Gene loops enhance transcriptional directionality

Abstract

Eukaryotic genomes are extensively transcribed, forming both messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs). ncRNAs made by RNA polymerase II often initiate from bidirectional promoters (nucleosome-depleted chromatin) that synthesize mRNA and ncRNA in opposite directions. We demonstrate that, by adopting a gene-loop conformation, actively transcribed mRNA encoding genes restrict divergent transcription of ncRNAs. Because gene-loop formation depends on a protein factor (Ssu72) that coassociates with both the promoter and the terminator, the inactivation of Ssu72 leads to increased synthesis of promoter-associated divergent ncRNAs, referred to as Ssu72-restricted transcripts (SRTs). Similarly, inactivation of individual gene loops by gene mutation enhances SRT synthesis. We demonstrate that gene-loop conformation enforces transcriptional directionality on otherwise bidirectional promoters.

Figure 1. Ssu72 inactivation abrogates FMP27 gene loop and enhances antisense transcription

A) Graphical representation of 3C interaction levels in ssu72-2 vs WT across FMP27. Red stars show significant 3C interaction. Positions of 3C primers are indicated as are RT-qPCR amplicons. For 3C analysis primer 1 (anchor) was combined sequentially with downstream primers 2-7. Error bars represent SEM. B) RT-qPCR analysis of FMP27 mRNA and ncRNA in ssu72-2 vs WT. Error bar represent SEM. C) Pol II profile (ChIP-seq) across FMP27 promoter region (12) in ssu72-2 vs WT.

Figure 2. Ssu72 inactivation leads to novel ncRNA transcription (SRTs)

A) I-IV. Genomic transcription across 28 kb of chromosomes 2 and 4 (x axis) for the Watson (W, top) and the Crick (C, bottom) strands. For the whole genome see http://steinmetzlab.embl.de/proudfoot_lab/index.html. Normalized signal intensities shown for profiled samples (y axis). Triplicate data shown for WT (1–3), ssu72-2 (1–3), Δrrp6 (1-3) and ssu72-2Δrrp6 (1–3) strains. Red vertical lines represent inferred transcript boundaries. Nucleosome positions (green tracks, darker for more significant scores; (25) and genome annotations are shown in the centre: annotated ORFs (blue boxes), ncRNAs (orange boxes), and TSS (arrows). (I) ncRNA0151, (II) ncRNA0397, (III) ncRNA0524 and (IV) ncRNA4353 represent Ssu72 repressed transcripts (SRTs) that are promoter-associated. B) Distribution of relative distances of 605 SRTs (upper panel) versus 1982 CUTs (lower panel) to their nearest ORF TSS (red line). Dashed lines (350 bp) indicate cut-off position used to define intergenic cryptic transcription sharing ORF promoters. C) Distributions of gene expression levels are shown for downstream (down) and upstream (up) ORFs of tandem genes (3 categories: downstream promoter pSRT (blue) or pCUT (purple) or no pncRNA (green). Downstream ORFs with pncRNAs are significantly higher expressed than those without (p=0.001 [pSRT] and p<2.2e-16 [pCUTs]) No significant association was found for upstream ORFs. D) Pol II profiles for ssu72-2 (red) and wild type (blue) around ORF promoter in tandem genes more than 400bp apart. Solid lines indicate median Pol II-occupancy and shaded areas 25-75 percentiles. Pol II occupancy increases upstream of TSS in ssu72-2.

Figure 3. pSRTs initiate from bidirectional promoters

A) I. Histone H4 acetylation (as a ratio with H3) compared over intergenic region between ORF TSS and divergent pSRT (blue) or pCUT (purple) TSS in wild type strains. Intergenic regions of pCUTs show higher H4 acetylation levels than those of pSRTs. II. ChIP analysis across the promoter regions of the indicated loci with WT and ssu72-2 derived chromatin using anti-H4ac. Ssu72 inactivation caused H4 acetylation increase at all 4 loci. Telomeric region (TELV1) used as negative control. Error bars represent SEM. B) Metagene analysis of Δrco1Δrrp6 vs Δrrp6 (green) and ssu72-2Δrrp6 vs Δrrp6 (blue) differential expression levels for all antisense ncRNAs that initiate between tandem genes in relative position to upstream gene TTS and the downstream gene TSS.

Figure 4. Gene loop disruption by PAS mutation enhances divergent transcription

A-C) I. Graphical representation of 3C interaction analyses for CYC1 plasmid (A), MSN5 (B) and mammalian β-globin gene construct (C) as in Fig. 1A. II, RT-qPCR analyses of mRNA versus pncRNAs. AIII. Northern blot for CYC1 transcripts. Note that the CYC1 mRNA is smaller with RCS replacement due to lack of pA tail. Also UTR1 downstream of CYC1 is inactive due to promoter deletion, denoted by crossed arrow. Error bars represent SEM. D) Gene loop defines transcription unit and promotes transcription directionality. Loss of gene loop in ssu72-2 or with PAS mutation increases antisense pncRNA transcription. Gene loops involving Ssu72 (green oval) may act to maintain nucleosome (yellow bubble; ac denotes H4Ac) positions, preventing association of a second PIC (red) leading to divergent transcription initiation.