HES-1 is a Hairy-related basic helix-loop-helix protein with three evolutionarily conserved regions known to define its function as a transcription repressor. The basic region, helix-loop-helix domain, and WRPW motif have been characterized for their molecular function in DNA binding, dimer formation, and corepressor recruitment, respectively. In contrast, the function conferred by a fourth conserved region, the helix 3-helix 4 (H-3/4) domain, is not known. To better understand H-3/4 domain function, we expressed HES-1 variants under tetracycline-inducible control in PC12 cells. As expected, the induced expression of moderate levels of wild-type HES-1 in PC12 cells strongly inhibited nerve growth factor-induced differentiation. This repression was dependent on the H-3/4 domain. Unexpectedly, expression of HES-1 also arrested cell growth, an effect that could be reversed upon down regulation of HES-1. Concomitant with growth arrest, there was a strong reduction in bromodeoxyuridine incorporation and PCNA protein levels, although not in cyclin D1 expression. HES-1, the Hairy and Enhancer of split homologue 1 (19,52), is a vertebrate member of a highly conserved family of Hairy-related basic helix-loop-helix (bHLH) proteins. Originally described in Drosophila melanogaster, Hairy-related proteins include Hairy (51), Deadpan (3), and the seven bHLH members of the Enhancer of split [E(Spl)] complex (14, 37). Members of this family are DNA-binding transcription repressors that antagonize the function of bHLH activators and repress neuronal development (reviewed in references 6, 21, 35, and 36). The Hairy-related proteins bind to specific DNA sites (class C sites or N-boxes) in target gene promoters by means of the conserved basic region (43,44,52,56,57,59). The DNAbinding function of Hairy has been shown to be essential for the transcriptional repression of its downstream target, achaete, a proneural bHLH activator gene (44, 59). Transcriptional repression of target promoters is thought to occur at least partly by recruitment of a corepressor protein, Groucho, via the WRPW tetrapeptide motif conserved in the C terminus of all family members (24,46,61). Indeed, a fusion of the WRPW motif to the Gal-4 heterologous DNA-binding protein is sufficient by itself to repress transcription (22,25). However, Hairy also binds to another corepressor, dCtBP (48, 65), suggesting that Hairy may have alternative repression functions in addition to the conserved Groucho recruitment mechanism.Additionally, some bHLH repressors do not share the requirement for intrinsic DNA-binding capability to repress neuronal development. A bHLH-deleted version of E(Spl) (m8) has been shown to repress neuronal development despite lacking intrinsic DNA-binding capability (24,41,43). Functional dissection of the E(Spl) protein in Drosophila highlighted the importance of the helix 3-helix 4 (H-3/4) domain (37) and the WRPW motif, as well as the intervening C-terminal region, for correct bristle development (24). The mechanism of repression did not appear ...

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HES-1 is a vertebrate homologue of the Drosophila basic helix-loop-helix (bHLH) protein Hairy, a transcriptional repressor that negatively regulates neuronal differentiation. HES-1 expression in neuronal precursors precedes and represses the expression of the neuronal commitment gene MASH-1, a bHLH activator homologous to the proneural Achaete-Scute genes in Drosophila. Down-regulation of HES-1 expression in developing neuroblasts may be necessary for the induction of a regulatory cascade of bHLH activator proteins that controls the commitment and progression of neuronal differentiation. Here we show that the differentiation of embryonic day-17 rat hippocampal neurons in culture was coincident with a decline in HES-1 expression and DNA binding. Therefore, we examined the effect of forced expression of HES-1 and MASH-1 upon nerve growth factor (NGF) -induced differentiation in TrkA transfected hippocampal neurons. Expression of HES-1 inhibited both the intrinsic and NGF-induced neurite outgrowth, whereas MASH-1 expression increased neurite outgrowth. Strikingly, the increased hippocampal differentiation observed with MASH-1 expression is completely blocked by coexpression of HES-1. Furthermore, both wild-type HES-1 and a non-DNA binding mutant of HES-1 repressed MASH-1-dependent transcription activation. These results suggest that down-regulation of HES-1 is necessary for autonomous, growth factor-induced and MASH-1-activated hippocampal differentiation.

HES-1 is a vertebrate homologue of the Drosophila basic helix-loop-helix (bHLH) protein Hairy, a transcriptional repressor that negatively regulates neuronal differentiation. HES-1 expression in neuronal precursors precedes and represses the expression of the neuronal commitment gene MASH-1, a bHLH activator homologous to the proneural Achaete-Scute genes in Drosophila. Down-regulation of HES-1 expression in developing neuroblasts may be necessary for the induction of a regulatory cascade of bHLH activator proteins that controls the commitment and progression of neuronal differentiation. Here we show that the differentiation of embryonic day-17 rat hippocampal neurons in culture was coincident with a decline in HES-1 expression and DNA binding. Therefore, we examined the effect of forced expression of HES-1 and MASH-1 upon nerve growth factor (NGF) -induced differentiation in TrkA transfected hippocampal neurons. Expression of HES-1 inhibited both the intrinsic and NGF-induced neurite outgrowth, whereas MASH-1 expression increased neurite outgrowth. Strikingly, the increased hippocampal differentiation observed with MASH-1 expression is completely blocked by coexpression of HES-1. Furthermore, both wild-type HES-1 and a non-DNA binding mutant of HES-1 repressed MASH-1-dependent transcription activation. These results suggest that down-regulation of HES-1 is necessary for autonomous, growth factor-induced and MASH-1-activated hippocampal differentiation.