Genomic study of RNA polymerase II and III SNAPc-bound promoters reveals a gene transcribed by both enzymes and a broad use of common activators

Abstract

SNAP(c) is one of a few basal transcription factors used by both RNA polymerase (pol) II and pol III. To define the set of active SNAP(c)-dependent promoters in human cells, we have localized genome-wide four SNAP(c) subunits, GTF2B (TFIIB), BRF2, pol II, and pol III. Among some seventy loci occupied by SNAP(c) and other factors, including pol II snRNA genes, pol III genes with type 3 promoters, and a few un-annotated loci, most are primarily occupied by either pol II and GTF2B, or pol III and BRF2. A notable exception is the RPPH1 gene, which is occupied by significant amounts of both polymerases. We show that the large majority of SNAP(c)-dependent promoters recruit POU2F1 and/or ZNF143 on their enhancer region, and a subset also recruits GABP, a factor newly implicated in SNAP(c)-dependent transcription. These activators associate with pol II and III promoters in G1 slightly before the polymerase, and ZNF143 is required for efficient transcription initiation complex assembly. The results characterize a set of genes with unique properties and establish that polymerase specificity is not absolute in vivo.

Figure 1. Pol II and III occupancy of snRNA genes.

(A) Bar graph showing POLR2B (dark blue), GTF2B (light blue), POLR3D (red), and BRF2 (orange) ChIP-seq scores (y axis) on SNAP_c-occupied genes and the few snRNA genes devoid of SNAP_c (x axis). Genes are ordered by decreasing POLR2B scores for the pol II and RPPH1 genes followed by increasing POLR3D scores for the pol III genes. (B) UCSC browser view of RPPH1 gene showing POLR2B, POLR3D, GTF2B, and BRF2 occupancy. Y axis: tag counts. (C) POLR2B (light grey) or POLR3D (dark grey) occupancy in cells not treated or treated with 50 µg/ml α-amanitin for 2 or 6 h, as indicated on the x axis. Upper two panels: results are shown as % of input. Lower two panels: POLR2B and POLR3D occupancy without α-amanitin was set at 1.

Figure 2. SNAP_c subunits occupancy and proximal promoter motifs.

(A) UCSC browser views of a pol III (tRNAU1) and a pol II (RNU4atac) gene showing occupancy by the factors indicated on the left. The chromosome coordinates are shown on top, the genes present in the region and their orientation at bottom. The y axis shows tag counts. (B) Two examples of non-annotated genomic regions showing occupancy by SNAP_c subunits, GTF2B, and POLR2B. (C) Box plot of BRF2, GTF2B, and SNAP_c subunit positions. For each gene, the position of the peak summit for each SNAP_c subunit relative to the TSS (set at 0) was determined. A median position (black bars in boxes, number in brackets on the y axis) was calculated. For the pol II genes, only the upper two tertiles of each SNAP_c subunit and GTF2B scores were included. The position for each gene is represented by a circle. (D) LOGOs of PSE and TATA box generated by WebLogo with the motifs identified with MEME (alignments in Figures S4 and S5). The top panel shows the PSE LOGO for pol II snRNA genes, the middle panel shows the PSE LOGO for pol III genes, and the bottom panel shows the TATA box LOGO for pol III genes.

Figure 3. RNA pol II and III occupancy within 3′ flanking regions.

(A) 3′ box LOGO generated by WebLogo with the motifs found within 100 bp downstream of the RNA coding sequence of Pol II genes (see alignment in Figure S6). The bracketed positions 5–7 of the LOGO correspond to the positions that are sometimes gaps in the alignment of Figure S6. (B) Graphical representation of POLR2B (in blue) and POLR3D (in red) tag accumulation past the 3′ end of the RNA-coding region of pol II and pol III genes, respectively. X axis: position around the 3′ end of the RNA coding regions (set at 0). Y axes: tag counts for POLR2B on the left and POLR3D on the right.

Figure 4. Activator occupancy and distal promoter motifs.

(A) UCSC browser view of three pol II (RNU4atac, U1-like-5, and Unknown-6) and one pol III (tRNAU1) gene showing occupancy by the factors indicated on the right of each panel. The chromosome coordinates are shown on top, the genes present in the region and their orientation at bottom. The y axis shows tag counts. (B) Promoter region (−400 to +1) of the four genes depicted in (A) with the positions of the GABPA (GA-motif), ZNF143 (SBS), and POU2F1 (octamer) binding sites found by MEME or MAST indicated. The positions of the PSE and TATA box are also shown, and the promoters were aligned according to the PSE position. The crossed-out motifs have either no corresponding peak of occupancy or are not the closest to the peak summit. The orientation of each motif is indicated with an arrow. (C) LOGOs of the ZNF143, POU2F1 (octamer) and GABP binding motifs generated by WebLogo with the motifs located closest to the corresponding factor peak summits (see alignments in Figures S9, S10, S11).

Figure 5. RNU1, RNU2, and RNU6 transcription and factor recruitment during mitosis to G1 phase transition.

(A) Time course of U1 and U6 reporter transcript and U2 and pre-U2 snRNA accumulation after mitosis release. The 5.8S RNA served as an internal control. The time after mitosis release is indicated above each panel. (B) Time course analysis of transcription factor recruitment on various promoter regions. ChIPs were performed at the times indicated (x axis) after mitosis release with antibodies directed against the factors indicated on top of each panel, and analyzed by real time PCR. The analyzed regions are indicated at the upper right of each panel. The control region (Ctrl) is 2 kb upstream of RNU1. The results are expressed relative to input DNA. Two sets of RNU1 primers were used: set U1A recognizes U1-1, U1-2, U1-3, U1-8, U1-like-3 loci and was used in the top panel; set U1B recognizes U1-2 and U1-3 loci and was used in the 3 lower panels. The RNU2 primers are specific for the RNU2 cluster in chr17_unknown, and the RNU6 primers for the U6-1 locus. (C) Real time PCR analysis of RNU1 (top panel, U1A primer set for POLR2B, GTF2B, SNAPC1 and POLR3D ChIPs; and U1B primer set for the other ChIPs) and RNU6 (bottom panel) promoters pulled down after ChIP with antibodies against the factors indicated below the panels either at mitosis (1 h after release) or in mid-G1 (7 h after release). The results are expressed relative to mitosis values, which were set at 1 for each factor. Means and error bars were calculated over triplicate PCR analyses. Each experiment was performed at least twice.

Figure 6. Depletion of endogenous ZNF143 reduces transcription factor recruitment on the U1 promoter in mid-G1.

(A) Immunoblot showing ZNF143 and Tubulin (control) levels during mitosis and mid-G1 phase after treatment with siRNA against Luciferase (Luc, control siRNA) or ZNF143. (B) Real time PCR analysis of RNU1 promoter pulled down after ChIP with antibodies against the factors indicated below the panel either after treatment of the cells with siRNA against Luciferase (siLuc, control siRNA) or siRNA against ZNF143 (siZNF143). The values obtained with the siZNF143 treatment are shown relative to those obtained with the siLuc treatment, which were set at 100%. Means and error bars were calculated over triplicate PCR analyses. Each experiment was performed at least twice. The U1A primer set was used for the POLR2B, GTF2B and SNAPC1 ChIPs, the U1B primer set for the other ChIPs.