An mRNA Capping Enzyme Targets FACT to the Active Gene To Enhance the Engagement of RNA Polymerase II into Transcriptional Elongation

Abstract

We have recently demonstrated that an mRNA capping enzyme, Cet1, impairs promoter-proximal accumulation/pausing of RNA polymerase II (Pol II) independently of its capping activity in Saccharomyces cerevisiae to control transcription. However, it is still unknown how Pol II pausing is regulated by Cet1. Here, we show that Cet1's N-terminal domain (NTD) promotes the recruitment of FACT (facilitates chromatin transcription that enhances the engagement of Pol II into transcriptional elongation) to the coding sequence of an active gene, ADH1, independently of mRNA-capping activity. Absence of Cet1's NTD decreases FACT targeting to ADH1 and consequently reduces the engagement of Pol II in transcriptional elongation, leading to promoter-proximal accumulation of Pol II. Similar results were also observed at other genes. Consistently, Cet1 interacts with FACT. Collectively, our results support the notion that Cet1's NTD promotes FACT targeting to the active gene independently of mRNA-capping activity in facilitating Pol II's engagement in transcriptional elongation, thus deciphering a novel regulatory pathway of gene expression.

Keywords: Cet1; FACT; RNA polymerase II; mRNA capping; transcription.

FIG 1

The NTD (aa 1 to 204) of Cet1 targets FACT to ADH1 independently of mRNA-capping activity. (A) (Top) Analysis of Pol II association with ADH1 in the cet1(Ts) (cet1-438) mutant (YSB717) and wild-type (YSB540) strains at the nonpermissive (left) and permissive (right) temperatures. Immunoprecipitations were performed using mouse monoclonal antibody 8WG16 (Covance) against the CTD of Rpb1. The ratio of immunoprecipitate to input in the autoradiogram was measured. The maximum ratio was set to 100, and other ratios were normalized with respect to 100. The normalized ratio (represented as normalized occupancy) is plotted in the form of a histogram. (Bottom) Schematic diagram showing the locations of different primer pairs at ADH1 for ChIP analysis. The numbers are presented with respect to the position of the first nucleotide of the initiation codon (+1). (B) Analysis of FACT (Spt16-Myc) association with ADH1 in the presence and absence of the NTD of Cet1. (Top) Yeast cells were grown in YPD medium at 30°C to an OD₆₀₀ of 1.0 prior to formaldehyde-based in vivo cross-linking. Immunoprecipitated DNA was analyzed by PCR using primer pairs targeted to different locations in ADH1. (Bottom) Schematic diagram of the different domains of Cet1. (C) Western blot analysis of Spt16 in the wild-type and cet1Δ204 strains. (D) ChIP analysis of FACT at ADH1 in the cet1(Ts) (cet1-448) mutant and its isogenic wild-type equivalent. Both wild-type and mutant cells were grown in YPD at 30°C to an OD₆₀₀ of 0.85 and then switched to 37°C for 90 min prior to cross-linking. (E) ChIP analysis of FACT at ADH1 in the cet1(Ts) (cet1-438) mutant and its isogenic wild-type equivalent. Yeast cells were grown as for panel D. (F) ChIP analysis of Spt16 at GAL1 in the wild-type and cet1Δ204 strains in galactose (Gal)- or raffinose (Raf)-containing growth medium. The error bars indicate SD.

FIG 2

Cet1 interacts with FACT in facilitating transcription. (A) Formaldehyde-based in vivo cross-linking and co-IP assay. Schematic outline of the experimental strategy (top) and IP (bottom). WB, Western blotting. (B) (Bottom) Analysis of interaction of Pob3 with Spt16. (Top) Schematic outline of the experimental strategy to analyze protein-protein interactions in vitro. (C and D) Analysis of interaction of Cet1 with FACT. (E) Cet1 does not interact with FACT in the absence of its NTD. (F) ChIP analysis of Rpb1 association with the coding sequence of ADH1 in the wild-type and spt16(Ts) mutant strains at the nonpermissive temperature. (G) RT-PCR analysis of ADH1 mRNA levels in the wild-type and spt16(Ts) mutant strains at the nonpermissive temperature. (H) Growth analysis of wild-type and cet1Δ204 strains in liquid medium (YPD). (I) Growth analysis of wild-type and cet1Δ204 strains in solid medium with or without 6-AU. The error bars indicate SD.

FIG 3

The NTD of Cet1 targets FACT to PMA1, PGK1, and PYK1. (A) Analysis of FACT (Spt16-Myc) association with PMA1 in the cet1(Ts) (cet1-438) mutant and its wild-type equivalent. (B) Analysis of FACT (Spt16-Myc) association with PMA1 in the presence and absence of the NTD of Cet1. (C) Analysis of FACT (Spt16-Myc) association with PMA1 in the cet1(Ts) (cet1-448) mutant and its wild-type equivalent. (D) ChIP analysis of FACT at the 5′ ORFs of PGK1 and PYK1 in the presence and absence of the NTD of Cet1. (E) ChIP analysis of FACT at the 5′ ORFs of PGK1 and PYK1 in the cet1(Ts) (cet1-448) mutant and its wild-type equivalent. (F) ChIP analysis of FACT at the 5′ ORFs of PGK1 and PYK1 in the cet1(Ts) (cet1-438) mutant and its wild-type equivalent. (G) RT-PCR analysis of PGK1, PYK1, and PMA1 mRNAs in the spt16(Ts) mutant and its wild-type equivalent. (H) ChIP analysis of Rpb1 at the PGK1, PYK1, and PMA1 coding sequences in the spt16(Ts) mutant and its wild-type equivalent. The error bars indicate SD.

FIG 4

Analysis of Cet1's NTD function in regulation of the levels of TBP, Pol II, and histone H3 at GAL1. (A and B) ChIP analysis of TBP and Pol II at the GAL1 core promoter after switching on GAL1 transcription in galactose-containing growth medium. (C to E) ChIP analysis of Rpb1 association with the promoter-proximal site and downstream coding regions (i.e., ORF1 and ORF2) of GAL1 after switching on GAL1 transcription. (F to I) Analysis of the Cet1 NTD's role in evicting histone H3 from GAL1 after switching on transcription. (J to M) Analysis of the Cet1 NTD's role in depositing histone H3 at GAL1 after switching off GAL1 transcription in dextrose-containing growth medium. The error bars indicate SD.

FIG 5

Cet1's NTD recruits the Paf1C via FACT to promote transcription. (A and B) Cet1's NTD enhances recruitment of the Paf1C to the promoter-proximal sites (or 5′ ORFs) of ADH1, PGK1, PMA1, and PYK1. (C) Analysis of Paf1 and actin levels in strains expressing PAF1 under the control of the GAL1 promoter (P_GAL1-PAF1-HA) after switching the carbon source in the growth medium from galactose (when total OD₆₀₀ was 0.6) to dextrose. (D and E) Analysis of Rpb1 association with the ADH1, PGK1, PYK1, and PMA1 coding sequences in the strains expressing PAF1 under the control of the GAL1 promoter (P_GAL1-PAF1-HA) after switching the carbon source in the growth medium from galactose to dextrose. (F) RT-PCR analysis of ADH1, PGK1, PYK1, and PMA1 mRNAs in strains expressing PAF1 under the control of the GAL1 promoter (P_GAL1-PAF1-HA) after switching the carbon source in the growth medium from galactose to dextrose. (G) Co-IP analysis between Cet1 and Paf1 without in vivo cross-linking by formaldehyde. (H) Analysis of Pob3 and Paf1 levels in strains expressing HA-tagged Pob3 under the control of the GAL1 promoter (P_GAL1-POB3-HA) after switching the carbon source in the growth medium from galactose to dextrose. (I) FACT enhances the recruitment of Paf1C to the promoter-proximal sites (or 5′ ORFs) of ADH1, PGK1, PMA1, and PYK1. (J and K) Pob3 is required for recruitment of Spt16 to the active gene. (J) Levels of Pob3 and Spt16 (by Western blot analysis) in the strain expressing HA-tagged Pob3 under the control of the GAL1 promoter (P_GAL1-POB3-HA) after switching the carbon source in the growth medium from galactose to dextrose. (K) ChIP analysis of Spt16 and Pob3 at the ADH1 coding sequence following shutdown of Pob3 expression in dextrose-containing growth medium for 4 h. The error bars indicate SD.

FIG 6

Cet1's NTD does not regulate recruitment of Spt5 or Spt6 to the promoter-proximal sites (or 5′ ORFs) of ADH1, PGK1, PYK1, and PMA1. (A and B) ChIP analysis of Spt6 at the 5′ ORFs of ADH1, PGK1, PYK1, and PMA1. (C and D) ChIP analysis of Spt5 at the 5′ ORFs of ADH1, PGK1, PYK1, and PMA1. (E and F) Analysis of Myc-tagged Spt16 and HA-tagged Paf1 association with the ADH1, PGK1, PYK1, and PMA1 coding sequences in the strain expressing HA-tagged Paf1 under the control of the GAL1 promoter (P_GAL1-PAF1-HA) and Myc-tagged Spt16 after switching the carbon source in the growth medium from galactose to dextrose for 4 h. The error bars indicate SD.

FIG 7

Schematic diagram showing how Cet1's NTD impairs promoter-proximal accumulation/pausing of Pol II. Cet1, which associates with Pol II via Ceg1 (35) following PIC formation and transcriptional initiation (61, 62), interacts with FACT via the NTD (Fig. 2A to E) and facilitates its targeting to the active gene in an mRNA-capping activity-independent manner (Fig. 1B to F and 3A to F). In addition, FACT also interacts with histones, and such interaction is responsible for chromatin association with FACT (15–17, 22, 23). Thus, Cet1's NTD and histones synergistically target FACT to the active gene. In the absence of Cet1's NTD, FACT targeting to the active gene is significantly decreased (Fig. 1B and 3B and D). Decreased association of FACT reduces Paf1C recruitment (Fig. 5H to K). Thus, Cet1's NTD facilitates targeting of the Paf1C via FACT (Fig. 5H to K) but not Spt5 and Spt6 (Fig. 6A to D). Paf1 promotes transcriptional elongation (33, 40) (Fig. 5D to F). Hence, loss of Cet1's NTD reduces recruitment of the Paf1C via FACT, leading to increased accumulation/pausing of Pol II at the promoter-proximal site. Like FACT, Bre1 (an E3 ubiquitin ligase for histone H2B ubiquitylation) is also involved in Paf1 recruitment, independently of Pol II (63–65). The double-headed arrows indicate interactions.