A role for Chd1 and Set2 in negatively regulating DNA replication in Saccharomyces cerevisiae

Abstract

Chromatin-modifying factors regulate both transcription and DNA replication. The yFACT chromatin-reorganizing complex is involved in both processes, and the sensitivity of some yFACT mutants to the replication inhibitor hydroxyurea (HU) is one indication of a replication role. This HU sensitivity can be suppressed by disruptions of the SET2 or CHD1 genes, encoding a histone H3(K36) methyltransferase and a chromatin remodeling factor, respectively. The additive effect of set2 and chd1 mutations in suppressing the HU sensitivity of yFACT mutants suggests that these two factors function in separate pathways. The HU suppression is not an indirect effect of altered regulation of ribonucleotide reductase induced by HU. set2 and chd1 mutations also suppress the HU sensitivity of mutations in other genes involved in DNA replication, including CDC2, CTF4, ORC2, and MEC1. Additionally, a chd1 mutation can suppress the lethality normally caused by disruption of either MEC1 or RAD53 DNA damage checkpoint genes, as well as the lethality seen when a mec1 sml1 mutant is exposed to low levels of HU. The pob3 defect in S-phase progression is suppressed by set2 or chd1 mutations, suggesting that Set2 and Chd1 have specific roles in negatively regulating DNA replication.

Figure 1.—

set2 and chd1 replication defects caused by yFACT mutations. (A) Tenfold dilutions of strains DY11848 (spt16-11), DY11852 (spt16-11 set2), DY11851 (spt16-11 chd1), DY11855 [spt16-11 H4(K5R, K12R)], DY11849 [spt16-11 H4(K5R, K12R) set2], and DY11853 [spt16-11 H4(K5R, K12R) chd1] were plated on complete medium for 5 days at 25° or for 3 days at either 30° or 33°. (B) Tenfold dilutions of strains DY150 (wild type), DY8690 (set2), DY6957 (chd1), DY8107 (spt16-11), DY8777 (spt16-11 set2), and DY9152 (spt16-11 chd1) were plated on complete or 50 mm HU medium for 2 days at 30°. (C) Tenfold dilutions of strains DY150 (wild type), DY8690 (set2), DY6957 (chd1), DY7379 [pob3(L78R)], DY8878 [pob3(L78R) set2], and DY9458 [pob3(L78R) chd1] were plated on complete or 50 mm HU medium for 3 days at 25°. (D) (Left) Tenfold dilutions of strains DY2860 (wild type), DY8898 (set2), DY10722 [pob3(Q308K)], and DY10723 [pob3(Q308K) set2] were plated at 30° on complete medium for 3 days or on 150 mm HU medium for 4 days. (Right) Tenfold dilutions of strains DY150 (wild type), DY9809 (chd1), DY10890 [pob3(Q308K)], and DY10897 [pob3(Q308K) chd1] were plated on complete or 50 mm HU medium for 2 days at 30°. (E) (Left) Tenfold dilutions of strains DY150 (wild type), DY8690 (set2), DY10308 [pob3(Q308K)], and DY12028 [pob3(Q308K) set2] were plated on complete medium at 30° for 2 days or at 35° for 3 days. (Right) Tenfold dilutions of strains DY150 (wild type), DY9809 (chd1), DY10890 [pob3(Q308K)], and DY10897 [pob3(Q308K) chd1] were plated on complete medium for 2 days at 30° or 35°.

Figure 2.—

chd1 and set2 mutations do not stabilize mutant yFACT proteins. Strains were grown to logarithmic phase at 25°, and the culture was split and then incubated for 3 hr at either 25° or 37°. Pob3 and Spt16 protein levels were determined by Western blotting. Identical gels stained with Coomassie blue verified equal protein loading. The blots were quantitated and normalized to the wild-type strain at that temperature. (A) The Pob3(L78R) protein has reduced abundance at both 25° and 37°. Strains DY150 (wild type), DY7379 [pob3(L78R)], DY9809 (chd1), DY9458 [pob3(L78R) chd1], DY8690 (set2), and DY8878 [pob3(L78R) set2] were used. The blot was probed with antibody to both Pob3 and Spt16, and the bottom band of the doublet in this experiment is a proteolytic fragment of Spt16. (B) The Pob3(Q308K) protein is at the same levels as wild type. Strains DY150 (wild type), DY10308 [pob3(Q308K)], DY10711 (chd1), DY10897 [pob3(Q308K) chd1], DY8795 (set2), and DY1228 [pob3(Q308K) set2] were used. In this experiment, only Pob3 was probed so both bands visualized are Pob3 protein. (C) The Spt16-11 protein has reduced abundance at 37°, but normal levels at 25°. Strains DY150 (wild type), DY8107 (spt16-11), DY10711 (chd1), DY12430 (spt16-11 chd1), DY8795 (set2), and DY8777 (spt16-11 set2) were used. The blot was probed with antibody to Spt16.

Figure 3.—

set2 and chd1 are additive in suppressing HU sensitivity of yFACT mutants. (A) Tenfold dilutions of strains DY150 (wild type), DY9809 (chd1), DY8690 (set2), DY9838 (chd1 set2), DY7379 [pob3(L78R)], DY9458 [pob3(L78R) chd1], DY8878 [pob3(L78R) set2], and DY9547 [pob3(L78R) chd1 set2] were plated at 25° on complete medium for 3 days or 150 mm HU medium for 6 days. (B) Tenfold dilutions of strains DY150 (wild type), DY9809 (chd1), DY8690 (set2), DY9838 (chd1 set2), DY8107 (spt16-11), DY12430 (spt16-11 chd1), DY8777 (spt16-11 set2), and DY9153 (spt16-11 chd1 set2) were plated at 30° on complete medium for 2 days or 120 mm HU medium for 4 days.

Figure 4.—

set2 and chd1 suppress HU sensitivity of DNA replication mutants. (A) Tenfold dilutions of strains DY5662 (wild type), DY10055 (set2), DY10058 (cdc2-1), and DY10097 (cdc2-1 set2) were plated at 25° on complete medium for 3 days or 100 mm HU medium for 6 days. (B) Tenfold dilutions of strains DY5662 (wild type), DY10055 (set2), DY10056 (ctf4), and DY10092 (ctf4 set2) were plated at 25° on complete medium for 2 days or 100 mm HU medium for 9 days. (C) Tenfold dilutions of strains DY150 (wild type), DY10711 (chd1), DY11082 (orc2-1), and DY11084 (orc2-1 chd1) were plated at 25° on complete medium for 2 days or 50 mm HU medium for 3 days. (D) Tenfold dilutions of strains DY150 (wild type), DY8825 (set2), DY12610 (nhp10), and DY12611 (nhp10 set2) were plated at 30° on complete medium for 2 days or 150 mm HU medium for 5 days. (E) Tenfold dilutions of strains DY150 (wild type), DY10675 (chd1), DY10665 (mec1 sml1), and DY10669 (mec1 sml1 chd1) were plated at 25° on complete medium for 2 days or 2 mm HU medium for 3 days.

Figure 5.—

RNR gene expression in pob3(L78R) and pob3(Q308K) mutants. (A) Strains DY150 (wild type) and DY7379 [pob3(L78R)] were grown at 25° in YPAD medium to OD₆₀₀ = 0.6, before a preinduction sample (“−”) was taken, and then HU was added to a concentration of 100 mm, and cultures were grown for an additional 2 hr before the postinduction sample (“+ HU”) was taken. Strains DY10641 (wild type) and DY10642 [pob3(Q308K)] were grown identically, expect at 30°. RNA was isolated from the samples and expression of RNR1, RNR2, RNR3, and RNR4 was determined by S1 nuclease protection, with tRNA serving as an internal control. (B) Graphs of RNR gene expression before and after HU induction using the quantitation from the S1 protection assay in supplemental Figure S2.

Figure 6.—

Deletion of CHD1 bypasses the MEC1 checkpoint. (A) DY6958 (chd1∷LEU2) was crossed to DY10112 (mec1∷TRP1 sml1∷HIS3), yielding DY10671 (mec1∷TRP1 chd1∷LEU2 SML1). DY10671 was then mated to wild-type strain DY1868, and haploid progeny are shown after 7 days of growth at 25°. Symbols indicate selected genotypes. (B) Strains DY9809 (chd1∷TRP1) and DY10689 (rad53∷HIS3 YEp-LEU2-RNR1) were mated and the haploid progeny from sporulating that diploid are shown after 5 days of growth. rad53 is lethal, but lethality can be suppressed by a multicopy plasmid with RNR1 (Desany et al. 1998). Viable rad53 chd1 strains were recovered both with and without the YEp-LEU2-RNR1 plasmid, and the presence or the absence of the YEp-LEU2-RNR1 plasmid did not affect the growth rate of the rad53 chd1 strains. Symbols indicate selected genotypes. (C) Tenfold dilutions of strains DY10112 (mec1 CHD1 sml1) and DY10671 (mec1 chd1 SML1) were plated on complete medium at 25° for 3 days. (D) Strains DY150 (wild type), DY9809 (chd1), and DY8690 (set2) were grown to logarithmic phase at 30° and HU was added to a concentration of 200 mm. Samples were taken before HU addition, and at 5-min intervals after, and examined on immunoblots probed with antibody to Rad53. Identical gels stained with Coomassie blue verified equal protein loading. The arrowhead indicates the position of phosphorylated Rad53. (E) HU (10 mm final concentration) was added to cultures of strains DY150 (wild type), DY10670 (mec1 sml1 chd1), DY10148 (mec1 sml1), and DY10150 (mec1 sml1 set2) growing at 30°, and at the indicated times samples were taken. Cells were sonicated and washed with water once before plating to determine the fraction of viable cells. The experiment was also conducted with sml1, sml1 chd1, and sml1 set2 strains, and the results were similar to that seen for wild type.

Figure 7.—

set2 and chd1 suppress the pob3 defect in S-phase progression. (A) Wild-type (DY150), pob3(L78R) (DY7379), pob3(L78R) chd1 (DY9458), and pob3(L78R) set2 (DY8878) cells were grown to log phase at 25°, arrested with α-factor for 2.5 hr, and released from the arrest by resuspending in fresh media containing protease at 25°. Samples were taken at 10-min intervals and DNA content was determined by flow cytometry. The 1C and 2C positions are indicated. (B) The same as in A, except cells released from the α-factor arrest at 34°. Strains DY150 (wild type), DY10308 [pob3(Q308K)], DY10897 [pob3(Q308K) chd1], and DY12028 [pob3(Q308K) set2] were used.