Binding Of TCF7L2 Research Articles

The expression of Lin28B was co-regulated by H3K4me2 and Wnt5a/β-catenin/TCF7L2

Lin28A and Lin28B are homologous RNA-binding proteins that participate in the development of primordial germ cells. The mechanisms underlying expression and regulation of Lin28A have been well documented, but such information for Lin28B is limited. In this study, a fragment of the Lin28B promoter was cloned, the pEGFP-pLin28B vector was constructed. DF-1 chicken fibroblasts were transfected and the expression of green fluorescent protein (GFP) was measured. Furtherly, Lin28B promoter of different lengths fragments was cloned using the chromosome-walking method and the fragments were ligated into the PGL3-Basic vector, and transfected into DF-1 cells. Results of dual-luciferase reporter assay showed that the core of the Lin28B promoter was included in the sequence from –1 431 to –1 034 bp. The binding sites of the transcription factor TCF7L2 was showed within this sequence by bioinformatics analysis. The promoter activity of Lin28B was downregulated (P<0.05) when the TCF7L2 binding site was mutated. Further experiments suggested that Lin28B promoter activity responded to the activation or inhibition of Wnt signaling. Results of chromatin immunoprecipitation and quantitative PCR showed that β-catenin-TCF7L2 may be enriched in the Lin28B promoter core area. In vivo and in vitro activation or inhibition of Wnt signaling significantly up- or down-regulated (P<0.05) Lin28B expression. H3K4me2 enriched in the promoter of Lin28B, which affected the regulation of Wnt signaling to Lin28B. In conclusion, our results showed that H3K4me2 and Wnt5a/β-catenin/TCF7L2 were the positive regulators of Lin28B expression. Findings of this study may lay a theoretical foundation for illuminating the mechanism underlying Lin28B expression.

A High-Throughput Genome-Integrated Assay Reveals Spatial Dependencies Governing Tcf7l2 Binding.

SUMMARYPredicting where transcription factors bind in the genome from their in vitro DNA-binding affinity is confounded by the large number of possible interactions with nearby transcription factors. To characterize the in vivo binding logic for the Wnt effector Tcf7l2, we developed a high-throughput screening platform in which thousands of synthesized DNA phrases are inserted into a specific genomic locus, followed by measurement of Tcf7l2 binding by DamID. Using this platform at two genomic loci in mouse embryonic stem cells, we show that while the binding of Tcf7l2 closely follows the in vitro motif-binding strength and is influenced by local chromatin accessibility, it is also strongly affected by the surrounding 99 bp of sequence. Through controlled sequence perturbation, we show that Oct4 and Klf4 motifs promote Tcf7l2 binding, particularly in the adjacent ~50 bp and oscillating with a 10.8-bp phasing relative to these cofactor motifs, which matches the turn of a DNA helix.

Artesunate inhibits atherosclerosis by upregulating vascular smooth muscle cells-derived LPL expression via the KLF2/NRF2/TCF7L2 pathway

Lipoprotein lipase (LPL) plays a central role in hydrolyzing triglyceride and its deficiency leads to atherosclerosis. Artesunate (ART), a derivative of artemisinin, has been demonstrated that ART reduces the formation of atherosclerotic plaques. However, it remains unclear whether ART-alleviated atherosclerotic lesion is involved in regulating lipid metabolism. ApoE-/- mice were fed a high-fat diet to form atherosclerotic plaques and then injected with artesunate or not. Oil Red O, HE and Masson staining were performed to assess atherosclerotic plaques. Both Western blot and qRT-PCR were applied to detect protein expression. The Luciferase reporter gene and Chromatin immunoprecipitation assays were used to assess the interaction between proteins. Immunofluorescence assay was performed to show the localization of target proteins. In vitro, our data shown that ART increased LPL expression and inhibition of NRF2 blocked the binding of TCF7L2 to LPL promoter region in VSMCs. Downregulated Klf2 could decrease the nuclear enrichment of NRF2, TCF7L2 and LPL expression. In vivo, ART decreased atherosclerotic plaque formation and increased VSMC counts and LPL expression within atherosclerotic plaques. We observed the reduced tendency of serum lipids, and increased in serum LPL activity in mice. In support of vitro data, the markedly increased KLF2, TCF7L2 and LPL expression have been detected in aorta. Our study suggests that ART may be a novel therapeutic drug for inhibition of atherosclerotic plaque formation. The molecular mechanism may involve in upregulation of LPL expression via the KLF2/NRF2/TCF7L2 pathway in VSMCs.

Coordinated Tcf7l2 regulation in a mouse model implicates Wnt signaling in fetal alcohol spectrum disorders.

Mouse models of fetal alcohol spectrum disorders (FASD) have repeatedly identified genes with long-term changes in expression, DNA methylation, noncoding RNA, and histone modifications in response to neurodevelopmental alcohol exposure. Articulation of FASD is achieved via alcohol's effect on gene expression, likely involving epigenetic regulation. The list of genes affected is large and heterogeneous, depending on experimental protocol. We present reanalysis and synthesis of results highlighting the Wnt transcription factor 7 like 2 (Tcf7l2) gene as uniquely compatible with hippocampal DNA methylation, histone modifications, and gene expression changes in a coordinated response to neurodevelopmental alcohol exposure. We data-mined the literature for Tcf7l2 alterations in response to prenatal alcohol exposure. Four studies identified changes in brain Tcf7l2 expression in different FASD models. Further, we performed an in silico TCF7L2 binding site analysis for FASD mouse model data sets. Seven of these published gene lists were significantly enriched for TCF7L2 binding, indicating potential functional relationships. Finally, TCF7L2 is involved in regulation of hundreds of genes, with a role in brain development, myelination, and neuronal function. Tcf7l2 may be involved in neurological defects associated with alcohol exposure via dysregulation of many genes through Wnt signaling. Further functional work is warranted to validate this model for FASD.

The LIM homeodomain protein ISL1 mediates the function of TCF7L2 in pancreatic beta cells.

Pancreatic β-cell Tcf7l2 deletion or its functional knockdown suggested the essential role of this Wnt pathway effector in controlling insulin secretion, glucose homeostasis and β-cell gene expression. As the LIM homeodomain protein ISL1 is a suggested Wnt pathway downstream target, we hypothesize that it mediates metabolic functions of TCF7L2. We aimed to determine the role of ISL1 in mediating the function of TCF7L2 and the incretin hormone GLP-1 in pancreatic β-cells. The effect of dominant negative TCF7L2 (TCF7L2DN) mediated Wnt pathway functional knockdown on Isl1 expression was determined in βTCFDN mouse islets and in the rat insulinoma cell line INS-1 832/13. Luciferase reporter assay and chromatin immunoprecipitation were utilized to determine whether Isl1 is a direct downstream target of Tcf7l2 TCF7L2DN adenovirus infection and siRNA-mediated Isl1 knockdown on β-cell gene expression were compared. Furthermore, Isl1 knockdown on GLP-1 stimulated β-catenin S675 phosphorylation and insulin secretion was determined. We found that TCF7L2DN repressed ISL1 levels in βTCFDN islets and the INS-1 832/13 cell line. Wnt stimulators enhanced Isl1 promoter activity and binding of TCF7L2 on Isl1 promoter. TCF7L2DN adenovirus infection and Isl1 knockdown generated similar repression on expression of β-cell genes, including the ones that encode GLUT2 and GLP-1 receptor. Either TCF7L2DN adenovirus infection or Isl1 knockdown attenuated GLP-1-stimulated β-catenin S675 phosphorylation in INS-1 832/13 cells or mouse islets and GLP-1 stimulated insulin secretion in INS-1 832/13 or MIN6 cells. Our observations support the existence of TCF7L2-ISL1 transcriptional network, and we suggest that this network also mediates β-cell function of GLP-1.

Characterization of Coding and Noncoding Variants for Human CKD Using Novel Strategies

Interpreting the mechanism of genetic variants is one of the greatest challenges impeding the analysis of the rapidly increasing volume of patients' genomic data, particularly for rare variants that do not reach GWAS significance due to low population frequencies. There are 153 GWAS hits for altered kidney function due to commonly inherited genetic variants, yet minimal knowledge of the mechanism of these variations is known at the current time. Utilizing a computational strategy to filter 3,921 genetic variants that are found within linkage disequilibrium of the GWAS hits we have nominated 4 damaging protein coding variants and more importantly 30 noncoding variants predicted to alter transcription factor based gene regulation. One of these noncoding variants, rs17319721 associated with risk of chronic kidney disease (CKD), is found near the SHROOM3 gene and is thought to play a functional role in podocytes of the kidney. In this study we show this variant falls upstream of a novel transcriptional start site that results in a shorter SHROOM3 isoform lacking the PDZ domain. This variant disrupts binding of TCF7L2 in podocytes and the single variant (rs17319721) alters transcription levels of SHROOM3 as determined by variant mutation into the genome using CRISPR/Cas9 technology.To begin deciphering the role of rare variants within human genomes we performed a full assessment of the ExAC database for &gt;60,000 sequenced human exomes, revealing several coding variants in SHROOM3 that are likely to alter function, including a coding variant (P1244L) we show to have association to CKD in humans. We determined that P1244L attenuates the interaction of SHROOM3 with 14‐3‐3 binding and alters signaling through the Hippo pathway, a known mediator of CKD. Animal studies show a loss of function in zebrafish kidney due to the variant, resulting in increased glomerular permeability. These data demonstrate multiple new SHROOM3‐dependent genetic and molecular mechanisms that regulate CKD and demonstrate a novel analytical pipeline with broad application to systematically characterizing risk mechanisms of CKD and other complex diseases.Support or Funding InformationSupport for this study was from NIH‐K01ES025435 (to JWP), NIH‐R01HL069321 (to HJJ), NIH‐R01EY014167 (to BL), the Collaborative Research Centre 1093 through the Deutsche Forschungsgemeinschaft (DFG, to CO), and HudsonAlpha Institute for Biotechnology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inference of hierarchical regulatory network of TCF7L2 binding sites in MCF7 cell line.

The TCF7L2 transcription factor (TF) is a member of Wnt signalling pathway, and may influence transcription of several genes by binding to distinct regulatory regions. Genome-wide studies have identified thousands of TCF7L2 binding sites and have revealed some associated TF partners. However, there is still a large uncharted region in the hierarchical regulatory network for TCF7L2 and the partner TFs in MCF7 cells. We analysed ChIP-seq data by searching for motifs in the enriched peak region based on TF-specific position weight matrix (PWM). We found association of FOXO1 and CAD with up-regulated genes, AP2α, PBF and AP1 with down-regulated genes. TCF7L2 and GATA3 were found to be associated with both up and down-regulated genes. Our study uncovers new TCF7L2 associated regulatory networks by mining ChIP-seq data in MCF7 cell, which may contribute to further study of the mechanisms related to Wnt pathway in breast cancer or other diseases.

The mechanisms of genome-wide target gene regulation by TCF7L2 in liver cells

In the liver Wnt-signaling contributes to the metabolic fate of hepatocytes, but the precise role of the TCF7L2 in this process is unknown. We employed a temporal RNA-Seq approach to examine gene expression 3–96 h following Tcf7l2 silencing in rat hepatoma cells, and combined this with ChIP-Seq to investigate mechanisms of target gene regulation by TCF7L2. Silencing Tcf7l2 led to a time-dependent appearance of 406 differentially expressed genes (DEGs), including key regulators of cellular growth and differentiation, and amino acid, lipid and glucose metabolism. Direct regulation of 149 DEGs was suggested by strong proximal TCF7L2 binding (peak proximity score > 10) and early mRNA expression changes (≤18 h). Indirect gene regulation by TCF7L2 likely occurred via alternate transcription factors, including Hnf4a, Foxo1, Cited2, Myc and Lef1, which were differentially expressed following Tcf7l2 knock-down. Tcf7l2-silencing enhanced the expression and chromatin occupancy of HNF4α, and co-siRNA experiments revealed that HNF4α was required for the regulation of a subset of metabolic genes by TCF7L2, particularly those involved in lipid and amino-acid metabolism. Our findings suggest TCF7L2 is an important regulator of the hepatic phenotype, and highlight novel mechanisms of gene regulation by TCF7L2 that involve interplay between multiple hepatic transcriptional pathways.

A Polymorphic Enhancer near GREM1 Influences Bowel Cancer Risk through Differential CDX2 and TCF7L2 Binding

SummaryA rare germline duplication upstream of the bone morphogenetic protein antagonist GREM1 causes a Mendelian-dominant predisposition to colorectal cancer (CRC). The underlying disease mechanism is strong, ectopic GREM1 overexpression in the intestinal epithelium. Here, we confirm that a common GREM1 polymorphism, rs16969681, is also associated with CRC susceptibility, conferring ∼20% differential risk in the general population. We hypothesized the underlying cause to be moderate differences in GREM1 expression. We showed that rs16969681 lies in a region of active chromatin with allele- and tissue-specific enhancer activity. The CRC high-risk allele was associated with stronger gene expression, and higher Grem1 mRNA levels increased the intestinal tumor burden in ApcMin mice. The intestine-specific transcription factor CDX2 and Wnt effector TCF7L2 bound near rs16969681, with significantly higher affinity for the risk allele, and CDX2 overexpression in CDX2/GREM1-negative cells caused re-expression of GREM1. rs16969681 influences CRC risk through effects on Wnt-driven GREM1 expression in colorectal tumors.

TCF7L2 Modulates Glucose Homeostasis by Regulating CREB- and FoxO1-Dependent Transcriptional Pathway in the Liver

Peripheral insulin resistance contributes to the development of type 2 diabetes. TCF7L2 has been tightly associated with this disease, although the exact mechanism was largely elusive. Here we propose a novel role of TCF7L2 in hepatic glucose metabolism in mammals. Expression of medium and short isoforms of TCF7L2 was greatly diminished in livers of diet-induced and genetic mouse models of insulin resistance, prompting us to delineate the functional role of these isoforms in hepatic glucose metabolism. Knockdown of hepatic TCF7L2 promoted increased blood glucose levels and glucose intolerance with increased gluconeogenic gene expression in wild-type mice, in accordance with the PCR array data showing that only the gluconeogenic pathway is specifically up-regulated upon depletion of hepatic TCF7L2. Conversely, overexpression of a nuclear isoform of TCF7L2 in high-fat diet-fed mice ameliorated hyperglycemia with improved glucose tolerance, suggesting a role of this factor in hepatic glucose metabolism. Indeed, we observed a binding of TCF7L2 to promoters of gluconeogenic genes; and expression of TCF7L2 inhibited adjacent promoter occupancies of CREB, CRTC2, and FoxO1, critical transcriptional modules in hepatic gluconeogenesis, to disrupt target gene transcription. Finally, haploinsufficiency of TCF7L2 in mice displayed higher glucose levels and impaired glucose tolerance, which were rescued by hepatic expression of a nuclear isoform of TCF7L2 at the physiological level. Collectively, these data suggest a crucial role of TCF7L2 in hepatic glucose metabolism; reduced hepatic expression of nuclear isoforms of this factor might be a critical instigator of hyperglycemia in type 2 diabetes.

Cell type-specific binding patterns reveal that TCF7L2 can be tethered to the genome by association with GATA3

BackgroundThe TCF7L2 transcription factor is linked to a variety of human diseases, including type 2 diabetes and cancer. One mechanism by which TCF7L2 could influence expression of genes involved in diverse diseases is by binding to distinct regulatory regions in different tissues. To test this hypothesis, we performed ChIP-seq for TCF7L2 in six human cell lines.ResultsWe identified 116,000 non-redundant TCF7L2 binding sites, with only 1,864 sites common to the six cell lines. Using ChIP-seq, we showed that many genomic regions that are marked by both H3K4me1 and H3K27Ac are also bound by TCF7L2, suggesting that TCF7L2 plays a critical role in enhancer activity. Bioinformatic analysis of the cell type-specific TCF7L2 binding sites revealed enrichment for multiple transcription factors, including HNF4alpha and FOXA2 motifs in HepG2 cells and the GATA3 motif in MCF7 cells. ChIP-seq analysis revealed that TCF7L2 co-localizes with HNF4alpha and FOXA2 in HepG2 cells and with GATA3 in MCF7 cells. Interestingly, in MCF7 cells the TCF7L2 motif is enriched in most TCF7L2 sites but is not enriched in the sites bound by both GATA3 and TCF7L2. This analysis suggested that GATA3 might tether TCF7L2 to the genome at these sites. To test this hypothesis, we depleted GATA3 in MCF7 cells and showed that TCF7L2 binding was lost at a subset of sites. RNA-seq analysis suggested that TCF7L2 represses transcription when tethered to the genome via GATA3.ConclusionsOur studies demonstrate a novel relationship between GATA3 and TCF7L2, and reveal important insights into TCF7L2-mediated gene regulation.

Requirement of TCF7L2 for TGF-β-dependent Transcriptional Activation of the TMEPAI Gene

The TGF-β and Wnt pathways are involved in cell fate and tumorigenicity. A recent report indicated that a TGF-β target gene, TMEPAI (transmembrane prostate androgen-induced RNA), is possibly also a downstream target of Wnt signaling. Although TMEPAI was believed to be involved in tumorigenicity because of its blockage of TGF-β signaling, how TGF-β and Wnt signals affect the activation of the TMEPAI gene is not well understood. Herein, we show that the TMEPAI promoter is regulated synergistically by TGF-β/Smad and Wnt/β-catenin/T cell factor (TCF) 7L2. The critical cis-element for dual signals, termed TGF-β-responsive TCF7L2-binding element (TTE), is located in intron 1 of the TMEPAI gene. TCF7L2, but not Smad proteins, bound to TTE, whereas the disruption of TTE by mutagenesis remarkably counteracted both TGF-β and TCF7L2 responses. The introduction of mutations in critical Smad-binding elements blocked the activation of the TMEPAI promoter by TCF7L2. Furthermore, our DNA-protein interaction experiments revealed the indirect binding of TCF7L2 to Smad-binding elements via Smad3 upon TGF-β stimulation as well as its TGF-β-dependent association with TTE. We demonstrate that the Wnt/β-catenin/TCF7L2 pathway is preferentially able to alter the transcriptional regulation of the TGF-β-target gene, TMEPAI.

BMP and WNT-Directed Transcription Factors TCF7L2/TCF4 and SMAD1 Bind to Distinct Hematopoietic-Specific Target Genes Depending on Cell Lineage

AbstractAbstract 3870The BMP and WNT signaling pathways are two highly conserved signaling pathways that cooperate in many developmental processes, ultimately through alteration of transcription via SMAD and TCF transcription factors. These pathways elicit pleiotropic outcomes across cell types, yet only a few cell-specific direct target genes are known for the signaling transcription factors that mitigate these effects. We took a genome-wide approach to define the binding sites of BMP and WNT-directed transcription factors in different hematopoietic lineages. Using heat-shock inducible transgenic fish lines that overexpress BMP2 or WNT8, we demonstrated accelerated marrow recovery following irradiation. Irradiation recovery was blunted by heat shock induced overexpression of the respective inhibitors Chordin and DKK1. Similar to the zebrafish regeneration results, competitive transplants with mouse bone marrow treated with the WNT agonist BIO led to enhanced chimerism. Inhibition of BMP diminished peripheral blood contribution even in the presence of WNT stimulation, suggesting a conserved and cell intrinsic interaction for these signaling pathways in adult stress hematopoiesis. To examine potential target genes that could account for the synergy, we performed chromatin immunoprecipitation with WNT- and BMP-activated transcription factors followed by sequencing (ChIP-seq) in K562 cells. ChIP-seq was performed with TCF7L2/TCF4, a mediator of the WNT pathway, and SMAD1, a mediator of the BMP signaling pathway, and >2000 binding sites were identified for each factor. Motif discovery revealed that the DNA sequences bound by TCF7L2 and SMAD1 were not only enriched for TCF and SMAD binding elements, respectively, but were also enriched for a GATA motif. Comparison of the TCF7L2 and SMAD1 bound genes with published ChIP-Seq data for GATA1 and GATA2 in K562 cells revealed that both signaling factors bind more than 40% of GATA1 bound genes and greater than 70% of GATA2 bound genes. Ingenuity and GSEA analysis revealed that genes important for erythropoiesis were among the genes co-bound by these factors. To evaluate the effect of cell lineage on signaling factor binding, ChIP-seq of TCF7L2 and SMAD1 in U937, a monocytic leukemia cell line, was performed. Motif discovery of sequences bound in U937 found enrichment for an ETS motif, which is bound by the key myeloid transcription factor Pu.1. In addition, TCF7L2 and SMAD1 bound genes in U937 overlapped genes bound by C/EBPalpha in U937 by greater than 70%. These genes are implicated in monocytic development. The overlap of binding between TCF7L2 in K562 and U937 was less than 15% and the overlap of SMAD1 binding sites between the cell lines was less than 10%, indicating a substantial influence of cell lineage on transcription factor binding. Confirmation of cell type selective binding of TCF7L2 and SMAD1 in vivo was accomplished by ChIP of the transcription factors in zebrafish nucleated erythrocytes. Binding of TCF7L2 and SMAD1 in these cells showed that these factors co-bind with GATA1 in many genes with established roles in erythropoiesis. Together our data suggest the co-binding of WNT- and BMP-specific transcription factors with master regulators of each hematopoietic cell type results in regulation of distinct blood genes based on lineage.(First two authors contributed equally to this work) Disclosures:Zon:FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.