ZFP423 coordinates Notch and bone morphogenetic protein signaling, selectively up-regulating Hes5 gene expression

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.

FIGURE 1.

Colocalization of Zfp423 and Hes5 in the medial cerebellar primordium. A, whole mount LacZ staining of an E10.5 embryo carrying a gene trap insertion in the Zfp423 locus. A′ and A″, Whole mount E10.5 embryos hybridized with Hes5 and Hes1, respectively. Cb, cerebellar primordium; Hb, hindbrain. Note Zfp423 expression at the border with the roof plate. B–E, E11.5 and E12.5 sagittal sections from medial and lateral territories of the cerebellar primordium, hybridized with Zfp423. Solid arrowhead: VZ; arrow: RL. B′–E′ and B″–E″) as above, hybridized with Hes5 and Hes1, respectively. Empty arrowhead in B″ indicates the isthmic organizer (IO). Notably, Hes5 is expressed in VZ and RL and sharply silenced in the IO, whereas Hes1 is transcribed in the RL and IO, and silenced in most of the VZ.

FIGURE 2.

Cooperative activation of Hes5 and Blbp gene expression by NICD and ZFP423 in cell lines. A, semiquantitative RT-PCR analysis of exogenous Nicd and Zfp423 levels in transfected P19 cells. Note increasing Zfp423 levels in lanes 4–6. This is representative of the amounts of Nicd and Zfp423 cDNAs used in B–F. B–G, quantitative RT-PCR analysis of P19 (B–E, G) and C2C12 (F) cells treated as indicated below. B and C, Nicd and Zfp423 coexpression in neuralized P19 cells up-regulates endogenous Hes5 and Blbp expression, respectively, to a level greater than cells transfected with Nicd alone. Induction of Hes5 and Blbp transcription is dependent on Zfp423 dosage. D and E, cotransfection of P19 cells with Nicd and Zfp423 fails to activate Hes1 and Nrarp gene expression to a level greater than Nicd alone. F, transfection of C2C12 cells with Nicd and Zfp423 induces Hes5 gene expression in a Zfp423 dose-dependent fashion. In this line, Hes5 expression is strictly dependent on the addition of exogenous Zfp423. G, Zfp423 RNA interference in P19 cells reduces endogenous Hes5 expression. H, Zfp423 RNA interference reduces Hes5 expression in P19 cells grown on Fc-Jagged1-coated plates compared with cells plated onto Fc-TRAIL-R4-coated plates. *, p ≤ 0.05; **, p < 0.01.

FIGURE 3.

Selective, cooperative activation of ESR1 gene expression by N^act and ZFP423 in Xenopus embryos. A and B, whole mount in situ hybridization analysis of Xenopus Zfp423 gene expression in stage 17 (frontal view) and stage 22 (lateral view, head to the left) embryos, respectively: the gene is widely expressed starting in the neural plate/tube first (arrow), and then moving to the cranial neural crest (arrowhead). C–E, whole mount in situ hybridization analysis of ESR1 gene expression in embryos injected unilaterally (bracket) with Zfp423 (600 pg) and/or N^act (100 pg), as indicated. LacZ (blue stain) serves as an indicator of the injected side. Note strong ESR1 expression after coinjection of N^act and Zfp423. F, embryo injected unilaterally (bracket) with 1 ng of Zfp423. G, embryo injected unilaterally (bracket) with 1 ng of Zfp423 and 400 pg of Delta^Stu, encoding a dominant negative Notch ligand. Note disappearance of ESR1 signal on the co-injected side. H and I, unilateral injection (bracket) of a Zfp423 morpholino oligonucleotide (H) and of a control morpholino (I) (see “Experimental Procedures”). Note disappearance of ESR1 signal on the injected side in embryos injected with Zfp423-specific morpholino unlike those injected with the control morpholino. J–L, Zfp423 overexpression selectively up-regulates ESR1 but not Hairy1 or Nrarp in Xenopus embryos. Whole mount in situ hybridization analysis of ESR1, Hairy1, and Nrarp gene expression in embryos injected unilaterally with Zfp423 (300 pg, arrow) in a dorsal blastomere. Note expanded ESR1 expression domain (J), but reduced/unaffected expression of Hairy1 (K) and Nrarp (L) on the injected side. Gfp was coinjected with Zfp423 as a lineage tracer (not shown).

FIGURE 4.

ZFP423 binds to and activates the most proximal 267 bp of the Hes5 promoter. A and B, promoter-reporter assays performed in C2C12 cells using a long and a short version of the Hes5 gene promoter fused to luciferase. The two variants of the Hes5 promoter are sketched above each histogram. A, 1-kb wt 5′-sequence responds to cotransfection with Zfp423 by up-regulating luciferase compared with levels reached with Nicd alone. B, likewise, a 267-bp proximal element is cooperatively activated by Nicd and Zfp423, albeit to a lower level with respect to the experiment in A. *, p < 0.05; **, p < 0.01. C, chromatin immunoprecipitation was conducted on neuralized P19 cells. Sheared chromatin immunoprecipitated with anti-ZFP423 was purified and amplified by quantitative PCR. a–d, primer pairs spanning the Hes5 gene (b is the primer pair spanning the BRE, see text). e, primer pair amplifying the syntenic gene Mrps15. Data are plotted as fold enrichment relative to the abundance of the Mrps15 qPCR product (e). ***, p < 0.0004.

FIGURE 5.

Molecular interaction between ZFP423 and NICD. In A, COS7 cells transfected with the indicated constructs, were subjected to immunoprecipitation using anti-GFP as an irrelevant antibody, or anti-Myc to precipitate 6xmycZFP423. Filters were cut and stained for ZFP423, NICD, and actin (unrelated protein). Only NICD coprecipitated in the fraction immunoprecipitated with anti-Myc. B, lysates of COS7 cells, transfected with the indicated constructs, were blotted and immunostained as shown. C, lysates shown in B were immunoprecipitated with the anti-Myc antibody. Filters were immunostained for ZFP423, NICD, and EBF3. Notably, NICD coprecipitated equally with ZFP423 in the presence or absence of EBF3.

FIGURE 6.

EBF TFs reduce ZFP423-NICD-mediated Hes5 gene expression in vitro and in vivo. A, RT-qPCR analysis of Hes5 gene expression in P19 cells transfected with the indicated constructs. Note that Ebf1, Ebf2, or Ebf3 alone does not affect basal Hes5 expression. B–D, whole mount in situ hybridization analysis of ESR1 gene expression in embryos injected unilaterally (bracket) with N^act (100 pg) and Zfp423 (600 pg) and/or XEbf2 (100 pg) or XEbf3 (100 pg). E and F, whole mount in situ hybridization analysis of ESR1 gene expression in embryos injected unilaterally with XEbf2 (100 pg) or XEbf3 (100 pg), as indicated. Note slightly increased ESR1-positive cells in XEbf2- and XEbf3-injected embryos (arrow). B–F, LacZ (blue stain) serves as an indicator of the injected side.

FIGURE 7.

ZFP423 cooperates with NICD and BMP signaling activation to promote Hes5 gene expression. Δ9–20 ZFP423 localizes partially in the cell nucleus but fails to activate Hes5 gene expression in cooperation with NICD. A, scheme of the in-frame deletion of exon 4 producing a protein devoid of Zn fingers 9–20, implicated in the interaction with SMAD proteins and the BMP-responsive element. B, immunofluorescence analysis of the subcellular localization of a Myc-tagged wt and Δ9–20 construct in COS7 cells. Note that the mutant protein has a nuclear localization, although in some cells it is also distributed in the cytoplasm. DAPI labels DNA. ovl, overlay. C, histogram illustrating the results of a promoter-reporter assay revealing the lack of a cooperative interaction between Zfp423 Δ9–20 and NICD in Hes5 gene activation. D, cooperative activation of Hes5 gene expression by BMP4 and Notch mediated by ZFP423. Real-time RT-qPCR analysis of RNA from C2C12 cells, mock transfected or transfected with either Nicd, Zfp423, or both. Cells were either left untreated or treated with BMP4 for 2 h. Transfection with Zfp423 strongly enhances the cooperative effect of NICD and BMP signaling on Hes5 gene expression. E, RNAs from the C2C12 cell lysates analyzed in D were subjected to RT-qPCR using primers specific for flagNICD. FlagNICD expression levels are comparable in the presence and absence of Zfp423. This excludes the possibility that differences in Hes5 expression depend on different levels of flag-Nicd in Zfp423-transfected versus Zfp423-untransfected samples. *, p < 0.05; **, p < 0.005.

FIGURE 8.

A working model of the interactions between ZFP423 and other networks in Hes5 gene activation. Hes5 expression is induced upon Notch signaling activation independently of ZFP423. ZFP423 boosts the effect of Notch signaling by forming a complex with NICD and by recruiting it onto the Hes5 promoter. The interaction between the BMP4 transducer SMAD1-SMAD4 and ZFP423 further potentiates this effect (solid arrow), although BMP4 signaling might also cooperate with NICD in the absence of ZFP423 (stippled arrow). In overexpression experiments, EBF/COE transcription factors antagonize Hes5 activation by competing with NICD for the interaction with ZFP423.