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Xenopus Zic3 (zinc finger in the cerebellum-3) is expressed in the dorsal neural tube of tailbud embryos and tadpoles. We have isolated a 3.1kb DNA fragment from the Xenopus laevis Zic3 locus that drives proper expression of a GFP reporter in transgenic frog tailbud embryos and tadpoles. This fragment contains regions that are highly similar among frogs, mice, and humans. One extremely conserved region contains a predicted Pbx binding site. We found that the transcription factors Pbx1b and Meis1 can bind this site and synergistically transactivate expression of a reporter containing the conserved region. Finally, we found that an intact Pbx site is essential for normal Zic3 promoter activity in transgenic frog embryos. Our data strongly suggest that a highly conserved region of the Zic3 promoter functions by direct interaction with Pbx1b and Meis1.
Fig. 1. Zic3 sequence flanking the coding region is highly conserved among species. Alignment of Xenopus, human, and mouse Zic3 genomic sequences.
(A) Summary of Multi-PIP Maker and ClustalW results. Bar represents Xenopus sequence. Regions of high identity with human (red) and mouse (blue)
sequences are represented. Regions of high identity with both human and mouse sequences are shown with blue and red crosshatching. Sequence is
numbered from the predicted start of transcription. The 247 bp conserved region is marked (*). (B) Detail of proximal 247 bp region of high similarity
among all three sequences. Red letters represent bases that are identical among all three species. The 53 bp conserved region (CR) nearly identical between
all three species is underlined. The predicted Pbx site is boxed. (For interpretation of the references to color in this figure legend, the reader is referred to
the web version of this paper.)
Fig. 2. Pbx1b and Meis1 physically interact with the Zic3 promoter conserved region. (A) Electrophoretic mobility shift assay (EMSA) using synthetic
Pbx1b and Meis1 proteins, radiolabelled conserved region (CR) probe, and unlabelled wild-type or mutated CR oligonucleotide competitors. Filled
arrowheads denote specific bands observed with Pbx1b or Meis1 protein. Open arrowheads denote specific complexes observed with co-translated Pbx1b
and Meis1. (B) EMSA using CR probe and tailbud embryo nuclear extract. The specific complexes observed are marked (a,b). (C) Immunoblot of proteins
extracted from EMSA gel. (i) Autoradiograph of wet gel from EMSA using embryo nuclear extract and CR probe. Bands excised from gel (a,b) are boxed.
(ii) Immunoblot of proteins excised from EMSA gel probed with antibody raised against Xenopus Meis1. Lane designations correspond to EMSA band
designations in left panel. (D) Immunoblot of epitope-tagged proteins extracted from EMSA gel. (i) Immunoblot of lysates prepared from embryos
overexpressing myc-tagged Pbx using antibodies raised against Xenopus Pbx1b or myc. (ii) EMSA using lysates prepared from embryos overexpressing
myc-Pbx1b. (iii) Autoradiograph of wet gel from EMSA using lysates of embryos injected with RNA encoding myc-Pbx1b RNA. (iv) Immunoblot of
proteins isolated from EMSA gel (iii) probed with antibody raised against the myc epitope tag. Lane designations correspond to EMSA band designations
in left panel. (E) Reporter activation by Pbx1b is dependent on an intact Pbx site. Pbx1b activates the 3· CRwt/Luc reporter, containing three copies of the
wild-type Zic3 CR, but not the 3· CRmut/Luc reporter, in which the putative Pbx sites in the Zic3 CR have been mutated. (F) Pbx1b and Meis1 cooperate
to activate a reporter containing the Zic3 conserved region. Luciferase reporter gene assay using lysates prepared from embryos injected with RNA
encoding Pbx1b and/or Meis1. Abbreviations: Compâcompetitor; CRâconserved region; FPâfree probe; IBâimmunoblot; Mâmutated CR
oligonucleotide; Wâwild-type CR oligonucleotide.
Fig. 3. The 3.1 kb Zic3 promoter is active in most endogenous Zic3 expression domains. The Zic33.1/GFP transgene was introduced into embryos
(A,B,D,E,G,H). Transgene expression was visualized by fluorescence (B) or wholemount in situ hybridization (WISH) using a GFP antisense riboprobe
(D,E,G,H). Endogenous Zic3 expression was visualized by WISH using a Zic3 antisense riboprobe (C,F,I). (A–C) At late tailbud stage, the transgene and
endogenous Zic3 are expressed in the dorsal brain and spinal cord. (D–F) At mid-gastrula stage, both the transgene and endogenous Zic3 gene are
expressed in involuting mesoderm (arrow). Endogenous Zic3, but not the transgene, is also expressed in the animal hemisphere (arrowhead). (G–I) At
early tailbud stages, the transgene and endogenous Zic3 are expressed in the neural tube (arrow), including the developing spinal cord and brain. Some
embryos exhibited expression of the transgene in regions flanking the neural tube (G, arrowhead).
Fig. 4. The conserved Pbx1b binding site is essential for Zic3 promoter function. Transgenic embryos were prepared using wild-type (A) or Pbx mutated
(B,C) Zic33.1/GFP transgene. In late tailbud embryos, the mutated transgene was expressed in an attenuated (B) or ubiquitous (C) pattern.
