Cap completion and C-terminal repeat domain kinase recruitment underlie the initiation-elongation transition of RNA polymerase II

Abstract

After transcription initiation, RNA polymerase (Pol) II escapes from the promoter and recruits elongation factors. The molecular basis for the initiation-elongation factor exchange during this transition remains poorly understood. Here, we used chromatin immunoprecipitation (ChIP) to elucidate the initiation-elongation transition of Pol II in the budding yeast Saccharomyces cerevisiae. We show that the early Pol II elongation factor Spt5 contributes to stable recruitment of the mRNA capping enzymes Cet1, Ceg1, and Abd1. Genome-wide occupancy for Cet1 and Ceg1 is restricted to the transcription start site (TSS), whereas occupancy for Abd1 peaks at ~110 nucleotides downstream, and occupancy for the cap-binding complex (CBC) rises subsequently. Abd1 and CBC are important for recruitment of the kinases Ctk1 and Bur1, which promote elongation and capping enzyme release. These results suggest that cap completion stimulates productive Pol II elongation.

Fig 1

The Spt5 CTR is involved in recruitment of capping enzymes. ChIP-qPCR analysis was performed to monitor capping enzyme and Pol II recruitment at three different gene regions of ADH1: TSS (5′), coding (open reading frame [ORF]), and terminator region (3′). Occupancies were calculated as fold enrichments over an ORF-free untranscribed region on chromosome V and are indicated on the y axes (see Materials and Methods). Error bars show standard deviations from three independent experiments of biological replicates, and the asterisk indicates factor occupancies that are significantly different (P < 0.05) between the wild-type and mutant conditions using Student's t test. (A) Cet1, Ceg1, Abd1, and Pol II occupancies for the wild type compared to Spt5ΔCTR cells are shown. (B) Abd1 and Pol II occupancies for the wild type compared to bur2Δ cells are shown. Abd1 fold enrichments relative to Pol II (Abd1/Pol II) were calculated by dividing Abd1 occupancies by Pol II occupancies. (C) Occupancies as described in panel B for Cet1. (D) Cet1/Pol II and Abd1/Pol II ChIP signals for the ORF region of ADH1 relative to the 5′ end.

Fig 2

Genome-wide occupancy profiling of the capping machinery. (A) Gene-averaged ChIP-chip profiles for capping enzymes and Pol II phosphorylated at serine 5 (S5P) residues of the CTD (upper panel) and for initiation factor TFIIB, CBC subunit CBP20, and elongation factors Bur1, Ctk1, and Spt5 (lower panel). Profiles in the lower panel were described previously (21) except that for CBP20. Occupancy profiles taken from the quality-filtered total gene set (1,140 genes) were cut around the TSS (250 bp upstream to 650 bp downstream; only genes of >680 bp were considered) and averaged using a 5% trimmed mean at each genomic position. Profiles for gene length classes are similar (see Fig. S2A in the supplemental material). ChIP-chip signal intensity is expressed as log₂ IP/input. For details refer to Materials and Methods. Dashed gray lines mark the peak positions of the averaged ChIP-chip profiles. (B) Gene-averaged ChIP-chip profiles as described for panel A but for medium-length genes (1,238 ± 300 bp; n = 339). Genes were aligned at their TSS and pA sites (47), scaled to median length, and averaged using a 5% trimmed mean at each genomic position. Profiles of other gene length classes are similar (see Fig. S2B). For details refer to Materials and Methods. (C) Gene-averaged ChIP-chip profiles as described in panel A for Abd1 in wild-type compared to Spt5ΔCTR cells (upper panel) and for Bur1 (lower panel). The dashed gray line marks the position where full recruitment of Bur1 is reached ∼250 bp downstream of the TSS.

Fig 3

Capping enzyme profile peak distances are independent of expression level and gene length. (A) Gene-averaged profiles for Ceg1 and Abd1 for genes in three expression level classes (for Cet1, see Fig. S2E in the supplemental material). The quality-filtered set of medium length genes (Fig. 2B) was partitioned into three groups: low (25% to 50% quantile), medium (50% to 75% quantile), and high (>75% quantile) expression levels (see Materials and Methods). (B) Box plots showing the gene-wise variation of peak distances between Cet1 and Abd1 for short (725 ± 213 bp; n = 266), medium (1,238 ± 300 bp; n = 339), and long (2,217 ± 679 bp; n = 299) genes. Mean distances are indicated by filled black circles. The distributions of peak distances between Ceg1 and Abd1 are similar (data not shown). For details see Materials and Methods.

Fig 4

CBP20 is involved in recruitment of kinases Bur1 and Ctk1. ChIP-qPCR analysis was performed to monitor changes in elongation factor and Pol II recruitment upon CBP20 deletion. Changes in levels of Ser-2 phosphorylated Pol II CTD (S2P) were also analyzed. Three gene regions of ADH1 (A) (Fig. 1) and PMA1 (B) (see Fig. S4B in the supplemental material) were investigated. Occupancies for wild-type (black bars) compared to cbp20Δ (gray bars) cells are shown. Occupancies were calculated as fold enrichments over an ORF-free untranscribed region on chromosome V and are indicated on the y axes (see Materials and Methods). Error bars show standard deviations from at least three independent experiments of biological replicates, and the asterisk indicates factor occupancies that are significantly different (P < 0.05) between the wild-type and mutant conditions using Student's t test.

Fig 5

Abd1 contributes to recruitment of the CBC, Bur1, and Ctk1. ChIP-qPCR analysis was performed to monitor changes in Bur1, Ctk1, CBP20, and Pol II recruitment upon Abd1 nuclear depletion using the anchor-away technique. Changes in levels of Ser-2-phosphorylated Pol II CTD (S2P) were also analyzed. Three gene regions of ADH1 (A) (Fig. 1) and PMA1 (B) (see Fig. S4B in the supplemental material) were investigated. Occupancies in the Abd1 anchor-away strain that was left untreated (black bars) or treated with rapamycin for 60 min (gray bars) are indicated. Occupancies were calculated as fold enrichments over an ORF-free untranscribed region on chromosome V and are indicated on the y axes (see Materials and Methods). Error bars show standard deviations from at least three independent experiments of biological replicates, and the asterisk indicates factor occupancies that are significantly different (P < 0.05) between the treated and untreated conditions using a paired-sample Student's t test.

Fig 6

Correlation-based network of occupancy profiles reflects different phases of the transcription cycle. A correlation analysis of genome-wide ChIP-chip profiles reported here and elsewhere (21, 23, 51) was performed. Pairwise Pearson correlation coefficients were calculated between concatenated gene profiles ranging each from the TSS minus 250 bp to the pA site plus 250 bp (see also Table S1 and Fig. S3 in the supplemental material) and were provided as a similarity metric to calculate a two-dimensional network using the GraphViz's Neato algorithm (see Materials and Methods). Solid lines represent known direct interactions between factors. Dashed lines represent known direct interactions between factors and the phosphorylated CTD of Pol II. Essential and nonessential factors are represented as boxes and circles, respectively. Pol II phosphoisoforms are represented as hexagons.

Fig 7

Model for the Pol II initiation-elongation transition. Pol II and its CTD (black) transcribe DNA (horizontal black line) from left to right to produce capped mRNA (gray). PIC, preinitiation complex; EC, elongation complex; IIA/B/D/E/F/H, initiation factors; S2/5P, phosphorylated serine 2 and 5 residues; m7, N7 methyl group of the cap; Paf1C, Paf1 complex. For details, see the text.