Transcriptional function of E2A, Ebf1, Pax5, Ikaros and Aiolos analyzed by in vivo acute protein degradation in early B cell development.

Early B cell lymphopoiesis depends on E2A, Ebf1, Pax5 and Ikaros family members. In the present study, we used acute protein degradation in mice to identify direct target genes of these transcription factors in pro-B, small pre-B and immature B cells. E2A, Ebf1 and Pax5 predominantly function as transcriptional activators by inducing open chromatin at their target genes, have largely unique functions and are essential for early B cell maintenance. Ikaros and Aiolos act as dedicated repressors to cooperatively control early B cell development. The surrogate light-chain genes Igll1 and Vpreb1 are directly activated by Ebf1 and Pax5 in pro-B cells and directly repressed by Ikaros and Aiolos in small pre-B cells. Pax5 and E2A contribute to V(D)J recombination by activating Rag1, Rag2, Dntt, Irf4 and Irf8. Similar to Pax5, Ebf1 also represses the cohesin-release factor gene Wapl to mediate prolonged loop extrusion across the Igh locus. In summary, in vivo protein degradation has provided unprecedented insight into the control of early B cell lymphopoiesis by five transcription factors.

Tbl1 promotes Wnt-ß-catenin signaling-induced degradation of the Tcf7l1 protein in mouse embryonic stem cells.

Activation of the Wnt-ß-catenin signaling pathway by CHIR99021, a specific inhibitor of GSK3ß, induces Tcf7l1 protein degradation, which facilitates the maintenance of an undifferentiated state in mouse embryonic stem cells (mESCs); however, the precise mechanism is still unclear. Here, we showed that the overexpression of transducin-ß-like protein 1 (Tbl1, also known as Tbl1x) or its family member Tblr1 (also known as Tbl1xr1) can decrease Tcf7l1 protein levels, whereas knockdown of each gene increases Tcf7l1 levels without affecting Tcf7l1 transcription. Interestingly, only Tbl1, and not Tblr1, interacts with Tcf7l1. Mechanistically, Tbl1 translocates from the cytoplasm into the nucleus in association with ß-catenin (CTNNB1) after the addition of CHIR99021 and functions as an adaptor to promote ubiquitylation of the Tcf7l1 protein. Functional assays further revealed that enforced expression of Tbl1 is capable of delaying mESC differentiation. In contrast, knockdown of Tbl1 attenuates the effect of CHIR99021 on Tcf7l1 protein stability and mESC self-renewal. Our results provide insight into the regulatory network of the Wnt-ß-catenin signaling pathway involved in promoting the maintenance of naïve pluripotency.

miR-10167-3p targets TCF7L1 to inhibit bovine adipocyte differentiation and promote bovine adipocyte proliferation.

MicroRNAs (miRNAs) are widely involved in various lipogenic processes, including adipocyte proliferation and differentiation, lipid droplet formation, and adipocyte-specific gene activation. The present study aimed to investigate the gene expression profiles of bovine preadipocytes under high miR-10167-3p expression using the RNA-seq technique and to verify the functions of its downstream target genes on the proliferation and differentiation of bovine preadipocytes. First, RNA-seq identified 573 differentially expressed genes (DEGs), of which 243 were downregulated and 330 were upregulated. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that 15.19% of the DEGs were enriched in pathways related to lipid metabolism. Meanwhile, dual-luciferase reporter gene assay verified the target-binding relationship between miR-10167-3p and TCF7L1. The function of TCF7L1 was assessed using several experiments in adipocytes with high TCF7L1 expression and RNA interference. The mRNA and protein expression of proliferation, differentiation, and apoptosis marker genes were detected using qPCR and western blot, respectively; lipid droplet synthesis was detected using oil red O, Nile red, and bodipy staining; adipocyte proliferation was detected by EdU; and apoptosis was detected using flow cytometry. The results revealed that TCF7L1 overexpression inhibited bovine preadipocyte differentiation and apoptosis and promoted their proliferation, with opposite results obtained with its RNA interference. These results may provide a reference for the subsequent investigation of the molecular mechanism of bovine fat deposition.

Characterization of a chromatin-associated TCF7L1 complex in human embryonic stem cells.

Human embryonic stem cells (hESCs) resemble the pluripotent epiblast cells found in the early postimplantation human embryo and represent the "primed" state of pluripotency. One factor that helps primed pluripotent cells retain pluripotency and prepare genes for differentiation is the transcription factor TCF7L1, a member of a small family of proteins known as T cell factors/Lymphoid enhancer factors (TCF/LEF) that act as downstream components of the WNT signaling pathway. Transcriptional output of the WNT pathway is regulated, in part, by the activity of TCF/LEFs in conjunction with another component of the WNT pathway, ß-CATENIN. Because TCF7L1 plays an important role in regulating pluripotency, we began to characterize the protein complex associated with TCF7L1 when bound to chromatin in hESCs using rapid immunoprecipitation of endogenous proteins (RIME).  Data are available via ProteomeXchange with identifier PXD047582. These data identify known and new partners of TCF7L1 on chromatin and provide novel insights into how TCF7L1 and pluripotency itself might be regulated.

Structural basis of the bHLH domains of MyoD-E47 heterodimer.

The basic helix-loop-helix (bHLH) family is one of the most conserved transcription factor families that plays an important role in regulating cell growth, differentiation and tissue development. Typically, members of this family form homo- or heterodimers to recognize specific motifs and activate transcription. MyoD is a vital transcription factor that regulates muscle cell differentiation. However, it is necessary for MyoD to form a heterodimer with E-proteins to activate transcription. Even though the crystal structure of the MyoD homodimer has been determined, the structure of the MyoD heterodimer in complex with the E-box protein remains unclear. In this study, we determined the crystal structure of the bHLH domain of the MyoD-E47 heterodimer at 2.05 Å. Our structural analysis revealed that MyoD interacts with E47 through a hydrophobic interface. Moreover, we confirmed that heterodimerization could enhance the binding affinity of MyoD to E-box sequences. Our results provide new structural insights into the heterodimer of MyoD and E-box protein, suggesting the molecular mechanism of transcription activation of MyoD upon binding to E-box protein.

VBP1 modulates Wnt/ß-catenin signaling by mediating the stability of the transcription factors TCF/LEFs.

The Wnt/ß-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the ß-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/ß-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/ß-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/ß-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/ß-catenin signaling pathway activity.

TCF7L1 suppresses primitive streak gene expression to support human embryonic stem cell pluripotency.

Human embryonic stem cells (hESCs) are exquisitely sensitive to WNT ligands, which rapidly cause differentiation. Therefore, hESC self-renewal requires robust mechanisms to keep the cells in a WNT inactive but responsive state. How they achieve this is largely unknown. We explored the role of transcriptional regulators of WNT signaling, the TCF/LEFs. As in mouse ESCs, TCF7L1 is the predominant family member expressed in hESCs. Genome-wide, it binds a gene cohort involved in primitive streak formation at gastrulation, including NODAL, BMP4 and WNT3 Comparing TCF7L1-bound sites with those bound by the WNT signaling effector ß-catenin indicates that TCF7L1 acts largely on the WNT signaling pathway. TCF7L1 overlaps less with the pluripotency regulators OCT4 and NANOG than in mouse ESCs. Gain- and loss-of-function studies indicate that TCF7L1 suppresses gene cohorts expressed in the primitive streak. Interestingly, we find that BMP4, another driver of hESC differentiation, downregulates TCF7L1, providing a mechanism of BMP and WNT pathway intersection. Together, our studies indicate that TCF7L1 plays a major role in maintaining hESC pluripotency, which has implications for human development during gastrulation.

Convergence of cMyc and ß-catenin on Tcf7l1 enables endoderm specification.

The molecular machinery that directs formation of definitive endoderm from pluripotent stem cells is not well understood. Wnt/ß-catenin and Nodal signalling have been implicated, but the requirements for lineage specification remain incompletely defined. Here, we demonstrate a potent effect of inhibiting glycogen synthase kinase 3 (GSK3) on definitive endoderm production. We find that downstream of GSK3 inhibition, elevated cMyc and ß-catenin act in parallel to reduce transcription and DNA binding, respectively, of the transcriptional repressor Tcf7l1. Tcf7l1 represses FoxA2, a pioneer factor for endoderm specification. Deletion of Tcf7l1 is sufficient to allow upregulation of FoxA2 in the presence of Activin. In wild-type cells, cMyc contributes by reducing Tcf7l1 mRNA, while ß-catenin acts on Tcf7l1 protein. GSK3 inhibition is further required for consolidation of endodermal fate via upregulation of Sox17, highlighting sequential roles for Wnt signalling. The identification of a cMyc/ß-catenin-Tcf7l1-FoxA2 axis reveals a de-repression mechanism underlying endoderm induction that may be recapitulated in other developmental and patho-logical contexts.

ß-catenin stimulates Tcf7l1 degradation through recruitment of casein kinase 2 in mouse embryonic stem cells.

Activation of the Wnt/ß-catenin signaling pathway by the inhibition of glycogen synthase kinase-3 (GSK-3) will induce Tcf7l1 protein degradation to effectively promote embryonic stem cell (ESC) self-renewal. However, the exact mechanism remains unclear. Here, we found that inhibition of casein kinase 2 (Csnk2) by TBB or DMAT was sufficient to block the reduction of the Tcf7l1 protein induced by CHIR99021, a specific inhibitor of GSK-3. Similarly, downregulation of Csnk2 increased the Tcf7l1 level. In contrast, overexpression of Csnk2 significantly decreased Tcf7l1 protein stability in mouse ESCs. Notably, Csnk2α1 controls Tcf7l1 turnover to a greater degree than the other two isoforms of Csnk2, Csnk2α2 and Csnk2ß, as Csnk2α1-overexpressing mouse ESCs exhibited the lowest level of Tcf7l1. Csnk2α1 interacted with and phosphorylated Tcf7l1. In addition, the association of Csnk2α1 and Tcf7l1 was enhanced by CHIR99021. Our study demonstrated, for the first time, that Csnk2 is involved in Tcf7l1 turnover mediated by the Wnt/ß-catenin signaling pathway. These results expand our understanding of the function and circuit of Wnt/ß-catenin signaling pathway in ESCs.

Tcf7l1 Acts as a Suppressor for the Self-Renewal of Liver Cancer Stem Cells and Is Regulated by IGF/MEK/ERK Signaling Independent of ß-Catenin.

Tcf7l1, which is a key effector molecule of the Wnt/ß-catenin signaling pathway, is highly expressed in various cancers, and it promotes tumor growth. In this study, we demonstrated that unlike its tumor-promoting effects in several other types of cancers, Tcf7l1 expression is downregulated in hepatocarcinoma compared with their adjacent nontumor counterparts. Underexpression of Tcf7l1 is correlated with poorer survival. In liver cancer stem cell (CSC) populations, Tcf7l1 expression is downregulated. Ectopic expression of Tcf7l1 attenuates the self-renewal abilities of liver CSCs. Mechanistically, Tcf7l1 regulates the self-renewal abilities of liver CSCs through transcriptional repression of the Nanog gene, and the effect is independent of ß-catenin. Moreover, we found that Tcf7l1 expression is controlled by extracellular insulin-like growth factor (IGF) signaling, and we demonstrated for the first time that IGF signaling stimulates Tcf7l1 phosphorylation and degradation through the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Overall, our results provide some new insights into how extracellular signals modulate the self-renewal of liver CSCs and highlight the inhibitory roles of Tcf7l1 in cancer. Stem Cells 2019;37:1389-1400.

Tcf7l1 protects the anterior neural fold from adopting the neural crest fate.

The neural crest (NC) is crucial for the evolutionary diversification of vertebrates. NC cells are induced at the neural plate border by the coordinated action of several signaling pathways, including Wnt/ß-catenin. NC cells are normally generated in the posterior neural plate border, whereas the anterior neural fold is devoid of NC cells. Using the mouse model, we show here that active repression of Wnt/ß-catenin signaling is required for maintenance of neuroepithelial identity in the anterior neural fold and for inhibition of NC induction. Conditional inactivation of Tcf7l1, a transcriptional repressor of Wnt target genes, leads to aberrant activation of Wnt/ß-catenin signaling in the anterior neuroectoderm and its conversion into NC. This reduces the developing prosencephalon without affecting the anterior-posterior neural character. Thus, Tcf7l1 defines the border between the NC and the prospective forebrain via restriction of the Wnt/ß-catenin signaling gradient.

Wnt suppressor and stem cell regulator TCF7L1 is a sensitive immunohistochemical marker to differentiate testicular seminoma from non-seminomatous germ cell tumor.

The accurate classification and proper identification of testicular germ cell tumors is imperative for treatment selection and clinical prognosis. Although such distinction can often be achieved by microscopic morphology alone, ancillary tests may at times be needed. T-cell factor 7 L1 (TCF7L1, also known as TCF3), a component of the Wnt signaling pathway, plays important roles in embryonic stem cell self-renewal and lineage specification. Here we examined the immunohistochemical expression and diagnostic utility of TCF7L1 in testicular germ cell tumors. Fifty cases of testicular germ cell tumors were collected, including 23 seminomas, 6 embryonal carcinomas, 1 teratoma, 1 choriocarcinoma, and 19 mixed germ cell tumors. The components of the mixed germ cell tumors were seminoma (n = 3), embryonal carcinoma (n = 18), yolk sac tumor (n = 9), teratoma (n = 15), and choriocarcinoma (n = 4). On immunohistochemistry of TCF7L1, only nuclear staining was considered positive. Staining was graded as negative (<5% of tumor cells stained), minimal (5-25% positive), focal (26-50%), and diffuse (>50%). All non-seminomatous components (n = 54) exhibited distinct nuclear expression of TCF7L1 (54/54; 100%). In contrast, no TCF7L1 expression was detected in the majority of seminomatous tumor component (24/26; 92%). Two seminomas (2/26; 8%) exhibited minimal weak nuclear staining (5% and 10%, respectively) for TCF7L1. In conclusion, TCF7L1, highly expressed in non-seminomatous testicular germ cell tumors, might be used as a marker for diagnosis of testicular germ cell tumors, two therapeutically different entities, for better patient management.

TCF7L1 indicates prognosis and promotes proliferation through activation of Keap1/NRF2 in gastric cancer.

Gastric cancer is one of the most common cancers worldwide and is the third leading cause of cancer-related deaths globally. Although significant progress has been made in the diagnosis and treatment for the cancer, less improvement has been made in overall survival rate. Thus, there is an urgent need for a better understanding of the biological aspects of the cancer. The transcription factor transcription factor 7-like 1 (TCF7L1) is an embryonic stem cell signature gene that is upregulated in multiple aggressive cancer types, but its role in gastric cancer has seldom been discussed. In the present study, by using the Cancer Genome Atlas dataset analysis, we demonstrated that patients with higher expression of TCF7L1 could be used to reflect prognosis. An examination of the mechanisms demonstrated that TCF7L1 could positively regulate antioxidant response in gastric cancer cells by positively regulating Keap1/NRF2 [Kelch-like ECH-associated protein 1/nuclear factor (erythroid-derived 2)-like 2] pathway. Collectively, our data demonstrated that TCF7L1 is a novel marker for predicting overall survival of gastric cancer and provided the possible underlying molecular mechanism.

Telomeric noncoding RNA promotes mouse embryonic stem cell self-renewal through inhibition of TCF3 activity.

Although long noncoding RNAs (lncRNAs) are emerging as new modulators in the fate decision of pluripotent stem cells, the functions of specific lncRNAs remain unclear. Here, we found that telomeric RNA (TERRA or TelRNA), one type of lncRNAs, is highly expressed in mouse embryonic stem cells (mESCs) but declines significantly upon differentiation. TERRA is induced by the Wnt/ß-catenin signaling pathway and can reproduce its self-renewal-promoting effect when overexpressed. Further studies revealed that T cell factor 3 ( TCF3) is a potential downstream target of TERRA and mediates the effect of TERRA in mESC maintenance. TERRA inhibits TCF3 transcription, while enforced TCF3 expression abrogates the undifferentiated state of mESCs supported by TERRA. Accordingly, the transcripts of the pluripotency genes Esrrb, Tfcp2l1, and Klf2, repressed by TCF3 in mESCs, are increased in TERRA-overexpressing cells. Our study therefore highlights the important role of TERRA in mESC maintenance and also uncovers a mechanism by which TERRA promotes self-renewal. These data will expand our understanding of the pluripotent regulatory network of ESCs.

TCF7L1 recruits CtBP and HDAC1 to repress DICKKOPF4 gene expression in human colorectal cancer cells.

The T-cell factor/Lymphoid enhancer factor (TCF/LEF; hereafter TCF) family of transcription factors are critical regulators of colorectal cancer (CRC) cell growth. Of the four TCF family members, TCF7L1 functions predominantly as a repressor of gene expression. Few studies have addressed the role of TCF7L1 in CRC and only a handful of target genes regulated by this repressor are known. By silencing TCF7L1 expression in HCT116 cells, we show that it promotes cell proliferation and tumorigenesis in vivo by driving cell cycle progression. Microarray analysis of transcripts differentially expressed in control and TCF7L1-silenced CRC cells identified genes that control cell cycle kinetics and cancer pathways. Among these, expression of the Wnt antagonist DICKKOPF4 (DKK4) was upregulated when TCF7L1 levels were reduced. We found that TCF7L1 recruits the C-terminal binding protein (CtBP) and histone deacetylase 1 (HDAC1) to the DKK4 promoter to repress DKK4 gene expression. In the absence of TCF7L1, TCF7L2 and ß-catenin occupancy at the DKK4 promoter is stimulated and DKK4 expression is increased. These findings uncover a critical role for TCF7L1 in repressing DKK4 gene expression to promote the oncogenic potential of CRCs.

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer.

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.

Tcf7l1 prepares epiblast cells in the gastrulating mouse embryo for lineage specification.

The core gene regulatory network (GRN) in embryonic stem cells (ESCs) integrates activities of the pro-self-renewal factors Oct4 (Pou5f1), Sox2 and Nanog with that of an inhibitor of self-renewal, Tcf7l1 (Tcf3). The inhibitor function of Tcf7l1 causes dependence on extracellular Wnt/ß-catenin signaling activity, making its embryonic role within the ESC GRN unclear. By analyzing intact mouse embryos, we demonstrate that the function of Tcf7l1 is necessary for specification of cell lineages to occur concomitantly with the elaboration of a three-dimensional body plan during gastrulation. In Tcf7l1(-/-) embryos, specification of mesoderm is delayed, effectively uncoupling it from the induction of the primitive streak. Tcf7l1 repressor activity is necessary for a rapid switch in the response of pluripotent cells to Wnt/ß-catenin stimulation, from one of self-renewal to a mesoderm specification response. These results identify Tcf7l1 as a unique factor that is necessary in pluripotent cells to prepare them for lineage specification. We suggest that the role of Tcf7l1 in mammals is to inhibit the GRN to ensure the coordination of lineage specification with the dynamic cellular events occurring during gastrulation.

The Sox4/Tcf7l1 axis promotes progression of BCR-ABL-positive acute lymphoblastic leukemia.

The transcription factor Sox4 plays an indispensable role in the development of early progenitor B cells from hematopoietic stem cells. However, its role in B-cell acute lymphoblastic leukemia, a malignant counterpart of normal progenitor B cells, is not fully understood. Here we show that SOX4 is highly expressed in human acute lymphoblastic leukemia cells. To systematically study the function of Sox4 in acute lymphoblastic leukemia, we established a genetically defined mouse leukemia model by transforming progenitor B cells carrying a floxed Sox4 allele and inducing deletion of the allele by the self-excising Cre recombinase. This model allowed us to work with two groups of leukemic cells that had either one copy or both copies of Sox4 deleted. We found that depletion of Sox4 in transformed cells in vitro reduced cell growth in vitro and the progression of leukemia in vivo. Moreover, depletion of Sox4 in leukemic cells in vivo prolonged the survival of the mice, suggesting that it could be a potential target in acute lymphoblastic leukemia therapy. Our microarray and bioChIP studies revealed that Tcf7l1 was the key gene directly regulated by Sox4. Knockdown of Tcf7l1 reduced cell proliferation, just as did knockout of Sox4, and ectopic expression of Tcf7l1 could reverse the effect of Sox4 knockout on cell proliferation. These data suggest that Sox4 and Tcf7l1 form a functional axis that promotes the progression of BCR-ABL-positive acute lymphoblastic leukemia.

TCF7L1 regulates colorectal cancer cell migration by repressing GAS1 expression.

Dysregulated Wnt/ß-catenin signaling is a common feature of colorectal cancer (CRC). The T-cell factor/lymphoid enhancer factor (TCF/LEF; hereafter, TCF) family of transcription factors are critical regulators of Wnt/ß-catenin target gene expression. Of the four TCF family members, TCF7L1 predominantly functions as a transcriptional repressor. Although TCF7L1 has been ascribed an oncogenic role in CRC, only a few target genes whose expression it regulates have been characterized in this cancer. Through transcriptome analyses of TCF7L1 regulated genes, we noted enrichment for those associated with cellular migration. By silencing and overexpressing TCF7L1 in CRC cell lines, we demonstrated that TCF7L1 promoted migration, invasion, and adhesion. We localized TCF7L1 binding across the CRC genome and overlapped enriched regions with transcriptome data to identify candidate target genes. The growth arrest-specific 1 (GAS1) gene was among these and we demonstrated that GAS1 is a critical mediator of TCF7L1-dependent CRC cell migratory phenotypes. Together, these findings uncover a novel role for TCF7L1 in repressing GAS1 expression to enhance migration and invasion of CRC cells.

Phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2.

Wnt pathways play essential roles in cell proliferation, morphogenesis, and cell fate specification during embryonic development. According to the consensus view, the Wnt pathway prevents the degradation of the key signaling component ß-catenin by the protein complex containing the negative regulators Axin and glycogen synthase kinase 3 (GSK3). Stabilized ß-catenin associates with TCF proteins and enters the nucleus to promote target gene expression. This study examines the involvement of HIPK2 (homeodomain-interacting protein kinase 2) in the regulation of different TCF proteins in Xenopus embryos in vivo. We show that the TCF family members LEF1, TCF4, and TCF3 are phosphorylated in embryonic ectoderm after Wnt8 stimulation and HIPK2 overexpression. We also find that TCF3 phosphorylation is triggered by canonical Wnt ligands, LRP6, and dominant negative mutants for Axin and GSK3, indicating that this process shares the same upstream regulators with ß-catenin stabilization. HIPK2-dependent phosphorylation caused the dissociation of LEF1, TCF4, and TCF3 from a target promoter in vivo. This result provides a mechanistic explanation for the context-dependent function of HIPK2 in Wnt signaling; HIPK2 up-regulates transcription by phosphorylating TCF3, a transcriptional repressor, but inhibits transcription by phosphorylating LEF1, a transcriptional activator. Finally, we show that upon HIPK2-mediated phosphorylation, TCF3 is replaced with positively acting TCF1 at a target promoter. These observations emphasize a critical role for Wnt/HIPK2-dependent TCF phosphorylation and suggest that TCF switching is an important mechanism of Wnt target gene activation in vertebrate embryos.