The Function Of Transcription Elongation In Hematopoietic Stem Cell Development

The specification of hematopoietic stem cells (HSCs) in vertebrate embryos is tightly regulated by RNA polymerase II (Pol II)-mediated transcription, which proceeds through multiple steps including initiation/activation, elongation and termination. Increasing evidences in recent years have demonstrated that transcriptionally engaged Pol II often pauses shortly after initiation by pausing factors DSIF and NELF, and its release requires positive elongation factors such as p-TEFb. Little is known about how pausing-to-elongation switch contribute to cell fate determination. In a zebrafish spt5 mutant, we detected a dramatic loss of runx1+ HSCs in the AGM region in 36-hour embryos. Spt5 is an essential subunit of DSIF, which pauses Pol II in early elongation stage but stimulates Pol II elongation upon phosphorylated by p-TEFb. The zebrafish mutant carries a single amino acid change in the Spt5 protein that disrupts the pausing function but leaves the stimulatory function intact, suggesting a requirement of Pol II pausing in HSC formation. Similarly, knocking down the other pausing factor NELF by morpholinos also greatly reduced the number of runx1+ HSCs in zebrafish embryos. This loss of HSCs is not caused by general developmental defects, as markers for vessels and aorta endothelium remain normal in mutants. We further demonstrated that reducing Pol II elongation by either p-TEFb inhibitors or morpholino knockdown of CDK9, the essential subunit of p-TEFb, could rescue the HSC defect in spt5 mutants. The rescue by cdk9 morpholino is dose-dependent. Low-dose morpholino that presumably caused partial knockdown of CDK9 was able to rescue spt5 mutants, whereas high-dose morpholino completely wiped out HSCs in both wild type and mutant embryos. In conclusion, our data suggested that HSC formation during embryonic development requires a balanced transcription that is regulated by both negative and positive elongation factors. Such mechanisms may also be employed in other tissues to regulate cell fate determination.