HES-1 repression of differentiation and proliferation in PC12 cells: role for the helix 3-helix 4 domain in transcription repression.
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. Expression of a HES-1 protein carrying the H-3/4 domain, but not the WRPW domain, still partially inhibited both proliferation and differentiation. Transcription assays in PC12 cells directly demonstrated that the H-3/4 domain can mediate DNA-binding-dependent transcription repression, even in the absence of corepressor recruitment by the WRPW motif. HES-1 expression strongly repressed transcription of the p21(cip1) promoter, a cyclin-cyclin-dependent kinase inhibitor up regulated during NGF-induced differentiation, and the H-3/4 domain is necessary for this repression. Thus, the H-3/4 domain of HES-1 contributes to transcription repression independently of WRPW function, inhibits neurite formation, and facilitates two distinct and previously uncharacterized roles for HES-1: the inhibition of cell proliferation and the direct transcriptional repression of the NGF-induced gene, p21.
Cell Biology and Genetics Graduate Program, Weill Medical College of Cornell University, New York, New York 10021, USA.
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Castella
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Sawai
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Nakao
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Wagner
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Caudy
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D002454:Cell Differentiation / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D018257:Helix-Loop-Helix Motifs; D018398:Homeodomain Proteins / Q000235:genetics*; D016716:PC12 Cells / Q000473:pathology* / Q000502:physiology*; D051381:Rats; D012097:Repressor Proteins; D000072056:Transcription Factor HES-1; D014158:Transcription, Genetic
D051792:Basic Helix-Loop-Helix Proteins; D018398:Homeodomain Proteins; D012097:Repressor Proteins; D000072056:Transcription Factor HES-1; C489735:Hes1 protein, rat