DNA Replication Origin Function Is Promoted by H3K4 Di-methylation inSaccharomyces cerevisiae

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 posttranslational modifications to support robust genome duplication. DNA replication initiates at discrete genomic loci termed "origins of replication." Each eukaryotic chromosome is replicated from many individual origins to ensure complete and precise genome duplication during each cell division cycle. Individual origins vary both in the likelihood that they will initiate replication, or "fire," in any given S phase and in the firing time within the S phase (Weinreich et al. 2004). Highly efficient origins fire in most cell cycles, whereas inefficient origins fire in only some cycles and are usually passively replicated by forks emanating from neighboring efficient origins. Although highly efficient origins that support initiation in most cell cycles have been identified in many genomes, the chromosomal determinants of origin location and function are still incompletely understood. Strikingly, while DNA sequence elements can be necessary, it is clear that sequence alone is insufficient to fully specify eukaryotic origin location and activity (Méchali 2010).Like all DNA-templated processes, replication occurs on chromatin. Recent progress in the field has demonstrated that the chromatin structure surrounding origins plays an essential role in controlling origin activity. For example, the positioning of nucleosomes near origins can either stimulate or inhibit origin function (Simpson 1990;Crampton et al. 2008;Berbenetz et al. 2010;Eaton et al. 2010). The major protein compon...

Selectivity of ORC binding sites and the relation to replication timing, fragile sites, and deletions in cancers

“…Given their near-universal presence at ORC2 sites, acetylated H3 and/or dimethylated H3K4 are excellent candidates to be recognized by ORC. H3K4 dimethylation promotes replication origin function in yeast (49), and H34K4me3 demethylation promotes replication origin firing (50), although the mechanism is unknown. However, the ORC subunits do not contain previously identified domains for interacting with these histone modifications.…”

Interaction of the Jhd2 Histone H3 Lys-4 Demethylase with Chromatin Is Controlled by Histone H2A Surfaces and Restricted by H2B Ubiquitination

“…Histone H3 lysine 4 (H3K4) 3 methylation plays an important role in transcriptional regulation (1,2) and other cellular processes (3)(4)(5)(6)(7). H3K4 methylation occurs in three forms or degrees because the lysine ⑀-amino group can be mono-, di-, or tri-methylated (indicated as H3K4me1, H3K4me2, and H3K4me3, respectively).…”

Catalysis-dependent stabilization of Bre1 fine-tunes histone H2B ubiquitylation to regulate gene transcription

“…One such histone PTM that has been well studied as a regulator of multiple DNA-templated processes is monoubiquitylation of histone H2B, which occurs at Lys123 (H2BK123ub1) in the budding yeast Saccharomyces cerevisiae (Robzyk et al 2000). This PTM functions in the context of transcriptional regulation (both initiation and elongation) (Henry et al 2003;Kao et al 2004;Xiao et al 2005;Pavri et al 2006;Fleming et al 2008;Chandrasekharan et al 2009Chandrasekharan et al , 2010 but has also been linked to other processes, including DNA replication (Rizzardi et al 2012;Trujillo and Osley 2012) and repair (Game and Chernikova 2009) and kinetochore function (Latham et al 2011).…”