Abstract
Zinc finger protein 423 encodes a 30 Zn-finger transcription factor involved in cerebellar and olfactory development. ZFP423 is a known interactor of SMAD1-SMAD4 and of Collier/Olf-1/EBF proteins, and acts as a modifier of retinoic acid-induced differentiation. In the present article, we show that ZFP423 interacts with the Notch1 intracellular domain in mammalian cell lines and in Xenopus neurula embryos, to activate the expression of the Notch1 target Hes5/ESR1. This effect is antagonized by EBF transcription factors, both in cultured cells and in Xenopus embryos, and amplified in vitro by BMP4, suggesting that ZFP423 acts to integrate BMP and Notch signaling, selectively promoting their convergence onto the Hes5 gene promoter.
Highlights
The Notch pathway exerts regulatory activities in a diverse array of developmental contexts
We demonstrate that ZFP423 interacts functionally and molecularly with the Notch intracellular domain (NICD) in mammalian cell lines and in Xenopus neurula embryos, to activate the expression of the Notch target Hes5/ESR1
In the E12.5 cerebellar primordium, Zfp423 is expressed at high levels flanking the midline, where it colocalizes with Hes5 (Fig. 1, BЈ–DЈ), an immediate transcriptional target of the Notch1 signaling pathway expressed in the VZ and rhombic lip (RL)
Summary
MRNA was injected into one dorsal blastomere of 16-cell stage embryos in the following amounts : Zfp423 (300 pg) and Gfp (200 pg). P19, HEK293, and COS7 cells were transfected with Lipofectamine2000 according to the manufacturer’s instructions (Invitrogen). Transfected P19 cells were sorted for GFP expression and lysed for RNA extraction with RNeasy MicroKit (Qiagen), according to the manufacturer’s instructions. Coating Assay—To generate a soluble form of Notch ligand, 293T cells grown in a 10-cm Petri dish were transfected using Fugene HD (Roche), according to the manufacturer’s instructions, with either 5 g of Fc-TRAIL-Receptor 4 (Fc-control) or 5 g of Fc-Jagged expression plasmids (Courtesy of Tom Kadesch, 51) After 48 h, the growth medium was collected and filtered through a 0.45-m syringe filter. Transfected P19 cells were sorted for GFP expression, and RNA was extracted with RNeasy MiniKit, according to the manufacturer’s instructions. All results are plotted as the mean Ϯ S.D
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