A positioned +1 nucleosome enhances promoter-proximal pausing

Abstract

Chromatin distribution is not uniform along the human genome. In most genes there is a promoter-associated nucleosome free region (NFR) followed by an array of nucleosomes towards the gene body in which the first (+1) nucleosome is strongly positioned. The function of this characteristic chromatin distribution in transcription is not fully understood. Here we show in vivo that the +1 nucleosome plays a role in modulating RNA polymerase II (RNAPII) promoter-proximal pausing. When a +1 nucleosome is strongly positioned, elongating RNAPII has a tendency to stall at the promoter-proximal region, recruits more negative elongation factor (NELF) and produces less mRNA. The nucleosome-induced pause favors pre-mRNA quality control by promoting the addition of the cap to the nascent RNA. Moreover, the uncapped RNAs produced in the absence of a positioned nucleosome are degraded by the 5'-3' exonuclease XRN2. Interestingly, reducing the levels of the chromatin remodeler ISWI factor SNF2H decreases +1 nucleosome positioning and increases RNAPII pause release. This work demonstrates a function for +1 nucleosome in regulation of transcription elongation, pre-mRNA processing and gene expression.

Figure 1.

Nucleosome mapping of constructs with different integrated sequences downstream the pause site of the mouse c-Myc. (A) Schematic representation of promoter and exon 1 of the mouse c-Myc gene showing the position in which 601, 5S or control (C) sequences were inserted. 601 and 5S were cloned in two different orientations. Distances (bp) of pause region and insertions from the transcription start site (TSS) are indicated below the gene. (B) Micrococcal nuclease mapping of promoter and 5′ region of the mouse c-Myc gene in the indicated cell lines. Cells were processed either for nuclei isolation or for DNA extraction and then samples were subjected to MNase digestion. Values represented correspond to the ratio between qPCR signals from chromatin samples and signals from naked DNA samples. Each value is located regarding the midpoint of the amplicon with respect to the TSS. Lines under the graphs denote locations of amplicons for qPCR analysis. c-Myc-C specific amplicons are represented as dotted lines and the rest are represented as black lines. Primer sequences are listed in Supplementary Table S1. Average and standard deviations from three independent experiments are displayed.

Figure 2.

Increased promoter-proximal RNAPII stalling under a strong +1 nucleosome positioning. (A) Schematic drawing of locations of amplicons used for ChIP and RT-qPCR experiments. This map is the same as the one in Figure 1A but at a different scale. The name of the amplicon indicates its midpoint position from the TSS (P2). Primer sequences are listed in Supplementary Table S1. (B and C) RNAPII ChIP analysis of mouse c-Myc in the indicated cell lines. Total RPB1 or Ser5 phosphorylated form of RPB1 were immunoprecipitated using N-20 (B) or 3E8 (C) antibodies, respectively. ChIP values were normalized to the values at −947 position. (D) c-Myc mRNA levels measured by RT-qPCR using the primers ChIP c-Myc-2007 (Supplementary Table S1). Values were normalized to GAPDH amplification and c-Myc-C signal was set to 1. c-Myc-wt mRNA levels are shown in a different scale. (B–D). Average and standard deviations from three independent experiments are displayed. P-values (student's t-test) for the different constructs comparing to c-Myc-C are indicated.

Figure 3.

Increased promoter-proximally paused polymerases under a strongly positioned +1 nucleosome. Nuclear run-on assay based on the incorporation of biotin-16-UTP in nascent transcripts of the mouse c-Myc gene in the indicated cell lines is represented. Each position indicates the midpoint of each amplicon used with respect to the TSS. Values are normalized to the ribosomal RNA 28S run-on signal and background from a control sample treated with UTP (without biotin) was subtracted. Primer sequences are listed in Supplementary Table S1. Average and standard deviations from three independent experiments are displayed.

Figure 4.

NELF-dependent promoter-proximal pausing induced by +1 nucleosome positioning. (A) RNAPII ChIP analysis of c-Myc-C (upper panel) and c-Myc-601F (bottom panel) genes under NELF-E knockdown conditions. Total RPB1 ChIP was performed as described in Figure 2B. (B) c-Myc mRNA levels after NELF-E siRNA treatment analyzed by RT-qPCR. Experiments and data analysis were carried out as described in Figure 2D. (C) NELF-E ChIP assay in c-Myc-601F and c-Myc-C genes as described in Figure 2B. Primer sequences are listed in Supplementary Table S1. (A–C) Average and standard deviations from three independent experiments are displayed (*P < 0.05; **P < 0.01, compared to control, by student's t-test).

Figure 5.

Increased mRNA capping in the c-Myc-601F gene. (A) K-121 immunoselection of capped c-Myc RNA in the indicated cell lines. For each IP reaction 2 μg of total RNA were used. c-Myc RNA levels were analyzed by RT-qPCR prior and subsequent to immunoselection. Primer pairs c-Myc-532, c-Myc-2007, GAPDH and 28S (Supplementary Table S1) were utilyzed. IP efficiency was the result of the ratio between the data obtained from immunoselected c-Myc or GAPDH RNAs and the total levels of each RNA prior immunoprecipitation. c-Myc values were normalized to GAPDH IP efficiency in each sample. (B) c-Myc pre-mRNA (upper panel) and mRNA (lower panel) levels in the indicated conditions were determined by RT-qPCR as described in Figure 2D. (A and B) Average signals and standard deviations from three independent experiments are displayed (*P < 0.05; **P < 0.01, between the indicated samples by student's t-test). Primer sequences are listed in Supplementary Table S1.

Figure 6.

Decreased +1 nucleosome positioning under SNF2H shortage. (A) c-Myc-C and c-Myc-601F mRNA levels in the indicated conditions analyzed by RT-qPCR. Values were normalized as in Figure 2D and c-Myc-C was set to 1. (B) RNAPII ChIP analysis of c-Myc-C (left panel) and c-Myc-601F (right panel) constructs under SNF2H knockdown conditions. Total RPB1 ChIP was performed as described in Figure 2B by using N-20 antibody. (C) Micrococcal nuclease mapping of promoter and 5′ region of the c-Myc-C (left panel) and c-Myc-601F (right panel) genes. Data were analyzed as described in Figure 1B. siControl value at position c-Myc-601F +203 was set to 1. (A–C) Average signals and standard deviations from three independent experiments are displayed (*P < 0.05; **P < 0.01, between the indicated samples by student's t-test). Primer sequences are listed in Supplementary Table S1.