Enhancers predominantly regulate gene expression in vivo via transcription initiation

Background

Transcription can be thought of as a series of biochemical steps. The promoter first must be made competent for RNA polymerase II (Pol II) binding, forming a pre-initiation complex. The complex pauses transiently 30-60 nucleotides downstream of the transcription start site (TSS), and post-translational modifications to Pol II subsequently results in pause release, leading to productive elongation of the transcript. Using the ratio of Pol II throughout the gene body to Pol II at the promoter proximal region as a proxy for levels of pausing (pausing index), it has been suggested that pause-release is an important step in gene regulation. While some or all of these steps are thought to be regulated, the relative contributions of each step to gene regulation or the rate-limiting step in transcription remains unclear. In addition, since enhancers are thought to activate transcription, it is important to understand which step enhancers regulate. To study these questions, the authors developed short capped RNA sequencing (scaRNA-seq) to simultaneously measure the TSS and TPS (transcription pause site) of genes.

Key findings

The primary claim of the preprint is that enhancers regulate gene expression primarily through transcription initiation, which can be either RNA polymerase II recruitment or simply initiating production of a transcript. To simultaneously assay pausing and initiation, the authors modified the Start-seq (Nechaev et al., 2010; Henriques et al., 2013) assay to develop scaRNA-seq. Briefly, scaRNA-seq measures transcripts up to 300nt (instead of around 100nt), and paired-end sequencing is done to identify the 3’ ends of the transcript, which corresponds to the TPS. In this way, all paused transcripts up to 300nt can be identified. By combining this information with nascent RNA-seq, the authors set up a model to distinguish whether pausing or initiation was changing, using the differentiation of mouse primary erythroid cells as a system (Figure 1a). When genes are activated/silenced during differentiation, changes in gene expression could be due to either pausing gain (quadrant A), initiation gain (quadrant B), initiation loss (quadrant C) or pausing loss (quadrant D). They found that more than 90% of the differentially expressed genes fell in quadrant B and C (Figure 1b), suggesting that changes in gene expression across differentiation are primarily due to changes in initiation.

To directly test whether the enhancers indeed regulate transcription initiation, the authors deleted well characterized enhancers and performed scaRNA-seq to study changes at their target genes. They chose the R1 and R2 enhancers (ΔR1R2), which regulate the α-globin genes and the HS2 and HS3 enhancers (ΔHS2HS3), which regulate the β-globin genes. They then generated homozygous mice containing these deletions and performed scaRNA-seq on primary erythroid cells derived from fetal liver. The scaRNA-seq results show that the number of scaRNAs are substantially decreased at both globin target genes, suggesting that transcription initiation is being downregulated in the enhancer mutants, not pausing. Because previous papers have used the pausing index to conclude that pausing is important at these genes, the authors also performed Pol II ChIP-seq in the enhancer deletions and showed an overall decrease in Pol II on the gene. They then calculated the pausing index and showed that they could recapitulate the increase in pausing in ΔR1R2 and ΔHS2HS3. These results highlight the need for using the right assay to measure pausing.

In performing and validating scaRNA-seq, the authors also found that annotated TSS positions appear to be skewed upstream of their actual positions, regardless of which annotation they checked. This suggested that there might have been some systematic bias in the methods that have been used to measure TSSs previously. TSS and TPS distributions both appear to be narrower than previously described, and the pause site appears to be enriched at 46bp rather than 86bp downstream of the TSS.

(A)

(B)

Figure 1: (A) Model to interpret scaRNA vs intronRNA data (Figure 1B from original preprint). (B) Differentially expressed genes appear to be due to changes in initiation (Figure 3A from original preprint).

What I liked

It is quite clear that transcription involves a couple of key biochemical reactions. However, what remains unclear is which steps are actively regulated to control gene expression or which step is rate-limiting. To understand these still open questions, studies often assess initiation and pausing at individual genes or use in vitro systems. The authors were quite clever to address this question genome-wide by tweaking methods that already exist. They also show that the pausing index is not a good measure of pausing, even though this has been used quite a bit in the field. This suggests that just because something is measurable does not mean it is always important, and underscores the need for using the right assay to address specific questions. The way that the preprint is structured also made it easy to read. By setting up the model at the beginning, interpreting the results was quite straightforward.

Future directions and questions

Since scaRNA-seq is a modification of the Start-seq protocol, is there any explanation for why the TSSs observed by scaRNA-seq and Start-seq are so different?

It is possible that enhancers can regulate more than one step in transcription. Since pausing comes after initiation, could this method be missing any regulation in pausing?

Pausing was originally discovered to be important in fast response genes, such as heat shock genes. In the differentiation model used in this preprint, genes need to be turned on permanently and relatively slowly compared to environmental response genes. It would be interesting to use this model to compare the differences between fast and slower responding genes.

References

Henriques, T., Gilchrist, D.A., Nechaev, S., Bern, M., Muse, G.W., Burkholder, A., Fargo, D.C., and Adelman, K. (2013). Stable Pausing by RNA Polymerase II Provides an Opportunity to Target and Integrate Regulatory Signals. Molecular Cell 52, 517–528.

Nechaev, S., Fargo, D.C., Santos, G. dos, Liu, L., Gao, Y., and Adelman, K. (2010). Global Analysis of Short RNAs Reveals Widespread Promoter-Proximal Stalling and Arrest of Pol II in Drosophila. Science 327, 335–338.

 

 

 

doi: https://doi.org/10.1242/prelights.15653

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