Roles for Ctk1 and Spt6 in regulating the different methylation states of histone H3 lysine 36

Abstract

Set2 (KMT3)-dependent methylation (me) of histone H3 at lysine 36 (H3K36) promotes deacetylation of transcribed chromatin and represses cryptic promoters within genes. Although Set2 is the only methyltransferase (KMTase) for H3K36 in yeast, it is not known if Set2 is regulated or whether the different methylation states at H3K36 are functionally distinct. Here we show that the N-terminal 261 residues of Set2 (Set2(1-261)), containing the SET KMTase domain, are sufficient for H3K36me2, histone deacetylation, and repression of cryptic promoters at STE11. Set2-catalyzed H3K36me2 does not require either Ctk1-dependent phosphorylation of RNA polymerase II (RNAPII) or the presence of the phospho-C-terminal domain (CTD) interaction (SRI) domain of Set2. This finding is consistent with a known correlation between H3K36me2 and whether a gene is on or off, but not the level of activity of a gene. By contrast, H3K36me3 requires Spt6, proline 38 on histone H3 (H3P38), the CTD of RNAPII, Ctk1, and the C-terminal SRI domain of Set2. We suggest that the C-terminal region of Set2, in conjunction with the phosphorylated CTD of RNAPII, influences the KMTase activity to promote H3K36me3 during transcription elongation.

FIG. 1.

The amino-terminal 261 residues of Set2, including the SET domain, are sufficient for H3K36me2. (A) Schematic of the three versions of Set2 analyzed in this work, showing the various domains and their requirement for H3K36me2 and H3K36me3. +++, WT levels; ++, moderate reduction; (+), occasionally seen; −, not detectable. (B and C) Western blots of whole-cell extracts prepared from the indicated strains cultured at 30°C and incubated with antibodies against histone H3, H3K36me3, H3K36me2, the 3FLAG epitope, or H3K4me2. (B) Strains engineered to express full-length Set2-3FLAG, or Set2-3FLAG derivatives containing the indicated regions, from the endogenous locus. (C) Plasmid-borne full-length Set2, or a truncated derivative, expressed from the ADH1 promoter in a set2Δ strain. (D) Chromatin immunoprecipitation to detect Set2-FLAG or derivatives at three positions on STE11, the coding region (CR) of MET16 and telomere 1L (TEL01L) in the strains indicated. Data are expressed as the percentage of input signal.

FIG. 2.

H3K26me2 is sufficient for histone deacetylation and repression of internally initiated transcripts at STE11. (A) Chromatin immunoprecipitation to detect H3K36me2 at three positions on STE11, the coding region (CR) of MET16, and telomere 1L (TEL01L) in the strains indicated. Data are presented as a percentage of the input signal and are shown normalized to the histone H3 signals. (B) Chromatin immunoprecipitation to detect H3K18ac at three positions on STE11 and telomere 1L (TEL01L) in the strains indicated. Data are presented as a percentage of input signal and shown normalized to the histone H3 signals. (C) Northern blots of total RNA isolated from the indicated strains, hybridized with a probe from the 3′ region of STE11 or RDN18 (18S rRNA). The full-length transcript and the three (1 to 3) short internally initiated transcripts are indicated.

FIG. 3.

Rpb1 CTD- and H3P38-dependent H3K36me3, but not H3K36me2. (A) Western blot of total protein isolated from an hht1-hhf1Δ hht2-hhf2Δ strain expressing HHT2-HHF2 or the P38V derivatives from a plasmid (lane 1, wild-type histone H3; lane 2, H3 with a P38V substitution; lane 3, set2Δ) incubated with the antibodies indicated. Anti-glyceraldehyde-6-phosphate dehydrogenase (αG6PDH) acted as a loading control. (B) Western blot of total protein extracts from strains carrying truncation of the CTD of Rbp1 (33) incubated with the indicated antibodies. (C) Western blots of total protein extracts from strains expressing SET2 from the GAL1 promoter integrated at the SET2 locus cultured in raffinose (lanes 3 and 5; YPR off) or galactose (lanes 4 and 6; YPG on) incubated with the indicated antibodies. Lanes 1 and 2 contain extract from the strains shown in which SET2 is expressed from the endogenous SET2 promoter. (D and E) Northern blots of total RNA isolated from the strains indicated below, hybridized with a probe from the 3′ region of STE11 or RDN18 (18S rRNA). The full-length transcript and the three (1 to 3) short internally initiated transcripts are indicated. (D) H3P38V substitution; (E) Rpb1 derivatives.

FIG. 4.

Spt6-dependent H3K36me3, but not H3K36me2, when Set2 protein is restored. (A) Spt6 influences the levels of Set2 protein. Western blots of total protein extracts from the strains indicated incubated with the antibodies shown. (B) Western blots of total protein extracts from strains expressing SET2 from the GAL1 promoter integrated at the SET2 locus in the FY2181 background (WT or spt6-1004) cultured in raffinose (lane 2; YPR off) or galactose (lane 3; YPG on) incubated with the indicated antibodies. Lane 1 contains extract from the WT parental background in which SET2 is expressed from the endogenous SET2 promoter. (C) Chromatin immunoprecipitation to detect Set2 at three positions on STE11, the coding region of MET16 (CR), and telomere 01L in the strains indicated, cultured at 30°C in raffinose (YPR off) or galactose (YPG on). Data are expressed as a percentage of the input signal. Note that the profile for Set2 at STE11 detected using the polyclonal antibodies is different from that with the FLAG epitope (see Fig. 1D). These differences are reproducible and have been observed by others using different epitope tags on Set2 (7).

FIG. 5.

Spt6 influences the ability of Set2 to trimethylate chromatin in vitro but does not influence Set2 association with RNAPII. (A) Schematic of Spt6 showing the regions missing in the two derivatives. spt6-1004-FLAG is in the FY2181 background in which the WT Spt6 is also FLAG tagged, and Spt6ΔC is in the FY118 background in which a stop codon and the ADH1 terminator has replaced the SPT6 sequences at positions 1250 (Spt6ΔC) or 1452 (WT). (B) Western blots of total protein extracts from the indicated strains incubated with the antibodies indicated. Strains were cultured at 30°C, the permissive growth temperature for spt6-1004. (C) Extracts from strains expressing FLAG-tagged Spt6 (WT FY2181) or Spt6-1004 were immunoprecipitated (IP) with anti-Set2 antibodies (lanes 1 and 2) or preimmune serum (lanes 3 and 4), and association with Ser5 P-CTD was assessed using H14 antibodies by Western blotting. Levels of Set2 in the IP were monitored to assess recovery. (D) Spt6 controls trimethylation of H3K36 by recombinant Set2. WT and spt6-1004 strains, with GAL1:SET2 at the SET2 locus, were grown in YPR (raffinose) to ensure repression of SET2 under the GAL1 promoter. Equal amounts of sonicated whole-cell extract were used in a HMT reaction in which the sonicated extracts were incubated with 50 μM SAM (all lanes) and with (lanes 2 and 3) or without (lanes 1 and 4) an equal amount of purified recombinant Set2 (rSet2) (16) at 30°C for 30 min. Aliquots of the reaction mixture were analyzed by Western blotting using antibodies raised against Set2, H3K36me2, H3K36me3, and histone H3. Due to the challenges in the use of the H3K36me2 antibody as described previously (17), representative blots from more than three experiments are shown. In each case, the independent replicates yielded equivalent results. Note that no H3K36me3 or H3K36me2 was detected without the addition of rSet2, despite the presence of SAM.

FIG. 6.

Ctk1 is required for H3K36me3 but not H3K36me2 when Set2 levels are restored. (A) Ctk1 influences the levels of Set2 protein. Western blot results shown are of total protein extracts from the strains indicated incubated with antibodies indicated. In this and subsequent experiments set2Δ and ctk1Δ are in the BY4741 background, the Euroscarf reference strain. (B) Western blots of total protein extracts from strains expressing SET2 from the GAL1 promoter in the BY4741 background (left panel) or the same background with ctk1Δ (right panel) cultured in raffinose (YPR off) or galactose (YPG on) incubated with the antibodies indicated. Lanes 1 and 4 contain extracts from the WT parental backgrounds in which SET2 is expressed from the endogenous promoter. (C) Chromatin immunoprecipitation to detect Set2 at three positions on STE11, the coding region of MET16 (CR), and telomere 01L in the strains indicated, cultured at 30°C in raffinose (YPR off) or galactose (YPG on). Data are expressed as a percentage of input signal. Note that the profile for Set2 at STE11 detected using the polyclonal antibodies is different from that with the FLAG epitope (see Fig. 1D). These differences are reproducible and have been observed by others using different epitope tags on Set2 (7).

FIG. 7.

H3K36me2 is sufficient to repress the cryptic promoters at STE11 and FLO8. Northern blots show total RNA isolated from the WT, spt6-1004, and ctk1Δ strains expressing SET2 from the GAL1 promoter induced with galactose (YPG on) or repressed with raffinose (YPR off), hybridized with a probe from the 3′ region of STE11 (A) or FLO8 (B). RDN18 (18S rRNA) acted as a loading control. For the experiment shown in panel B, WT and spt6-1004 strains were heat shocked at 39°C for 60 min to inactive Spt6-1004 function. The full-length transcript and short internally initiated transcripts at each gene are indicated.