DNA replication origin function is promoted by H3K4 di-methylation in Saccharomyces 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 post-translational modifications to support robust genome duplication.

Figure 1

COMPASS and H3K4 are required for robust growth of the temperature-sensitive cdc6-1 replication mutant. (A) COMPASS complex model adapted from Takahashi et al. (2011). An asterisk denotes a complex member that genetically interacts with cdc6-1. (B) Wild-type (BY4741), cdc6-1 (yLF058), set1Δ (yLF062), and cdc6-1 set1Δ (yLF063) were transformed with an empty vector [pGLx2], a vector producing LexA-tagged wild-type Set1 [pGLx2-SET1], or catalytically dead Set1 [pGLx2-set1-H1017K] from a GAL1 promoter construct or a vector producing normal Cdc6 [pRS316-CDC6] from the CDC6 promoter as indicated. Fivefold serial dilutions were spotted onto SC-URA containing 1% galactose/2% raffinose and grown at the indicated temperatures for 4 days. (C) The cdc6-1 mutation was introduced into the H3-H4 “shuffle” strain (DY7803) transformed with HHT2 or hht2-K4R plasmids. Fivefold serial dilutions were spotted onto YPD and grown at the indicated temperatures for 3 days. (D) Fivefold serial dilutions of wild-type (YMS196), cdc6-1 (JCY332), cdc6-1 swd1Δ (yLF114), cdc6-1 bre2Δ (yLF120), bre2Δ (yLF060), or swd1Δ (yLF061) were spotted onto YPD and grown at the indicated temperatures for 3 days.

Figure 2

H3K4 methylation is required for robust growth of multiple replication mutants. (A) Fivefold serial dilutions of the meiotic progeny from the cross of cdc7-1 (TSQ880), cdc7-4 (TSQ131), or cdc45-27 (TSQ694) with set1Δ (yLF062) were spotted onto YPD and grown at the indicated temperatures for 3 days. (B) Fivefold serial dilutions of wild-type (RUY121), ORC6-rxl CDC6-ΔNT (RUY028), swd1Δ (yLF051), and ORC6-rxl CDC6-ΔNT swd1Δ (yLF049) were spotted onto YP containing 2% dextrose (no re-replication) or galactose (re-replication induced) and grown for 2 days at 30°.

Figure 3

H3K4 methylation is present at replication origins. Chromatin immunoprecipitation experiments were performed on asynchronous wild-type (BY4741) or set1Δ (yLF062) strains grown at 30°. Immunoprecipitates using antibodies to total histone H3, di-methylated H3K4 (H3K4me2) in A and tri-methylated H3K4 (H3K4me3) in B were analyzed by quantitative PCR for chromosomal DNA fragments from a region near telomere VI-R and two replication origins, ARS315 and ARS822. Error bars represent the standard deviations of n ≥ 3 biological replicates. Significant enrichment of H3K4 methylation at origins compared to telomere VI-R was determined using the Student’s unpaired t-test (*P < 0.05).

Figure 4

H2BK123 mono-ubiquitination promotes robust growth of the temperature-sensitive cdc6-1 replication mutant. (A) Wild-type (BY4741), bre1Δ (yLF151), cdc6-1 (yLF058), and cdc6-1 bre1Δ (yLF150) were transformed with either an empty vector [pRS315], a vector expressing BRE1 [pRS315-9xMyc-BRE1], or bre1-H665A [pRS315-9xMyc-bre1-H665A] from the native BRE1 promoter. Fivefold serial dilutions were spotted onto SCD-LEU and grown for 3 days at the indicated temperatures. (B) Fivefold serial dilutions of wild-type (YMS196), cdc6-1 (JCY332), rad6Δ (yLF154), and cdc6-1 rad6Δ (yLF117) were spotted onto YPD and grown for 3 days at the indicated temperatures. As previously reported, the rad6Δ strain is cold sensitive at temperatures <30° (McDonough et al. 1995). (C) Wild-type (RUY121), ORC6-rxl CDC6-ΔNT (RUY028), bre1Δ (yLF052), and ORC6-rxl CDC6-ΔNT bre1Δ (yLF050) were spotted onto YP containing 2% dextrose or 1% galactose (re-replication induced) and grown for 3 days at 30°.

Figure 5

H3K4 methylation is required for efficient origin-dependent mini chromosome maintenance. (A) Plasmid loss rates of a single-origin-bearing plasmid [YCpLac33] were measured in a wild-type strain (BY4741) and in strains lacking SET1 (yLF089), BRE1 (yLF151), or DOT1 (YNL037). Loss rates are reported per cell division. (B) Plasmid loss rates of a single-origin-bearing plasmid [YCpLac111] were measured in the histone “shuffle” strain (DY7803) transformed with plasmids expressing wild-type HHT2, hht2-K4R, or hht2-K79R. (C) Plasmid loss rates of plasmids bearing either a single origin [YCpLac33] or three origins [YCpLac33 + 2X ARS209] were measured. For all experiments, the average loss rates were obtained from at least three independent transformants, and the error bars indicate standard deviations. Statistics were performed using the Student’s unpaired t-test (*P < 0.05).

Figure 6

H3K4 di-methylation promotes efficient mini-chromosome maintenance. (A) Plasmid loss rates of the single-origin-bearing plasmid [YCpLac33] were measured for wild-type (BY4741), swd1Δ (yLF061), bre2Δ (yLF060), and spp1Δ (yLF153). (B) Immunoblot analysis of whole-cell extracts (from strains shown in A) using antibodies to total H3, H3K4me1, H3K4me2, and H3K4me3. (C) Plasmid loss rates of the single-origin-bearing plasmid [YCpLac111] were measured for the H2B “shuffle” strain (FY406) transformed with plasmids expressing HTB1 [pZS145], htb1-K123R [pZS146], htb1-R119D [pZS473], or htb1-R119A [pZS145-R119A]. (D) Immunoblot analysis of whole-cell extracts (from strains shown in C) using antibodies specific for total H2B, H3, and H3K4 methylation as in B. All plasmid loss data represent the mean and standard deviation of at least three independent transformants. Statistics were performed using the Student’s unpaired t-test (*P < 0.05).

Figure 7

H3K4 di-methylation is sufficient for robust growth of the cdc6-1 mutant. (A) Wild-type (BY4741), cdc6-1 (yLF058), set1Δ (yLF062), and cdc6-1 set1Δ (yLF063) were transformed with an empty vector [pGLx2], a vector producing LexA-tagged Set1 [pGLx2-SET1], or Set1 lacking the RRM domain [pGLx2-set1-ΔRRM] from the GAL1 promoter. Fivefold serial dilutions were spotted onto SC-URA containing 1% galactose and grown at the indicated temperatures for 5 days. (B) Immunoblot analysis of whole-cell extracts from either wild-type (BY4741) or set1Δ (yLF062) strains transformed with empty vector [pGLx2] or vectors producing LexA-tagged normal (“WT”) Set1 [pGLx2-SET1], catalytically dead (“CD”) Set1 [pGLx2-set1-H1017K], or Set1 lacking the RRM domain (“RRM”) [pGLx2-set1-ΔRRM] from the GAL1 promoter. Blots were probed with antibodies specific for LexA, total H3, H3K4me3, H3K4me2, and H3K4me1. A single asterisk represents a likely degradation product unique to the positive control construct; a double asterisk represents a nonspecific band detected by the H3K4me1 antibody.

Figure 8

Two models by which H3K4 di-methylation could promote efficient DNA replication. The recruitment model suggests either direct or indirect recruitment of replication factors by H3K4 di-methylation, while the accessibility model suggests recruitment of a HAT that acetylates nucleosomes near the origin, resulting in an open chromatin state that allows replication factors to access and bind the origin.