Investigation of the posttranscriptional regulator BRF1 in embryonic stem cells by inducible RNA interference

Pluripotency in murine embryonic stem (ES) cells is maintained by a hierarchy of transcription factors (Nanog, Oct4, Sox2, etc.) but nothing is known to date if ES cell self-renewal or differentiation may also involve mechanisms that act posttranscriptionally at the level of mRNA turnover. Around 8% of all transcipts contain in their 3'-untranslated region a so-called AU-rich element (ARE), a destabilizing motif, which is a key target for proteins regulating dynamic mRNA turnover control, and many of these transcripts code for growth and cell cycle regulatory genes. Our longstanding interest in the laboratory is the ARE-mRNA regulator Brf1 (Zfp36L1), and we were interested to see if this protein is also involved in ES cell pluripotency or differentiation. In early experiments we found Brf1 to be expressed in undifferentiated ES cells (CCE, CGR-8) and under positive regulation by the LIF (leukemia inhibitory factor) and Stat3 axis. As LIF removal or targeting of Stat3 by siRNA led to downregulation of Brf1, we wished to explore the consequences of only downregulating Brf1.
To do so, we developed a cassette system where a small hairpin RNA (shRNA) of choice can be introduced into a defined frt (flip-recombinase target)-site by co-transfection with the recombinase, and which can, in addition, be induced by doxycycline. CCE ES cells already expressing the Tet-repressor (TR) for doxycycline inducibility were transfected with a construct expressing both GFP and neomycin resistance markers linked to a frt-site, allowing Flp-recombinase mediated integration of the plasmid encoding the inducible shRNA. For systems control, we targetet Stat3 by shRNA and observed in response to doxycycline, as exptected, the morphological and biochemical (Oct-4, Fgf4, Rex-1) signs of differentiation of ES cells even in presence of LIF, thus verifying the validity of the system.
Addition of doxycycline to clones with induvible Brf1 shRNA led to a distinct alteration of the morphology of plated embryoid bodies formed in hanging drops. Unexpectedly, downregulation of Brf1 strongly stimulated the formation of cardiac markers (Nkx2.5, Gata4) as well as the formation of beating bodies observed around day 9. To trigger these changes and to enhance cardiomyocyte formation, 4 days of doxycycline addition following LIF removal was sufficient.
A DNA microarray chip analysis was conducted to identify ARE-bearing transcripts with altered expression upon LIF removal and Brf1 downregulation by RNA interference. Based on our hypothesis, we hoped to identify ARE-bearing transcripts that would be increased by these changes. Unfortunately we could not identify any such transcripts suggesting that the effect may be subtle, or could be masked by the simultaneous changes in transcriptional regulation that accompanies these changes.
In a series of preliminary experiments to establish if posttranscriptional regulation is operating in undifferentiated ES cells, an EGFP reporter linked to the destabilizing ARE from the IL3 gene was transduced into CCE cells. The amount of EGFP expresion, as a marker for steady-state mRNA levels, was consistently lower in cells transduced with a reporter bearing the wild-type ARE when compared to cells bearing a non-functional mutant ARE suggesting reporter mRNA destabilization. In addition, when the reporter was expressed in cells bearing the Brf1 shRNA, a slight but significant increase in EGFP expression was observed upon Brf1 downregulation. These results suggest that posttranscriptional regulation of mRNAs is also active in murine ES cells.
Taken together, these findings raise the possibility that Brf1 could be a novel potential regulator of cardiomyocyte formation and suggest that posttranscriptional mechanisms may play an important role in early development. In addition, the inducible RNA interference system developed for this study can be used to investigate any gene of interest and its role in ES cell development.