Differential Induction of HNF-3 Transcription Factors during Neuronal Differentiation

Abstract

We have investigated the regulation of transcription factors HNF-3α and HNF-3β during the retinoic acid-mediated differentiation of mouse P19 cells. Retinoic acid treatment converts P19 stem cells into neurons and astrocytes and we have clearly shown that gene expression of both HNF-3α and HNF-3β is activated during this process. HNF-3α transcription was detected 2 h after addition of retinoic acid and took place in the absence ofde novoprotein synthesis. This suggests that HNF-3α is a primary target for retinoic acid action. HNF-3α induction displays a biphasic profile and HNF-3α mRNA reaches maximal levels at 2 and 6 days postdifferentiation. Additional experiments strongly suggest that the second peak is due to HNF-3α induction in postmitotic neurons. P19 stem cells, on the other hand, do not contain any detectable HNF-3α mRNA. According to our studies, the retinoic acid-mediated induction of HNF-3α occurs at the level of transcriptional initiation and is conferred by distal promoter sequences. In comparison to HNF-3α, HNF-3β induction is a subsequent event and detectable levels of HNF-3β mRNA materialize approximately 1 day after addition of retinoic acid to P19 stem cells. Time course studies firmly demonstrate that HNF-3β mRNA peaks at about 2 days postdifferentiation and then declines to virtually unreadable levels. This temporal pattern is consistent with HNF-3β being a secondary target for retinoic acid. In analogy to HNF-3α, HNF-3β activation also takes place at the level of transcriptional initiation. Recent studies implicate HNF-3α and HNF-3β in early mammalian neurogenesis. The detection of HNF-3α/β activation during P19 cell differentiation provides us with a convenient cell culture system to elucidate the induction mechanism and the precise role of both transcriptional regulators in the formation of neuronal cells.