Regulation of RNA polymerase III transcription during differentiation

Abstract

The differentiation of F9 embryonal carcinoma (EC) cells into parietal endoderm (PE)-like cells provides a tractable model for studying molecular events during early and inaccessible stages of murine development. It has been previously shown that PE formation is accompanied by a ~10-fold decrease in RNA polymerase (po1) III transcription. This down-regulation was attributed to a reduction in the activity of the basal po1 III factor TFIIIB; however, the mechanism involved was not established. The work presented here was aimed at elucidating the precise changes that lead to a decrease in po1 III transcriptional activity during F9 differentiation. The protein levels of all three components of the TFIIIB complex, Brf1, Bdp1 and TBP, were found to decrease. In the case of Brf1 and Bdp1, this decrease did not reflect a change in their stability, as proteasome inhibition did not have an effect on protein abundance. On the other hand, a specific down-regulation of the mRNAs encoding Brf1 and Bdp1 was observed. Temporal analysis of F9 differentiation, however, revealed that the decline in TFIIIB levels follows that in po1 III transcription in the same extracts. Furthermore, over-expression of Brf1 did not result in an increase in po1 III transcription in PE cells. These data point to TFIIIB playing a secondary role in the down-regulation of po1 III during differentiation. TFIIIB activity, and consequently po1 III transcription, can be regulated by a number of proteins. For example, c-Myc activates po1 III transcription, while the retinoblastoma protein (pRb) is a repressor. Time course analysis revealed that c- Myc down-regulation and pRb up-regulation closely follows the decline in po1 III transcription during differentiation. Furthermore, recombinant c-Myc efficiently restored transcription in PE extracts. These two proteins, therefore, provide likely candidates for the primary regulatory mechanism of po1 III transcription in response to differentiation, through their effect on TFIIIB