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Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast - PubMed

  • ️Wed Jan 01 2003

Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast

Tiaojiang Xiao et al. Genes Dev. 2003.

Abstract

Histone methylation is now realized to be a pivotal regulator of gene transcription. Although recent studies have shed light on a trans-histone regulatory pathway that controls H3 Lys 4 and H3 Lys 79 methylation in Saccharomyces cerevisiae, the regulatory pathway that affects Set2-mediated H3 Lys 36 methylation is unknown. To determine the functions of Set2, and identify factors that regulate its site of methylation, we genomically tagged Set2 and identified its associated proteins. Here, we show that Set2 is associated with Rbp1 and Rbp2, the two largest subunits of RNA polymerase II (RNA pol II). Moreover, we find that this association is specific for the interaction of Set2 with the hyperphosphorylated form of RNA pol II. We further show that deletion of the RNA pol II C-terminal domain (CTD) kinase Ctk1, or partial deletion of the CTD, results in a selective abolishment of H3 Lys 36 methylation, implying a pathway of Set2 recruitment to chromatin and a role for H3 Lys 36 methylation in transcription elongation. In support, chromatin immunoprecipitation assays demonstrate the presence of Set2 methylation in the coding regions, as well as promoters, of genes regulated by Ctk1 or Set2. These data document a new link between histone methylation and the transcription apparatus and uncover a regulatory pathway that is selective for H3 Lys 36 methylation.

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Figures

Figure 1
Figure 1

Set2 interacts with RNA pol II. (A) Purification of Set2-3Flag. Wild-type (WT) or genomically tagged Set2 whole-cell extracts (WCE) were incubated with anti-Flag resin, and the resulting bound proteins eluted with 3×Flag peptide. Eluted proteins were resolved by 8% SDS-PAGE and examined by Coomassie staining. Arrows indicate the protein identity of bands that were examined by mass spectrometry; sizes of the molecular weight markers are shown. (B) Interaction of Set2 with hyperphosphorylated RNA pol II. WCE from wild-type or Set2-3Flag cells were immunoprecipitated with anti-Flag beads followed by immunoblotting with antibodies directed against unmodified CTD (8WG16 [α-CTD]), Ser 2-phosphorylated CTD (H5 [α-Ser2P]), Ser 5 phosphorylated CTD (H14 [α-Ser5P]), or α-Flag. These WCEs (Inputs) were also examined by immunoblot analysis with the antibodies described above to monitor the presence of these proteins. (C) Interaction of Set2 with RNA pol II is phosphorylation dependant. Flag immunoprecipitations (IPs) from wild-type or Set2-3Flag WCEs were separated and treated with either buffer only or buffer plus λ phosphatase (PPase) followed by immunoblotting with the H5 [Ser2P], H14 [Ser5P], and 8WG16 [α-CTD] antibodies. (D) Set2 coimmunoprecipitations with hyperphosphorylated RNA pol II. Wild-type or Set2-3Flag WCEs were first immunoprecipitated with the H5 [Ser2P] or H14 [Ser5P] antibodies, followed by immunoblot analysis with α-Flag. Results with both antibodies were identical and only the H5 [Ser2P] results are shown. We note that with our immunoprecipitations using CTD antibodies, we were able to efficiently detect Ser 2 phosphorylation from H14 [Ser5P] immunoprecipitations (and vise versa), indicating the heterogeneity of phosphorylation that exists on RNA pol II in vivo (data not shown). Thus, whereas Set2 may be interacting preferentially with Ser 2-phosphorylated CTD (see Fig. 5 and text), it is likely that both phosphorylation states would be detected in these assays.

Figure 2
Figure 2

The C terminus of Set2 interacts with RNA pol II. (A) Schematic representation of the Set2-Flag constructs used to probe for RNA pol II interaction. The SET domain along with its flanking Cys-rich domains (C), WW domain (WW), and coiled-coil motif (CC) are shown. (B) set2Δ cells were transformed with either vector only or the indicated Set2-Flag constructs and WCEs were prepared. These extracts were immunoprecipitated with the α-Flag antibody followed by immunoblot analysis with the H5 [α-Ser2P], H14 [α-Ser5P], or α-Flag antibodies. Sizes of the molecular weight markers are shown, and asterisks indicate the location of expected Set2-Flag products. All input extracts showed equivalent levels of hyperphosphorylated RNA pol II (data not shown).

Figure 3
Figure 3

Set2 methylates at the coding regions, as well as promoters, of genes. Chromatin immunoprecipitation assays were used to examine the H3 Lys 36 methylation status on several genes in vivo. Whole-cell extracts prepared from formaldehyde-fixed wild-type or set2Δ cells were sonicated to shear their chromatin and then immunoprecipitated with either the H3 Lys 36 methylation-specific antibody [α-Me(Lys 36)H3], the H3 Lys 4 methylation-specific antibody [α-Me(Lys 4)H3], the CTD Ser 5 phosphorylation-specific antibody (H14 [α-Ser5P]), or the Ctk1-phosphorylated CTD antibody (α-Ctk1-PCTD). DNA from the enriched precipitates was isolated and used in PCR reactions with promoter or coding-specific primer pairs for the genes indicated. Primer pairs (labeled as A–D) include their sequence location relative to the translation initiation site. Although we show several genes positive for the presence of H3 Lys 36 methylation, other genes or distinct regions of chromatin examined (HIS3, rDNA, and telomeres) showed no enrichment for H3 Lys 36 methylation above background levels observed in set2Δ (data not shown).

Figure 4
Figure 4

The CTD is required for H3 Lys 36 methylation. (A) Yeast nuclear extracts prepared from nondeleted CTD (Z26 and Z27), or the indicated CTD deletion strains were probed with antibodies against either methylated lysine residues at H3 Lys 36, Lys 4, and Lys 79 or acetylated H3. C3, C23, and V26 represent CTDs of ∼10, 12, and 18 repeats, respectively (for review, see Nonet et al. 1987). Asterisk indicates partial N-terminal H3 breakdown products that are typically observed. (B) Expression levels of SET2 or ACT1 (used as a control) were not changed in the CTD tail-deletion strains. Shown are reverse transcription PCR reactions with (+) or without (−) reverse transcriptase (RTase).

Figure 5
Figure 5

The CTD kinase Ctk1 is essential for Set2 methylation. (A) Yeast nuclear extracts prepared from wild-type or the indicated deletion strains were probed with antibodies against either methylated lysine residues at H3 Lys 36, Lys 4, and Lys 79 or acetylated H3. (B) Nuclear extracts isolated from wild-type or ctk1Δ strains containing either the vector control plasmid (ctk1Δ + Vector) or wild-type CTK1 expression plasmid (ctk1Δ + CTK1) were probed with antibodies against either methylated lysine residues at H3 Lys 36 and Lys 4 or acetylated H3. (C) Expression levels of SET2 or ACT1 (used as a control) were not changed in ctk1Δ. Shown are RT–PCR reactions with (+) or without (−) reverse transcriptase (RTase). (D) The Ctk1 complex is essential for H3 Lys 36 methylation. Nuclear extracts isolated from wild-type or ctk1Δ, ctk2Δ, ctk3Δ, or sse2Δ strains were probed with antibodies against either methylated lysine residues at H3 Lys 36, Lys 4, and Lys 79 or acetylated H3. Asterisks indicate partial N-terminal H3 breakdown products that are typically observed. Sse2 was identified recently as a new component of the Ctk1 complex (Gavin et al. 2002).

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References

    1. Berger SL. Histone modifications in transcriptional regulation. Curr Opin Genet Dev. 2002;12:142–148. - PubMed
    1. Bernstein BE, Humphrey EL, Erlich RL, Schneider R, Bouman P, Liu JS, Kouzarides T, Schreiber SL. Methylation of histone H3 Lys 4 in coding regions of active genes. Proc Natl Acad Sci. 2002;99:8695–8700. - PMC - PubMed
    1. Briggs SD, Bryk M, Strahl BD, Cheung WL, Davie JK, Dent SY, Winston F, Allis CD. Histone H3 lysine 4 methylation is mediated by Set1 and required for cell growth and rDNA silencing in Saccharomyces cerevisiae. Genes & Dev. 2001;15:3286–3295. - PMC - PubMed
    1. Briggs SD, Xiao T, Sun ZW, Caldwell JA, Shabanowitz J, Hunt DF, Allis CD, Strahl BD. Gene silencing: Trans-histone regulatory pathway in chromatin. Nature. 2002;418:498. - PubMed
    1. Cho EJ, Kobor MS, Kim M, Greenblatt J, Buratowski S. Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain. Genes & Dev. 2001;15:3319–3329. - PMC - PubMed

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