G4 motifs correlate with promoter-proximal transcriptional pausing in human genes

Abstract

The RNA Pol II transcription complex pauses just downstream of the promoter in a significant fraction of human genes. The local features of genomic structure that contribute to pausing have not been defined. Here, we show that genes that pause are more G-rich within the region flanking the transcription start site (TSS) than RefSeq genes or non-paused genes. We show that enrichment of binding motifs for common transcription factors, such as SP1, may account for G-richness upstream but not downstream of the TSS. We further show that pausing correlates with the presence of a GrIn1 element, an element bearing one or more G4 motifs at the 5'-end of the first intron, on the non-template DNA strand. These results suggest potential roles for dynamic G4 DNA and G4 RNA structures in cis-regulation of pausing, and thus genome-wide regulation of gene expression, in human cells.

Figure 1.

Inverse correlation between Pol II binding and G-richness at the TSS. Graph of the frequency Pol II binding sites (1), CpG dinucleotides, and the frequency of G-richness in the interval −1000 to +1000 around the TSS. G-richness was defined as the frequency within each set of genes of 100 nt sequences containing the G4 DNA signature motif, G_≥3N_xG_≥3N_xG_≥3N_xG_≥3 (4).

Figure 2.

G4 motifs are enriched near promoters of paused genes. (A) Graph of the frequency of G-richness in genes defined as paused from the NCI-60 database in the interval −1000 to +1000 around the TSS. (B) Graph of the frequency of G-richness in genes in which Pol II is stably associated with the TSS in the absence of gene expression in the interval −1000 to +1000 around the TSS. This data set derives from analysis of primary resting human CD4⁺ T cells (1), and corresponds to the same data set for which genome-wide analysis Pol II position is presented in Figure 1. (C) Graph of the frequency of G-richness in the interval −1000 to +1000 around the TSS in genes carrying bivalent chromatin marks H3K4me3 and H3K27me3, as determined by analysis of primary resting human CD4⁺ T cells (1).

Figure 3.

Strand-biased G-richness downstream of the TSS at paused genes. Graph of the frequency of G-richness of the non-template (dark lines) and template (pale lines) strands in the interval −1000 to +1000 around the TSS for genes in the NCI-60 database classified as paused (left) or non-paused (center), and RefSeq genes (right).

Figure 4.

Transcriptional regulatory motifs do not account for G-richness near TSS of paused genes. Graph of the frequency of G-richness of non-template (dark lines) and template (pale lines) strands in the interval −1000 to +1000 around the TSS for paused genes (left), non-paused genes (centre) and all human RefSeq genes (right), with the following motifs masked: (A) SP1 motifs. (B) CpG motifs. (C) CpG, SP1, MAZ, KLF, EKLF, EGR-1 and AP-2 motifs.

Figure 5.

GrIn1 elements correlate with pausing. (A) Graph of the frequency of non-template strand G-richness within 1 kb downstream of the TSS for paused, non-paused genes and RefSeq genes (top). (B) Graph of the frequency of non-template strand G-richness as in Panel A, but with hnRNP A (UAGGGU/A) and hnRNP H (GGGA) motifs masked. (C) Graph of the frequency of non-template strand G-richness as in Panel B, with CpG motifs, hnRNP A (UAGGGU/A) and hnRNP H (GGGA) motifs masked.

Figure 6.

Regulation of transcriptional pausing at G4 motifs. Model of dynamic nucleic acid structures that may contribute to pausing upon transcription of a G-rich region. Mechanisms that contribute to pausing may include: (i) G4 DNA formed behind an advancing polymerase may be recognized by factors that promote pausing, (ii) G4 DNA structure formed in a ‘pioneering’ round of transcription may serve as a roadblock during the next round of transcription, (iii) a G4 RNA structure in the nascent transcript may communicate a pause to the transcription complex, as occurs in prokaryotes and (iv) a stable co-transcriptional RNA/DNA hybrid may promote pausing, via signals transmitted through the RNA processing apparatus.