The RNA polymerase II kinase Ctk1 regulates positioning of a 5' histone methylation boundary along genes

Abstract

In yeast and other eukaryotes, the histone methyltransferase Set1 mediates methylation of lysine 4 on histone H3 (H3K4me). This modification marks the 5' end of transcribed genes in a 5'-to-3' tri- to di- to monomethyl gradient and promotes association of chromatin-remodeling and histone-modifying enzymes. Here we show that Ctk1, the serine 2 C-terminal domain (CTD) kinase for RNA polymerase II (RNAP II), regulates H3K4 methylation. We found that CTK1 deletion nearly abolished H3K4 monomethylation yet caused a significant increase in H3K4 di- and trimethylation. Both in individual genes and genome-wide, loss of CTK1 disrupted the H3K4 methylation patterns normally observed. H3K4me2 and H3K4me3 spread 3' into the bodies of genes, while H3K4 monomethylation was diminished. These effects were dependent on the catalytic activity of Ctk1 but are independent of Set2-mediated H3K36 methylation. Furthermore, these effects are not due to spurious transcription initiation in the bodies of genes, to changes in RNAP II occupancy, to changes in serine 5 CTD phosphorylation patterns, or to "transcriptional stress." These data show that Ctk1 acts to restrict the spread of H3K4 methylation through a mechanism that is independent of a general transcription defect. The evidence presented suggests that Ctk1 controls the maintenance of suppressive chromatin in the coding regions of genes by both promoting H3K36 methylation, which leads to histone deacetylation, and preventing the 3' spread of H3K4 trimethylation, a mark associated with transcriptional initiation.

FIG. 1.

Ctk1 kinase activity regulates H3K4 methylation. (A) Shown is a sample of 56 of the 384 yeast deletion strains screened for H3K4 monomethylation defects. WCEs prepared from asynchronous, logarithmically growing deletion mutants were resolved by 15% SDS-PAGE and transferred onto a PVDF membrane prior to immunoblotting with anti-histone H3 K4 monomethylation-specific antibody (α-H3K4me1). The arrow in the top blot indicates extracts from an rtf1 mutant which encodes a component of the PAF transcription elongation complex previously identified as a regulator of H3K4 mono-, di-, and trimethylation. Arrows in the bottom three blots denote the members of the CTDK-1 complex that were found to regulate H3K4 monomethylation in this study. (B) Characterization of the effect of ctk1Δ on H3K4 methylation states. Increasing amounts (twofold/lane) of either WT (BY4741) or ctk1Δ WCE were resolved by 15% SDS-PAGE and transferred to PVDF prior to immunoblotting with the specified antibodies. Arrows indicate a point in the titration where the H3K4 di- and trimethylation increases are readily observed. (C) Quantification of ctk1Δ effects on the relative proportions of H3 that were H3K4 mono-, di-, and trimethylated. Immunoblots in panel B were quantified with a densitometer, and the results were plotted as the fold changes relative to the WT level. (D) Ctk1 catalytic activity is required to regulate H3K4 methylation. Normalized WCEs from WT (WZY42) or ctk1Δ (YTX045) cells transformed with the empty vector, a CTK1 expression vector, a mutant that produces catalytically dead Ctk1 [ctk1 (D324N)], or a ctk1 mutant that does not abolish its catalytic activity [ctk1 (T338A)] were immunoblotted as described above with the specified antibodies. (E) Synthetic genetic interactions of the CTDK-1 complex. An E-MAP (40) was created by SGA technology (48) and used to cross Nat^r strains harboring individual deletions of genes encoding two members of the CTDK-1 complex (ctk1Δ and ctk2Δ) with a transcription-targeted array of 384 Kan^r deletion strains to create sets of Nat^r/Kan^r haploid double mutants. Growth rates of the double mutants were assessed by automated image analysis of colony size (40), and lines in the diagram connect genes with significant synthetic growth defects. For the degrees of these synthetic interactions, see Table S1 in the supplemental material.

FIG. 2.

CTK1 deletion results in a spread of H3K4 di- and trimethylation along the coding regions of genes. (A) Schematic representation of the FMP27 locus and the relative locations of the PCR primers used. (B) Results of a ChIP experiment monitoring the distribution of H3K4 mono-, di-, and trimethylation; H3K79 trimethylation; and histone H3 in the WT and ckt1Δ backgrounds. Asterisks denote the location of an internal control band specific to a region of chromosome V lacking an ORF. (C) The degree of enrichment achieved in the ChIP indicated is plotted on the y axis. The primer pair used to assay enrichment is indicated on the x axis. Values are normalized to a histone H3 ChIP to account for differences in underlying nucleosome occupancy (see Materials and Methods). Error bars represent the standard error of the mean of three independent replicates. Note that for this and the subsequent panels, ratio values are log₂ transformed to ensure an accurate representation of ratios less than 1. Therefore, every change of 1 U represents a 2-fold change in the abundance of the indicated histone methylation event (a change of 4 U would indicate a 16-fold change and so on). Also, because of differences among the antibodies, the absolute values reported on the y axis apply only to ChIPs performed with the same antibody. (D to F) Same as in A to C except that the FIR1 gene was analyzed.

FIG. 3.

H3K4 methylation spreading in CTK1 deletion strains cannot be explained by defects in Set2/H3K36 methylation, increased cryptic initiation, or defects in RNAP II occupancy. (A) Schematic representation of the FMP27 locus and the relative locations of PCR primers used. (B) Results of a ChIP experiment monitoring the distribution of H3K4me3, H3K14ac, and histone H3 in the WT, ckt1Δ, and set2Δ backgrounds. Asterisks denote the location of an internal control band specific to a region of chromosome V lacking an ORF. (C) The fold enrichment achieved in the ChIP indicated in the mutants over the WT is plotted on the y axis. The primer pair used to assay enrichment is indicated on the x axis. Values are normalized to a histone H3 ChIP to account for differences in underlying nucleosome occupancy (see Materials and Methods). Error bars represent the standard error of the mean of three independent replicates. Results similar to those shown in panels B and C were also obtained with several other genes examined, including PMA1, ADH1, FIR1, and STE11 (data not shown). (D) set2 deletion does not affect H3K4 methylation levels globally. Increasing amounts of either WT or set2Δ WCE were resolved by 15% SDS-PAGE and transferred to PVDF prior to immunoblotting with the specified antibodies. (E) H3K4 methylation spreading observed in ctk1Δ cells cannot be explained by an increase in cryptic initiation or altered levels of transcription. Total RNAs purified from WT, set2Δ, or ctk1Δ cells were used for Northern blot analysis and probed with radiolabeled PCR products corresponding to the 3′ ends of the specified genes. FL denotes the full-length product, and short denotes smaller RNA species that were observed. WT cells naturally exhibit some additional smaller mRNA products for the FMP27 gene. Lanes were loaded with equivalent amounts of total RNA (assayed by ethidium staining [not shown]). STE11 was included as a positive control for cryptic initiation upon deletion of SET2 (4). (F) RNAP II occupancy levels and distribution are not affected by deletion of CTK1 and SET2. A ChIP experiment monitoring the distribution of RNAP II and Ser5 CTD phosphorylated RNAP II in ckt1Δ and set2Δ along the FMP27 gene (see Materials and Methods) was performed. Shown are the fold enrichment values measured at the FMP27 promoter and at increasing distances from the ATG codon (see the code at the far right). The primer pairs used in this panel are distinct from those shown in panel A. RNAP II levels were monitored with the 8WG16 (anti-CTD) antibody; nearly identical results were obtained with a different RNAP II antibody to the N terminus of Rpb1 (sc25758, Santa Cruz; not shown).

FIG. 4.

Transcriptional stress does not affect global levels of H3K4 methylation. (A) Set1 levels are not altered in ctk1Δ strains. WT (W303mycSet1) or ctk1Δ (YTX046) WCEs from strains harboring an N-terminal nine-myc tag on Set1 were loaded onto an 8% SDS-PAGE gel and immunoblotted with the specified antibodies. Glucose-6-phosphate dehydrogenase (G6PDH) and RNAP II were probed to ensure equivalent loading, and the anti-Ser2 CTD phosphospecific antibody (Upstate) was used to verify the expected loss of this modification in ctk1Δ cells. (B) Strains containing a Flag-tagged version of H2B that also harbor either a WT or a temperature-sensitive allele of KIN28 (kin28-ts16-HA) were shifted for 2 h at 37°C prior to harvesting cell pellets. A portion of these pellets was directly used for an H2B ubiquitylation assay (a positive control for loss of Kin28 kinase activity), and the remainder of each pellet was used to prepare WCEs to examine the H3K4 methylation status with the specified antibodies. Although H2B ubiquitylation is abolished in the KIN28 mutant, H3K4 methylation is more stable and would be expected to persist during the 2-h heat inactivation (33). (C) An rpb1-1 temperature-sensitive allele was shifted to 37°C for 2 h prior to cell harvesting, cell extraction, and immunoblot analyses with the specified antibodies. (D) WT (BY4741) cells containing the pRS316 vector were grown at 30°C in SC-Ura to an OD₆₀₀ of 1.0 prior to the addition of 100 μg/ml 6-azauracil (6-AU). These cells were pelleted, and WCE was prepared and used for immunoblot analysis with the specified antibodies. Temp, temperature.

FIG. 5.

H3K4me3 spreading in ctk1Δ cells occurs genome-wide on the ORFs of RNAP II-regulated genes. (A) Distribution of z scores following H3K4me3 ChIPs from WT WCE. Shown are ORFs (red), 3′ UTRs (blue), unidirectional promoters (black), and bidirectional promoters (green). z scores were normalized to histone H3 distribution as described in Materials and Methods. Promoters are clearly distinguished from ORFs and 3′ UTRs. (B) Same as in panel A, but H3K4me3 ChIPs were performed with WCE from ctk1Δ cells. (C) A moving average (window size = 40, step size = 1) of normalized z scores of H3K4me3 ChIP-chip data in WT (black), set2Δ (blue), and ctk1Δ (red) cells plotted as a function of ORF length. (D) A moving average (window size = 40, step size = 1) of H3K4me3 ChIP-chip z scores in WT (black), set2Δ (blue), and ctk1Δ (red) cells plotted as a function of the distance from the transcription start site among genes with coding regions greater than 1 kb in length and for which data were available on chromosome III (69 genes). A high-resolution microarray covering chromosome III was used for this analysis (see Materials and Methods), and the data plotted in this case were not normalized to histone occupancy. (E) Colors (scale at bottom) represent the median z scores of all data points recorded from all arrayed elements in the indicated Saccharomyces Genome Database functional class (labeled on the right, number of arrayed elements analyzed in parentheses). Data were normalized to histone H3 distribution and derived from three independent ChIP experiments (see Materials and Methods).

FIG. 6.

Role of Ctk1 in the control of chromatin structure. Shown is a model that describes a novel function for Ctk1 in the regulation of histone modifications in the coding regions of RNAP II-regulated genes. In addition to Ctk1 directing the association of Set2 to RNAP II, which results in the establishment of H3K36 methylation and deacetylated chromatin in the bodies of genes, our data reveal that this kinase also acts to prevent the spread of H3K4 trimethylation from its defined position at the 5′ end. We speculate that while Set1 is normally recruited to the 5′ end in a Ser5 CTD phosphorylation- and PAF-dependent manner, the phosphorylation of Ser2 on the CTD by Ctk1 acts to block persistent Set1 association along genes. While direct phosphorylation of Ser2 on the CTD is likely responsible, we do not rule out other possible mechanisms such as direct phosphorylation of COMPASS by Ctk1 or RNAP II kinetic changes upon initial transcription.