Pourebrahim R , Houtmeyers R , Ghogomu S , Janssens S , Thelie A , Tran HT , Langenberg T , Bellefroid E , Cassiman JJ , Tejpar S .

	FIGURE 1. ZIC2 negatively regulates the transcriptional activity of the β-catenin·TCF4 complex in 293T cells. A, activity of the TOPflash or FOPflash reporter in 293T cells transfected with ZIC2 expression vector in different amounts. B, TOPflash reporter activity of 293T cells transfected with ZIC2-expressing plasmid (20 ng) and increasing amounts of ΔN90-β-catenin-expressing plasmid (5–50 ng). The Western blot shows the levels of overexpressed proteins under each condition. C, TOPflash reporter activity of 293T cells treated with 20 mm LiCl for 12 h after transfection with the indicated plasmids.
	FIGURE 2. ZIC2 binding to TCF4. Cell lysates of 293T cells transfected with ZIC2-FLAG- and β-catenin-expressing plasmids together with TCF4-expressing vector (A) or without TCF4 (B) were subjected to immunoprecipitation (IP) using anti-FLAG antibody. C, schematic representation of LEFΔN-β-cateninΔN53 fusion protein. The graph shows the TOPflash reporter activity of 293T cells after transfection with the expression plasmids as indicated. D, Western blot showing the levels of the indicated proteins after FLAG-tagged ZIC2 overexpression in 293T cells.
	FIGURE 3. Mapping the ZIC2·TCF4 interaction domains. A, schematic representation of the different TCF4 mutants. The HMG domain and C-terminal region (CR) are represented as dark gray boxes. The β-catenin-interacting domain (β-Cat) is represented as a light gray box. B, Western blot showing transient coexpression of FLAG-tagged TCF4, deletion mutants, and ZIC2 using the indicated antibodies. C, immunoprecipitated proteins using anti-ZIC2 antibody (or IgG as a mock) were subjected to Western blot analysis for FLAG detection. D, immunoblot of in vitro protein binding assay. FLAG-tagged TCF4 and its truncated forms were purified with anti-FLAG beads (with IgG serving as a mock) and incubated with in vitro translated ZIC2, and the precipitated complex was subsequently subjected to immunoblotting using antibody against ZIC2. E, schematic representation of ZIC2 and its deletion constructs. Zinc finger domains are represented as dark gray boxes. F indicates FLAG. F, Western blot showing the protein levels of overexpressed full-length ZIC2, ZIC2 deletions, and TCF4 using the indicated antibodies. G, proteins immunoprecipitated from the lysates in F using anti-FLAG antibody (or IgG as a mock) were analyzed by Western blotting using anti-TCF4 antibody.
	FIGURE 4. Complex of ZIC2·TCF4 bound on DNA. A, EMSA was done by incubating a cell extract of 293T cells transfected with different expression plasmids as indicated with biotin-labeled probes containing wild-type or mutant TCF4-binding sequence. The lower panels represent Western blots of the corresponding samples as indicated. B, EMSA using 293T cells cotransfected with full-length TCF4 and its deletions together with ZIC2-expressing plasmid. Where indicated, the samples were preincubated with antibody against TCF4. C, DNA affinity precipitation of 293T cells transfected with the indicated expression vectors was performed as described under “Experimental Procedures.” The lysates were incubated with wild-type or mutant biotinylated probes corresponding to the TCF4-binding sequence, and the precipitated DNA·protein complexes were analyzed by Western blotting using the indicated antibodies. A probe with a mutant TCF-binding sequence was used as a control. D, 293T cells were transfected with ZIC2 and TCF4, and ChIP was performed with the indicated antibodies, followed by quantitative PCR using primer pairs spanning the human AXIN2 TCF4-binding site or an unrelated AXIN2 sequence as negative control. The ApoE promoter served as a positive control for ZIC2 binding. Results are presented as percentage of immunoprecipitated DNA over the input. E, luciferase reporter activity of the Wnt reporter (TOPflash) in 293T cells transfected with the expression vectors as indicated along with the β-catenin (DN90) expression vector.
	FIGURE 6. Zic2 morphants display increased Wnt reporter activity in the brain. Lateral views of stage 43 tadpoles injected with 40 ng of mismatch morpholino (MO) show normal development (A) and modest GFP expression (B). Embryos injected with Zic2 morpholino displayed loss of pigmented cells in the head and trunk (C) and markedly increased GFP expression (D), especially in the forebrain (open white arrowhead), the midbrain-hindbrain boundary (arrow), the dorsal side of the hindbrain (closed white arrowhead), and the eyes (closed yellow arrowhead). Note that the intestine shows autofluorescence (asterisk). The left-most tadpole is non-transgenic (NT). E–G, embryo injected unilaterally with Zic2 morpholino together with a red fluorescent LYSAMINE-labeled control morpholino. The injected side shows a marked increase in TOP-GFP expression, especially visible in the midbrain-hindbrain boundary (arrow).

Aoki, Nuclear endpoint of Wnt signaling: neoplastic transformation induced by transactivating lymphoid-enhancing factor 1. 1999, Pubmed

Aoki, Nuclear endpoint of Wnt signaling: neoplastic transformation induced by transactivating lymphoid-enhancing factor 1. 1999, Pubmed
Aruga, Mouse Zic1 is involved in cerebellar development. 1998, Pubmed
Aruga, Role of BMP, FGF, calcium signaling, and Zic proteins in vertebrate neuroectodermal differentiation. 2011, Pubmed , Xenbase
Aubert, Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. 2002, Pubmed
Belenkaya, pygopus Encodes a nuclear protein essential for wingless/Wnt signaling. 2002, Pubmed , Xenbase
Bellefroid, X-MyT1, a Xenopus C2HC-type zinc finger protein with a regulatory function in neuronal differentiation. 1996, Pubmed , Xenbase
Benedyk, odd-paired: a zinc finger pair-rule protein required for the timely activation of engrailed and wingless in Drosophila embryos. 1994, Pubmed
Billin, Beta-catenin-histone deacetylase interactions regulate the transition of LEF1 from a transcriptional repressor to an activator. 2000, Pubmed
Brannon, XCtBP is a XTcf-3 co-repressor with roles throughout Xenopus development. 1999, Pubmed , Xenbase
Brown, Holoprosencephaly due to mutations in ZIC2, a homologue of Drosophila odd-paired. 1998, Pubmed
Brown, Zic2 is expressed in pluripotent cells in the blastocyst and adult brain expression overlaps with makers of neurogenesis. 2009, Pubmed , Xenbase
Brown, In vitro analysis of partial loss-of-function ZIC2 mutations in holoprosencephaly: alanine tract expansion modulates DNA binding and transactivation. 2005, Pubmed
Buckles, Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation. 2004, Pubmed
Cui, Xwnt-8b: a maternally expressed Xenopus Wnt gene with a potential role in establishing the dorsoventral axis. 1995, Pubmed , Xenbase
Elms, Zic2 is required for neural crest formation and hindbrain patterning during mouse development. 2003, Pubmed
Gebbia, X-linked situs abnormalities result from mutations in ZIC3. 1997, Pubmed
Heeg-Truesdell, Neural induction in Xenopus requires inhibition of Wnt-beta-catenin signaling. 2006, Pubmed , Xenbase
Heisenberg, A mutation in the Gsk3-binding domain of zebrafish Masterblind/Axin1 leads to a fate transformation of telencephalon and eyes to diencephalon. 2001, Pubmed
Houart, Establishment of the telencephalon during gastrulation by local antagonism of Wnt signaling. 2002, Pubmed
Houston, Maternal Xenopus Zic2 negatively regulates Nodal-related gene expression during anteroposterior patterning. 2005, Pubmed , Xenbase
Idogawa, Poly(ADP-ribose) polymerase-1 is a component of the oncogenic T-cell factor-4/beta-catenin complex. 2005, Pubmed
Idogawa, Ku70 and poly(ADP-ribose) polymerase-1 competitively regulate beta-catenin and T-cell factor-4-mediated gene transactivation: possible linkage of DNA damage recognition and Wnt signaling. 2007, Pubmed
Inoue, Zic2 and Zic3 synergistically control neurulation and segmentation of paraxial mesoderm in mouse embryo. 2007, Pubmed
Ishiguro, ZIC2-dependent transcriptional regulation is mediated by DNA-dependent protein kinase, poly(ADP-ribose) polymerase, and RNA helicase A. 2007, Pubmed
Kiecker, A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus. 2001, Pubmed , Xenbase
Kong, Dickkopf (Dkk) 1 promotes the differentiation of mouse embryonic stem cells toward neuroectoderm. 2009, Pubmed
Lagutin, Six3 repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development. 2003, Pubmed
Mahmoudi, The leukemia-associated Mllt10/Af10-Dot1l are Tcf4/β-catenin coactivators essential for intestinal homeostasis. 2010, Pubmed
Mann, Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. 1999, Pubmed
McGrew, Analysis of Xwnt-4 in embryos of Xenopus laevis: a Wnt family member expressed in the brain and floor plate. 1992, Pubmed , Xenbase
Merzdorf, The zic1 gene is an activator of Wnt signaling. 2006, Pubmed , Xenbase
Mizugishi, Myogenic repressor I-mfa interferes with the function of Zic family proteins. 2004, Pubmed
Moens, Constructing the hindbrain: insights from the zebrafish. 2002, Pubmed
Moon, WNT and beta-catenin signalling: diseases and therapies. 2004, Pubmed
Niehrs, Head in the WNT: the molecular nature of Spemann's head organizer. 1999, Pubmed
Roose, The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors. 1998, Pubmed , Xenbase
Salero, Transcription factors Zic1 and Zic2 bind and transactivate the apolipoprotein E gene promoter. 2001, Pubmed
Sen, Odd paired transcriptional activation of decapentaplegic in the Drosophila eye/antennal disc is cell autonomous but indirect. 2010, Pubmed
Snider, Inhibition of Tcf3 binding by I-mfa domain proteins. 2001, Pubmed , Xenbase
Tran, Wnt/beta-catenin signaling is involved in the induction and maintenance of primitive hematopoiesis in the vertebrate embryo. 2010, Pubmed , Xenbase
Vleminckx, The C-terminal transactivation domain of beta-catenin is necessary and sufficient for signaling by the LEF-1/beta-catenin complex in Xenopus laevis. 1999, Pubmed , Xenbase
Warr, Zic2-associated holoprosencephaly is caused by a transient defect in the organizer region during gastrulation. 2008, Pubmed
Wolda, Overlapping expression of Xwnt-3A and Xwnt-1 in neural tissue of Xenopus laevis embryos. 1993, Pubmed , Xenbase
Yang, ZIC2 and Sp3 repress Sp1-induced activation of the human D1A dopamine receptor gene. 2000, Pubmed