there are additional histone genes that encode variant histones, such as centromeric histone H3, that are expressed outside of S phase. It is likely that one problem that arises when replication-dependent histones are overexpressed or expressed outside of S phase is that they disrupt the proper incorporation of the variant histones into chromatin. In this issue of Developmental Cell, Takayama et al., 2010Takayama Y. Mamnun Y.M. Trickey M. Dhut S. Masuda F. Yamano H. Toda T. Saitoh S. Dev. Cell. 2010; 18 (this issue): 385-396Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar report the mechanism by which histone gene transcription is inactivated at the end of S phase in Schizosacchromyces pombe, resulting in rapid disappearance of histone mRNA and cessation of histone synthesis. In both budding and fission yeast, histone mRNA levels are controlled primarily at the level of transcription and the mRNAs have very short half-life, resulting in their rapid appearance and disappearance when the rate of histone gene transcription changes. A major regulator of histone gene expression is the corepressor HIRA in both budding and fission yeast, (Spector et al., 1997Spector M.S. Raff A. DeSilva H. Lee K. Osley M.A. Mol. Cell. Biol. 1997; 17: 545-552Crossref PubMed Scopus (114) Google Scholar, Blackwell et al., 2004Blackwell C. Martin K.A. Greenall A. Pidoux A. Allshire R.C. Whitehall S.K. Mol. Cell. Biol. 2004; 24: 4309-4320Crossref PubMed Scopus (61) Google Scholar). Several years ago, Takahashi and coworkers (Chen et al., 2003Chen E.S. Saitoh S. Yanagida M. Takahashi K. Mol. Cell. 2003; 11: 175-187Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) showed that in S. pombe, histone gene transcription required the transcription activator, Ams2, and that the Ams2 protein was cell cycle regulated, being present primarily in S phase. Ams2 is necessary for histone gene expression since mutation of HIRA does not restore histone gene expression in Ams2 mutants. Here, they show that Ams2 is unstable when expressed in G2 cells but stable in S phase and that degradation was mediated by ubiquitination. They further identified the ubiquitin ligase as one of the SCF E3 ligases, containing the F-box protein Pof3, one of the three F-box proteins involved in cell cycle regulation in S. pombe (Katayama et al., 2002Katayama S. Kitamura K. Lehmann A. Nikaido O. Toda T. Mol. Biol. Cell. 2002; 13: 211-224Crossref PubMed Scopus (39) Google Scholar). Changes in electrophoretic mobility of Ams2 during the cell cycle led to the demonstration that a highly phosphorylated form of Ams2 was likely recognized by the SCF complex. One kinase essential for the degradation of Ams1 is the Hsk-Dfp kinase, which has no obvious homolog in metazoans or budding yeast. The Dfp subunit is cell cycle regulated, present in S phase where it is essential for entry into S phase and also present in G2 as a different phosphoisoform (Brown and Kelly, 1999Brown G.W. Kelly T.J. Proc. Natl. Acad. Sci. USA. 1999; 96: 8443-8448Crossref PubMed Scopus (75) Google Scholar). Whether the specificity of Hsk1-Dfp is different between S and G2 phases or whether there are other kinases also required for degradation of Ams2 is not known. Failure to degrade Ams2 at the end of S phase or constitutive expression of Ams2 from a strong promoter results in growth inhibition, persistent expression of histone mRNA, and chromosome instability. An Hsk ts mutant also causes severe chromosome instability (Snaith et al., 2000Snaith H.A. Brown G.W. Forsburg S.L. Mol. Cell. Biol. 2000; 20: 7922-7932Crossref PubMed Scopus (70) Google Scholar), consistent with degradation of Ams2 being required for proper chromosome transmission. In cells where Ams2 is not degraded, analysis of the centromeric chromatin by micrococcal nuclease digestion and chromatin immunoprecipitation demonstrated that the core centromeric chromatin structure was disrupted. These centromeres incorporated not only the usual histone H3-Cenp, but also canonical histone H3 and excess H4, suggesting an increased number of nucleosomes and hence a denser packing of nucleosomes as a result of inappropriate histone expression. The observation that mutants in Hsk1-Dfp also have defects in centromeres (see also Snaith et al., 2000Snaith H.A. Brown G.W. Forsburg S.L. Mol. Cell. Biol. 2000; 20: 7922-7932Crossref PubMed Scopus (70) Google Scholar) thus expands Hsk1-Dfp function to include not only the initiation of histone gene expression and DNA replication at entry into S phase but also the termination of histone gene expression in G2. This study emphasizes the importance of production of proper amounts of histone protein to successful replication and transmission of the chromosomes. There are multiple mechanisms to ensure that there is not overproduction of histone protein. In addition to the regulation of histone protein synthesis by both transcription activators and repressors, there is a pathway of histone protein degradation that is necessary to prevent overaccumulation of histones; disruption of this pathway results in chromosome instability (Gunjan and Verreault, 2003Gunjan A. Verreault A. Cell. 2003; 115: 537-549Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar) analogous to the results discussed above. Multiple mechanisms also regulate histone mRNA levels in metazoans. Here, the replication-dependent histone mRNAs differ from other mRNAs in that they are not polyadenylated, and the unique 3′ end of histone mRNAs is a major cis element for cell cycle regulation in mammalian cells (Marzluff et al., 2008Marzluff W.F. Wagner E.J. Duronio R.J. Nat. Rev. Genet. 2008; 9: 843-854Crossref PubMed Scopus (458) Google Scholar). In addition to regulation of histone gene transcription, formation of the 3′ end of histone mRNA is regulated, and processing is inhibited at the end of S phase. At the end of S phase, the half-life of histone mRNA is also greatly reduced, rapidly shutting down histone protein synthesis. A cell cycle-regulated factor necessary for histone pre-mRNA processing, the stem-loop-binding protein (SLBP) has an expression pattern similar to Ams2 in S. pombe, being expressed only in S phase cells. Like Ams1, SLBP is degraded at the end of S phase as a result of phosphorylation, in this case by cyclin A/Cdk1 which is active at the end of S phase (Koseoglu et al., 2008Koseoglu M.M. Graves L.M. Marzluff W.F. Mol. Cell. Biol. 2008; 28: 4469-4479Crossref PubMed Scopus (54) Google Scholar). Inappropriate synthesis of the replication-dependent histone proteins outside of S phase likely also causes problems with chromatin structure in metazoans. In Drosophila, blocking histone pre-mRNA processing by mutations in SLBP results in production of polyadenylated histone mRNAs, which are not degraded rapidly when DNA replication is inhibited. These mutations exhibit genetic instability as well as affecting heterochromatin formation and cell proliferation (Salzler et al., 2009Salzler H.R. Davidson J.M. Montgomery N.D. Duronio R.J. PLoS ONE. 2009; 4: e8168https://doi.org/10.1371/journal.pone.0008168Crossref PubMed Scopus (14) Google Scholar), consistent with production of histone protein in G2 phase disrupting normal chromatin structure. These studies point out the importance of producing the right amount of histones at the correct time, and this paper clearly demonstrates the defect in chromatin structure caused by overexpression of histones in G2 phase. Given the critical role of histone variants in epigenetic regulation of chromatin structure, it is likely also critical to properly balance the synthesis of canonical histones and histone variants in S phase, and to avoid production of histones in G1 phase. There are hints of sophisticated regulatory mechanisms in Drosophila that act to balance the production of canonical histones and variant histones. Knockdown of variant histone H2av or mutants lacking the H2av gene, have defects in the biosynthesis of the canonical histones, suggesting there is a mechanism to maintain proper balance of synthesis of the two types of histone protein during S phase (Marzluff et al., 2008Marzluff W.F. Wagner E.J. Duronio R.J. Nat. Rev. Genet. 2008; 9: 843-854Crossref PubMed Scopus (458) Google Scholar). Hsk1- and SCFPof3-Dependent Proteolysis of S. pombe Ams2 Ensures Histone Homeostasis and Centromere FunctionTakayama et al.Developmental CellMarch 16, 2010In BriefSchizosaccharomyces pombe GATA factor Ams2 is responsible for cell cycle-dependent transcriptional activation of all the core histone genes peaking at G1/S phase. Intriguingly, its own protein level also fluctuates concurrently. Here, we show that Ams2 is ubiquitylated and degraded through the SCF (Skp1-Cdc53/Cullin-1-F-box) ubiquitin ligase, in which F box protein Pof3 binds this protein. Ams2 is phosphorylated at multiple sites, which is required for SCFPof3-dependent proteolysis. Hsk1/Cdc7 kinase physically associates with and phosphorylates Ams2. Full-Text PDF Open Access