Sivaramakrishnan, Priya et al. (2021) International Worm Meeting "Transcription rates in the early embryo"

Sivaramakrishnan, Priya, MURRAY, JOHN ISAAC, Watkins, Cameron

[International Worm Meeting, 2021]

Rapid changes in gene expression are necessary for cells to respond to environmental, immunological or developmental signals. As a critical first step, high transcription synthesis rates are required during these responses. In the rapidly dividing C. elegans embryo, where cell cycles are as short as 15 minutes, temporal constraints on transcription are particularly severe. Early cell fate specification genes have to be transcribed at very high rates to accumulate to threshold levels within short developmental time windows. Studies in other labs have shown that reducing the normally high expression levels of some endodermal regulators results in aberrant intestinal fate specification. We hypothesize that high rates of transcription could be required for the fate decisions in several additional early cells. We estimated transcription rates in the early embryo by analyzing single cell RNA sequencing data and found that maximal rates vary across cell types, lineages, and embryonic stages. We have validated some of these by single molecule RNA fluorescence in situ hybridization (smFISH) and identified features of rapidly transcribed genes such as short gene length, shorter and fewer introns, and lack of trans-splicing. We found several motifs enriched in the core promoters of high-rate genes including the initiator element (inr), sites for the SP1 transcription factor, and binding sites for lineage-specific regulators. Using the high-rate mesoderm specification gene ceh-51 as a test case, we measured the effect of these promoter elements on transcription rates using CRISPR-mediated promoter mutagenesis and smFISH. Our analysis shows significant redundancies built into the ceh-51 core promoter with modest contributions to rate from each motif, suggesting that control of transcription rates is surprisingly robust. We are currently designing experiments to test for a direct impact of transcription rate on cell fate. Ultimately, we hope to understand how transcription parameters such as bursting are influenced by rate control and how this contributes to gene expression precision underlying the invariant C. elegans embryonic lineage.

Sivaramakrishnan, Priya et al. (2019) International Worm Meeting "Control of transcription rates in the Caenorhabditis elegans embryo."

MURRAY, JOHN ISAAC, Sivaramakrishnan, Priya

[International Worm Meeting, 2019]

Embryonic cell fate decisions rely on transcription programs that are coordinated in both space and time. Temporal coordination is particularly important in the rapidly dividing C. elegans embryo, where developmental regulators need to be transcribed quickly enough to instruct fate decisions before the next cell cycle. This requires embryonic transcription to be very accurate. But transcription is known to be noisy, occurring in bursts followed by periods of quiescence. Thus, how transcriptional noise is prevented or reduced to allow for developmental robustness is unclear. Several early zygotic regulators in C. elegans are transcribed at very high rates. Analysis of single molecule RNA FISH (smFISH) data shows that the mRNA levels of the endoderm regulator end-3 increase from zero to 600 transcripts in one cell within a 15-minute cell cycle. We hypothesize that high transcription rates of critical embryonic regulators can promote developmental robustness. To determine whether such high rates occur for other genes, we used single-cell RNA sequencing (scRNA-seq) data from 84,625 embryonic cells to measure maximum transcript levels at single cell resolution. We identified 20 candidate genes with high transcript abundance above a set threshold. We are using smFISH to validate the high expression of these genes and to measure the noise associated with their transcription. The expression of one candidate gene, ceh-51, a muscle-specific transcription factor is similar to end-3, producing 600 transcripts per cell. To estimate transcriptional bursting during ceh-51 expression, we targeted smFISH probes to ceh-51 introns to label sites of nascent transcription. Surprisingly, we detect transient accumulation of nuclear intron-containing transcripts within the nucleus outside of transcriptionally active sites. This suggests that transcription and splicing may be decoupled for ceh-51, which we are exploring further. scRNA-seq identifies genes with high transcript abundance in single cells, but does not directly measure transcription rates. Hence, we are combining the metabolic labeling of mRNAs with scRNA-seq to detect recent transcription events and quantify genome-wide transcription rates during embryonic development. Our approach and methodologies can be applied to identify universal mechanisms that support rapid and temporally precise transcription.