Amino-terminal Enhancer Of Split Research Articles

SIX1 Activation Is Involved in Cell Proliferation, Migration, and Anti-inflammation of Acute Ischemia/Reperfusion Injury in Mice.

Nephrogenic proteins are re-expressed after ischemia/reperfusion (I/R) injury; however, the role of these proteins is still unknown. We found that sine oculis homeobox 1 (SIX1), a developmentally regulated homeoprotein, is reactivated in tubular epithelial cells after I/R injury associated with cell proliferation/migration and anti-inflammation. We demonstrated that SIX1 promoted cell proliferation by upregulating cyclin and glycolytic genes, and might increase cell migration by upregulating the expression of matrix metalloproteinase 9 (MMP9) directly or indirectly in the cell model. Notably, SIX1 targeted the promoters of the amino-terminal enhancer of split (AES) and fused in sarcoma (FUS), which are cofactors of nuclear factor-κB (NF-κB) subunit RELA, and then inhibited the transactivation function of RELA. The expression of monocyte chemotactic protein-1 (MCP-1) was decreased by the SIX1-mediated NF-κB pathway. Our results showed that the expression of cyclin, glycolytic genes, and MMP9 were significantly increased, and the infiltration of monocytes/macrophages (Mophs) was suppressed in SIX1 overexpression kidney at 1, 2, and 3 days after reperfusion. The overexpression of SIX1 resulted in reducing kidney damage from I/R injury in mice by promoting cell proliferation and migration and by inhibiting inflammation. Our study provides evidence that SIX1 involved in cell proliferation, migration, and anti-inflammation in the I/R model, which might be a potential therapeutic target that could be used to ameliorate kidney damage.

Genetic alternations and immune characteristics in patients with small cell lung cancer

Genetic alternations and immune characteristics in patients with small cell lung cancer

The CK1δ/ε-AES axis regulates tumorigenesis and metastasis in colorectal cancer.

Background: Amino-terminal enhancer of split (AES) has been identified as a tumor and metastasis suppressor in some cancers including colorectal cancer (CRC), but very little is known about the regulation of AES expression.Methods: Bioinformatics analysis was used to investigate the expression patterns of AES, CK1δ and CK1ε. The co-immunoprecipitation, GST pull-down, Western Blot, real-time PCR and immunohistochemistry were performed to study the mechanism underlying the regulation of AES expression by CK1δ/ε. The biological function was assessed by in vitro colony formation, transwell, sphere formation, tumor organoids, in vivo tumor metastasis model and patient-derived colorectal tumor xenografts (PDTX) model.Results: A strong inverse relationship was observed between the expression of AES and the expression of CK1δ/ε. Mechanically, AES could interact with CK1δ/ε and SKP2 using its Q domain. SKP2 mediated the ubiquitination and degradation of AES in a CK1δ/ε-dependent manner. CK1δ/ε phosphorylated AES at Ser121 and accelerated the SKP2-mediated ubiquitination and degradation of AES. In colon cancer cells, CK1δ/ε antagonized the effect of wild-type AES but not that of its mutant (S121A) on Wnt and Notch signaling, leading to an increase in the expression of Wnt target genes and Notch target genes. By downregulating the expression of AES, CK1δ/ε enhanced anchorage-independent growth, migration, invasion and sphere formation in colon cancer cells. CK1δ/ε also promoted the growth of APCmin/+ colorectal tumor organoids and liver metastasis in colon cancer mouse models through the regulation of AES degradation. Furthermore, CK1 inhibitor SR3029 treatment suppressed tumor growth via stabilizing AES in APCmin/+ colorectal tumor organoids and patient-derived colorectal tumor xenografts (PDTX).Conclusions: Our results revealed that the CK1δ/ε-AES axis is important for CRC tumorigenesis and metastasis, and targeted inhibition of this axis may be a potential therapeutic strategy for CRC.

Hypothalamus and pituitary transcriptome profiling of male and female Hong Kong grouper (Epinephelus akaara)

Hong Kong grouper (Epinephelus akaara) is an important commercially cultured marine fish in Asia, and a protogynous hermaphrodite with the “diandry” pattern. In order to explore the gene expression patterns of hypothalamus and pituitary between male and female Hong Kong grouper, we used RNA-seq technology to investigate transcriptomes of both tissues in immature and mature male and female adults. This produced 227,227,148 and 215,858,948 high quality reads from hypothalamus and pituitary, which were jointly assembled into 199,203 unigenes. Among them, 30,786 unigenes were mapped to known genes. Differential expression analysis revealed 275 unigenes that were differentially expressed between immature male and female adults and 561 between mature male and female adults. According to annotation and KEGG information, these differentially expressed genes (DEGs) were involved in development, metabolism, and regulation of transcription. One DEG, amino-terminal enhancer of split (AES), a member of the Groucho/transducin-like enhancer of split family of transcriptional regulators that played important roles in neurogenesis, segmentation, and sex determination, was significantly upregulated in male individuals in both immature and mature adult comparisons, indicating it may be involved in male reproductive function during development. Our report, for the first time, uses RNA-seq technology to investigate transcriptomes of both hypothalamus and pituitary in teleost fish, and provides a basis for further studies of molecular mechanism of sex determination and development in Hong Kong grouper.

AML1-ETO requires enhanced C/D box snoRNA/RNP formation to induce self-renewal and leukaemia.

Leukaemogenesis requires enhanced self-renewal, which is induced by oncogenes. The underlying molecular mechanisms remain incompletely understood. Here, we identified C/D box snoRNAs and rRNA 2'-O-methylation as critical determinants of leukaemic stem cell activity. Leukaemogenesis by AML1-ETO required expression of the groucho-related amino-terminal enhancer of split (AES). AES functioned by inducing snoRNA/RNP formation via interaction with the RNA helicase DDX21. Similarly, global loss of C/D box snoRNAs with concomitant loss of rRNA 2'-O-methylation resulted in decreased leukaemia self-renewal potential. Genomic deletion of either C/D box snoRNA SNORD14D or SNORD35A suppressed clonogenic potential of leukaemia cells invitro and delayed leukaemogenesis invivo. We further showed that AML1-ETO9a, MYC and MLL-AF9 all enhanced snoRNA formation. Expression levels of C/D box snoRNAs in AML patients correlated closely with invivo frequency of leukaemic stem cells. Collectively, these findings indicate that induction of C/D box snoRNA/RNP function constitutes an important pathway inleukaemogenesis.

Expression of metastasis suppressor gene AES driven by a Yin Yang (YY) element in a CpG island promoter and transcription factor YY2.

We recently found that the product of the AES gene functions as a metastasis suppressor of colorectal cancer (CRC) in both humans and mice. Expression of amino‐terminal enhancer of split (AES) protein is significantly decreased in liver metastatic lesions compared with primary colon tumors. To investigate its downregulation mechanism in metastases, we searched for transcriptional regulators of AES in human CRC and found that its expression is reduced mainly by transcriptional dysregulation and, in some cases, by additional haploidization of its coding gene. The AES promoter‐enhancer is in a typical CpG island, and contains a Yin‐Yang transcription factor recognition sequence (YY element). In human epithelial cells of normal colon and primary tumors, transcription factor YY2, a member of the YY family, binds directly to the YY element, and stimulates expression of AES. In a transplantation mouse model of liver metastases, however, expression of Yy2 (and therefore of Aes) is downregulated. In human CRC metastases to the liver, the levels of AES protein are correlated with those of YY2. In addition, we noticed copy‐number reduction for the AES coding gene in chromosome 19p13.3 in 12% (5/42) of human CRC cell lines. We excluded other mechanisms such as point or indel mutations in the coding or regulatory regions of the AES gene, CpG methylation in the AES promoter enhancer, expression of microRNAs, and chromatin histone modifications. These results indicate that Aes may belong to a novel family of metastasis suppressors with a CpG‐island promoter enhancer, and it is regulated transcriptionally.

The Anoikis Effector Bit1 Inhibits EMT through Attenuation of TLE1-Mediated Repression of E-Cadherin in Lung Cancer Cells.

The mitochondrial Bcl-2 inhibitor of transcription 1 (Bit1) protein is part of an anoikis-regulating pathway that is selectively dependent on integrins. We previously demonstrated that the caspase-independent apoptotic effector Bit1 exerts tumor suppressive function in lung cancer in part by inhibiting anoikis resistance and anchorage-independent growth in vitro and tumorigenicity in vivo. Herein we show a novel function of Bit1 as an inhibitor cell migration and epithelial–mesenchymal transition (EMT) in the human lung adenocarcinoma A549 cell line. Suppression of endogenous Bit1 expression via siRNA and shRNA strategies promoted mesenchymal phenotypes, including enhanced fibroblastoid morphology and cell migratory potential with concomitant downregulation of the epithelial marker E-cadherin expression. Conversely, ectopic Bit1 expression in A549 cells promoted epithelial transition characterized by cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Specific downregulation of E-cadherin in Bit1-transfected cells was sufficient to block Bit1-mediated inhibition of cell motility while forced expression of E-cadherin alone attenuated the enhanced migration of Bit1 knockdown cells, indicating that E-cadherin is a downstream target of Bit1 in regulating cell motility. Furthermore, quantitative real-time PCR and reporter analyses revealed that Bit1 upregulates E-cadherin expression at the transcriptional level through the transcriptional regulator Amino-terminal Enhancer of Split (AES) protein. Importantly, the Bit1/AES pathway induction of E-cadherin expression involves inhibition of the TLE1-mediated repression of E-cadherin, by decreasing TLE1 corepressor occupancy at the E-cadherin promoter as revealed by chromatin immunoprecipitation assays. Consistent with its EMT inhibitory function, exogenous Bit1 expression significantly suppressed the formation of lung metastases of A549 cells in an in vivo experimental metastasis model. Taken together, our studies indicate Bit1 is an inhibitor of EMT and metastasis in lung cancer and hence can serve as a molecular target in curbing lung cancer aggressiveness.

Repression of HNF1α-mediated transcription by amino-terminal enhancer of split (AES)

HNF1α (Hepatocyte Nuclear Factor 1α) is one of the master regulators in pancreatic beta-cell development and function, and the mutations in Hnf1α are the most common monogenic causes of diabetes mellitus. As a member of the POU transcription factor family, HNF1α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge in their functional complex formation. In this study, we identified the Groucho protein AES (Amino-terminal Enhancer of Split) as a HNF1α-specific physical binding partner and functional repressor of HNF1α-mediated transcription, which has a direct link to glucose-stimulated insulin secretion in beta-cells that is impaired in the HNF1α mutation-driven diabetes.

Characterization of Aes nuclear foci in colorectal cancer cells.

Amino-terminal enhancer of split (Aes) is a member of Groucho/Transducin-like enhancer (TLE) family. Aes is a recently found metastasis suppressor of colorectal cancer (CRC) that inhibits Notch signalling, and forms nuclear foci together with TLE1. Although some Notch-associated proteins are known to form subnuclear bodies, little is known regarding the dynamics or functions of these structures. Here, we show that Aes nuclear foci in CRC observed under an electron microscope are in a rather amorphous structure, lacking surrounding membrane. Investigation of their behaviour during the cell cycle by time-lapse cinematography showed that Aes nuclear foci dissolve during mitosis and reassemble after completion of cytokinesis. We have also found that heat shock cognate 70 (HSC70) is an essential component of Aes foci. Pharmacological inhibition of the HSC70 ATPase activity with VER155008 reduces Aes focus formation. These results provide insight into the understanding of Aes-mediated inhibition of Notch signalling.

The anoikis effector Bit1 displays tumor suppressive function in lung cancer cells.

The mitochondrial Bit1 (Bcl-2 inhibitor of transcription 1) protein is a part of an apoptotic pathway that is uniquely regulated by integrin-mediated attachment. As an anoikis effector, Bit1 is released into the cytoplasm following loss of cell attachment and induces a caspase-independent form of apoptosis. Considering that anoikis resistance is a critical determinant of transformation, we hypothesized that cancer cells may circumvent the Bit1 apoptotic pathway to attain anchorage-independence and tumorigenic potential. Here, we provide the first evidence of the tumor suppressive effect of Bit1 through a mechanism involving anoikis induction in human lung adenocarcinoma derived A549 cells. Restitution of Bit1 in anoikis resistant A549 cells is sufficient to induce detachment induced-apoptosis despite defect in caspase activation and impairs their anchorage-independent growth. Conversely, stable downregulation of Bit1 in these cells significantly enhances their anoikis resistance and anchorage-independent growth. The Bit1 knockdown cells exhibit significantly enhanced tumorigenecity in vivo. It has been previously shown that the nuclear TLE1 corepressor is a putative oncogene in lung cancer, and we show here that TLE1 blocks Bit1 mediated anoikis in part by sequestering the pro-apoptotic partner of Bit1, the Amino-terminal Enhancer of Split (AES) protein, in the nucleus. Taken together, these findings suggest a tumor suppressive role of the caspase-independent anoikis effector Bit1 in lung cancer. Consistent with its role as a tumor suppressor, we have found that Bit1 is downregulated in human non-small cell lung cancer (NSCLC) tissues.

A PROP1-binding factor, AES cloned by yeast two-hybrid assay represses PROP1-induced Pit-1 gene expression

PROP1 mutation causes combined pituitary hormone deficiency (CPHD). Several mutations are located in a transactivation domain (TAD) of Prop1, and the loss of TAD binding to cofactors is likely the cause of CPHD. PROP1 cofactors have not yet been identified. In the present study, we aimed to identify the PROP1-interacting proteins from the human brain cDNA library. Using a yeast two-hybrid assay, we cloned nine candidate proteins that may bind to PROP1. Of those nine candidates, amino-terminal enhancer of split (AES) was the most abundant, and we analyzed the AES function. AES dose-dependently decreased the PROP1-induced Pit-1 reporter gene expression. An immunoprecipitation assay revealed the relationship between AES and PROP1. In a mammalian two-hybrid assay, a leucine zipper-like motif of the AES Q domain was identified as a region that interacted with TAD. These results indicated that AES was a corepressor of PROP1.

Suppression of RND3 activity by AES downregulation promotes cancer cell proliferation and invasion

Amino-terminal enhancer of split (AES) is a member of the Groucho/TLE family. Although it has no DNA-binding site, AES can regulate transcriptional activity by interacting with transcriptional factors. Emerging evidence indicates that AES may play an important role in tumor metastasis, but the molecular mechanism is still poorly understood. In this study, we found that knockdown of AES by RNA interference (RNAi) downregulated RND3 expression at the mRNA and protein levels in MDA-MB-231 and HepG2, two cancer cell lines. Furthermore, luciferase assays showed that overexpression of AES significantly enhanced RND3 promoter activity. Moreover, inhibition of AES both in MDA-MB-231 and HepG2 cells by RNAi significantly promoted cell proliferation, cell cycle progression and invasion, consistent with the effects of RNAi-mediated RND3 knockdown in these cells. For the first time, data are presented showing that alteration of the malignant behavior of cancer cells by AES is related to RND3 regulation, and these findings also provide new insights into the mechanism of AES action in regulating tumor malignancy.

Induction of Apoptosis and Anoikis by Bit1 in Pancreatic Cancer Cells

Pancreatic cancer is a highly aggressive disease with a very high mortality rate among all human cancers. The poor prognosis is in part due to intrinsic resistance to the apoptosis-inducing effects of radio- and chemotherapy. To find alternative cell death pathways that can bypass the apoptotic resistance of pancreatic cancer cells, we examined the role of the novel anoikis effector Bit1 (Bcl-2 inhibitor of transcription) in the survival and apoptotic resistance of pancreatic cancer cells. Bit1 is a mitochondrial protein that induces a caspase-independent apoptosis upon its release into the cytosol following loss of integrin-mediated attachment to extracellular matrix (anoikis). In this report, we observed that ectopic expression of Bit1 in the cytosol reduced viability and induced caspase-independent apoptosis in human pancreatic cancer cell lines, Miapaca-2 and PANC-1. While increased expression of mitochondrial Bit1 in these cells did not induce apoptosis under attached conditions, detachment significantly induced higher level of apoptosis in mitochondrial Bit1-transfected cells than in control transfected cells. Conversely, downregulation of endogenous Bit1 in PANC-1 cells further enhanced their anoikis resistance. Furthermore, exogenous expression of mitochondrial Bit1 in Miapaca-2 cells inhibited their anchorage-independent growth and enhanced their sensitivity to etoposide-mediated apoptosis. Mechanistically, we found that the Bit1 apoptosis function is in part dependent on the groucho related Amino-terminal Enhancer of Split (AES) expression and is abrogated by the transcriptional corepressor TLE1 protein. Consistent with our in vitro findings that Bit1 is an effector of apoptosis in pancreatic tumor cells, we find that Bit1 is significantly downregulated in a fraction of advanced stages of human pancreatic carcinoma tissues. Taken together, these findings indicate that the Bit1-apoptotic pathway can be targeted to trigger cell death in pancreatic cancer cells and implicate Bit1 as a novel therapeutic agent in attenuating pancreatic chemoresistance.

Amino-terminal Enhancer of Split (AES) Interacts with the Oncoprotein NUP98-HOXA9 and Enhances Its Transforming Ability

NUP98-HOXA9 is the prototype of NUP98 fusion oncoproteins that cause acute myeloid leukemia. It consists of an N-terminal FG-rich portion of the nucleoporin NUP98 fused to the homeodomain region of the homeobox protein HOXA9, and acts as an aberrant transcription factor. To identify interacting partners of NUP98-HOXA9, we used a cytoplasmic yeast two-hybrid assay to avoid the nonspecific trans-activation that would occur with the traditional yeast two-hybrid assay due to the transactivating properties of NUP98-HOXA9. We identified amino-terminal enhancer of split (AES), a transcriptional regulator of the transducin-like enhancer/Groucho family as a novel interaction partner of NUP98-HOXA9. The interaction was confirmed by in vitro pulldown and co-immunoprecipitation assays and was shown to require the FG repeat region of NUP98-HOXA9. Immunofluorescence analysis showed that AES localizes primarily to the interior of the nucleus. AES also showed a strong interaction with wild-type NUP98. AES augmented the transcriptional activity of NUP98-HOXA9. In the presence of NUP98-HOXA9, AES caused an increase in long-term proliferation of primary human CD34+ cells with a marked increase in the numbers of primitive cells. These effects of AES were not observed in the absence of NUP98-HOXA9. AES knockdown diminished the transcriptional and proliferative effects of NUP98-HOXA9. AES caused a shift away from the erythroid lineage in cells expressing NUP98-HOXA9. These data establish AES as an interacting partner of NUP98-HOXA9 and show that it cooperates with NUP98-HOXA9 in transcriptional regulation and cell transformation.

Abstract C33: N-terminal fragment of Bit1 apoptotic protein induces autophagy in tumor cells

Abstract Bit1 is a proapoptotic mitochondrial protein associated with anoikis, cell death resulting from the loss of cell attachment to extracellular matrix. Upon cell detachment, Bit1 is released into the cytoplasm, where it complexes with the Groucho family protein amino-terminal enhancer of split, and triggers cell death through a caspase-independent pathway. Bit1 consists of 179 amino acids; the N-terminal 62 amino acid mitochondrial localization domain, a transmembrane domain, and the C-terminal peptidyl-tRNA hydrolase domain. In the present study, we localized the Bit1 cell death domain (BCDD) to the N-terminal 62 amino acids of Bit1 by introducing truncated Bit1 proteins into the cytoplasm with cDNAtransfection and protein transduction. BCDD was more potent than the full-length Bit1 protein, when equivalent amounts of cDNA was transfected. Treatment of cultured prostate and breast cancer cells with recombinant proteins in which BCDD was fused with two cell-internalizing peptides, one of which is tumor specific, also caused cell death. Bit1 and BCDD shared the characteristics previously attributed to Bit1-induced cell death. In addition, we found that the cells exhibited the signs of autophagy. These results define the cell death domain in Bit1 and reveal autophagy as the main mechanism of Bit1-induced cell death. Tumor-targeted exogenous BCDD may have potential as an anticancer agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C33.

AML1/ETO induces self-renewal in hematopoietic progenitor cells via the Groucho-related amino-terminal AES protein

AbstractThe most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein, which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis, we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells, as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients, even in the absence of t(8;21). On a functional level, knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly, self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies, serial replating capacity of primary cells, and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML.

Molecular interactions of the oncogene NUP98-HOXA9 (153.31)

Abstract NUP98-HOXA9 is the prototype of the fusion oncoproteins that result from NUP98 gene rearrangements and are associated with acute myeloid leukemia. It consists of an N-terminal FG-rich portion of the nucleoporin NUP98 fused to a C-terminal portion of the homeobox protein HOXA9, and acts as an aberrant transcription factor. Using a yeast two-hybrid assay we have identified several interacting partners of NUP98-HOXA9 including Dynein Light Chain 1 (DYNLT1) and Amino-terminal Enhancer of Split (AES). We showed that the FG repeat region of NUP98 is critical for these interactions. In addition, we found that DYNLT1 localizes to the nuclear periphery, interacts with other nucleoporins and these interactions are dependent on the C-terminal of DYNLT1. Knockdown of DYNLT1 resulted in reduction of the ability of NUP98-HOXA9 to activate its target promoters. AES localizes primarily to the interior of the nucleus and showed a strong interaction with NUP98 but not with other FG repeat nucleoporins. AES augmented the transcriptional activity of NUP98-HOXA9. In the presence of NUP98-HOXA9, AES caused an increase in long-term proliferation of primary human CD34+ cells with an increase in the numbers of primitive cells. AES caused a shift away from the erythroid lineage in primary CD34+ cells, thus counteracting the effects of NUP98-HOXA9 on differentiation. These data establish that DYNLT1 and AES interact and cooperate with NUP98-HOXA9 in transcriptional regulation and cell transformation.

AES/GRG5: More than just a dominant‐negative TLE/GRG family member

The human Transducin-like Enhancer of Split (TLE) and mouse homologue, Groucho gene-related protein (GRG), represent a family of conserved non-DNA binding transcriptional modulatory proteins divided into two subgroups based upon size. The long TLE/GRGs consist of four pentadomain proteins that are dedicated co-repressors for multiple transcription factors (TF). The second TLE/GRG subgroup is composed of the Amino-terminal Enhancer of Split (AES) in humans and its mouse homolog GRG5 (AES/GRG5). In contrast to the dedicated co-repressor function of long TLE/GRGs, AES/GRG5 can both positively or negatively modulate various TF as well as non-TF proteins in a long TLE/GRG-dependent or -independent manner. Therefore, AES/GRG5 is a functionally dynamic protein that is not exclusively defined by its role as a long TLE/GRG antagonist. AES/GRG5 may function in various developmental and pathological processes but the functional characteristics of endogenous AES/GRG5 in a physiologically relevant context remains to be determined. Developmental Dynamics 239:2795–2805, 2010. © 2010 Wiley-Liss, Inc.

Loss-of-function mutations in the Transcription Factor 7 (T cell factor-1) gene in hepatogastrointestinal cancers

Inappropriate activation of the Wnt signaling pathway has been repeatedly implicated in the tumorigenesis of colon, liver, and gastric cancers. There is accumulating evidences that transcriptional factor 7 (TCF7; also called T cell factor 1) might also be one of the tumor suppressor genes in the Wnt pathway. We performed PCR-based sequencing analysis of the TCF7 gene in 234 alimentary tract cancers. The TCF7 mutants detected in this study were functionally analyzed after they were generated by a QuickChange site-directed mutagenesis kit. We detected 7 somatic mutations in the TCF7 gene, including 4 missense, 2 frameshift, and one 28-bp deletion. In a yeast two-hybrid assay, most of the mutants showed varying degrees of decreased binding to an amino-terminal enhancer of split (AES), a truncated form of Groucho-related protein lacking WD40 repeats. To determine whether mutant TCF7 proteins had decreased DNA binding, we performed electrophoretic mobility shift assays, and the 2 frameshift mutants were shown to have no DNA binding activity. Furthermore, luciferase reporter assays revealed that TCF7 mutants in the presence of AES failed in the AES-dependent transcriptional repression of the reporter gene. In addition, human embryonic kidney 293 cells transfected with TCF7 mutants expressed high levels of cyclin D1, up to 6 times more than cells transfected with wild-type TCF7. Therefore, the TCF7 mutations detected in this study seem to be “loss-of-function mutations“ caused by loss of TCF7 repressor activity through decreased binding to Groucho-related protein and/or DNA, thereby contributing to neoplastic transformation by causing accumulation of cylin D1.

Differential Modulation of TCF/LEF-1 Activity by the Soluble LRP6-ICD

The canonical Wnt/β-catenin (Wnt) pathway is a master transcriptional regulatory signaling pathway that controls numerous biological processes including proliferation and differentiation. As such, transcriptional activity of the Wnt pathway is tightly regulated and/or modulated by numerous proteins at the level of the membrane, cytosol and/or nucleus. In the nucleus, transcription of Wnt target genes by TCF/LEF-1 is repressed by the long Groucho/TLE co-repressor family. However, a truncated member of the Groucho/TLE family, amino terminal enhancer of Split (AES) can positively modulate TCF/LEF-1 activity by antagonizing long Groucho/TLE members in a dominant negative manner. We have previously shown the soluble intracellular domain of the LRP6 receptor, a receptor required for activation of the Wnt pathway, can positively regulate transcriptional activity within the Wnt pathway. In the current study, we show the soluble LRP6 intracellular domain (LRP6-ICD) can also translocate to the nucleus in CHO and HEK 293T cells and in contrast to cytosolic LRP6-ICD; nuclear LRP6-ICD represses TCF/LEF-1 activity. In agreement with previous reports, we show AES enhances TCF/LEF-1 mediated reporter transcription and further we demonstrate that AES activity is spatially regulated in HEK 293T cells. LRP6-ICD interacts with AES exclusively in the nucleus and represses AES mediated TCF/LEF-1 reporter transcription. These results suggest that LRP6-ICD can differentially modulate Wnt pathway transcriptional activity depending upon its subcellular localization and differential protein-protein interactions.