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Abstract [Objectives] This study was conducted to investigate characteristics of the human TCF7L2 gene promoter. [Methods] The 2 000 bp sequence of the 5’ regulatory region of the human TCF7L2 gene was obtained from the UCSC genome database. The promoter， transcription factor binding sites， CpG islands， SNPs and so on were analyzed by a variety of online softwares. [Results] The bioinformatics analysis results showed there were at least 5 potential promoters in the positive-sense strand of the 2 000 bp sequence， among which -242--192 bp， -853--803 bp might contain core promoters. A TATA box and a CpG island with a length of 499 bp were found. 241， 944 and 1 035 （positive-sense strand） transcription factor binding sites were predicted by the AliBaba2.1， PROMO and JASPAR softwares， respectively. 207 common transcription factor binding sites in the conserved region of human and mouse homologous TCF7L2 gene promoter were identified with CONREAL program， involving 66 kinds of transcription factors. Two SNPs were found in the promoter region. [Conclusions] The promoter of the human TCF7L2 gene was analyzed by bioinformatics， and the promoter characteristics were obtained.
Key words TCF7L2; Promoter; Transcription factor; Transcription factor binding sites; Bioinformatics
Received： July 3， 2020 Accepted： September 9， 2020
Supported by the Diabetes Special Fund Project of Hubei University of Science and Technology （2016-18XZ12）.
Siyuan BAO （1976-）， male， P. R. China， senior experimentalist， master， devoted to research about molecular biology.
*Corresponding author. E-mail： baosyuan@163.com.
Type 2 diabetes is a slowly progressive disease， mostly in developing countries. At present， the number of diabetic patients in China is about 121 million， of which type 2 diabetes （T2D） patients account for about 90%[1]. The pathogenesis of type 2 diabetes is very complicated， and genetic factors and environmental factors together determine the occurrence of type 2 diabetes[2]. Searching for diabetes susceptibility factors and genes and preventing and treating diabetes at the molecular level are the current priorities. TCF7L2 is currently one of the genes with the highest degree of association with T2D found in the genome-wide association study （GWAS）. TCF7L2， also known as T cell transcription factor 4 （TCF4）， is located on human chromosome 10q25.3. It is abundantly expressed in human pancreatic β cells and adipose tissue. Its expression product is a key protein of the Wnt signaling pathway and has a regulatory effect on the function of pancreatic β cells. In studies on different races， it has been confirmed that single nucleotide polymorphisms （SNPs） at multiple sites are associated with T2D. Several recent studies have confirmed that TCF7L2 rs7903146 C>T is related to the development of T2D[3-7]. Mari Cassol Ferreira et al.[8] showed that the existence of TCF7L2 rs7903146 T allele in T2D patients was related to the increase in postprandial insulin， proinsulin and C-peptide secretion; and after exenatide acetate treatment， the plasma insulin and C peptide peaks of T allele carriers after meals were significantly lower than those of patients who did not carry the T allele. Li et al.[9] found that individuals with the T allele of the TCF7L2 rs12255372G/T gene polymorphism may be more susceptible to T2D in the Chinese population. Li et al.[10] found that the TCF7L2 rs290481 T>C polymorphism was significantly related to the occurrence of T2D in the Han population in eastern China， and the T→C mutation of rs290481 promoted the increase of FPG level. Xia et al.[11] used genome editing technology to reveal that the rs7903146 region of TCF7L2 seems to be the interaction region between the ACSL5 promoter and TCF7L2， and the deletion of this region in two human colon cell lines significantly reduced the expression of ACSL5; and given that ACSL5 is involved in lipid biosynthesis， it is speculated that inhibiting ACSL5 enzyme activity can significantly improve insulin sensitivity. Dan et al.[12] pointed out that miR-181a-5p regulated TGFβ/Smad and Wnt signaling pathways by directly targeting Smad7 and TCF7L2， and promoted 3T3-L1 preadipocyte differentiation and adipogenesis. The expression level of TCF7L2 is closely related to the dedifferentiation of β cells. Down-regulation of TCF7L2 expression can increase the dedifferentiation of β cells induced by high glucose， and overexpression of TCF7L2 can reduce the dedifferentiation of β cells induced by high glucose[13]. TCF7L2 is involved in the regulation of glucose metabolism and may become a potential therapeutic target for patients with prediabetes[14]. Although TCF7L2 has been deeply and meticulously explored， the exact mechanism by which TCF7L2 and their respective SNPs change the metabolic process and affect the susceptibility to type 2 diabetes remains to be determined. It has not yet reached a consensus on how TCF7L2 increases the prevalence of T2D. To clarify these problems， it is necessary to understand the expression regulation mechanism of the TCF7L2 gene itself， and study it from the perspective of network regulation. The human TCF7L2 gene promoter sequence has not been recorded in the NCBI database， and the bioinformatics analysis of the TCF7L2 promoter has not been reported. In order to obtain the promoter sequence and characteristics of the human TCF7L2 gene， we used bioinformatics research methods to screen out potential promoter sequences from the genome database， and analyzed transcription factors and their binding sites， CpG islands， SNPs， etc.， laying a theoretical foundation for the study on the expression regulation of TCF7L2 gene and its mechanism of participating in the pathogenesis of T2D.
Materials and Methods
Materials
The human TCF7L2 gene is located at 10q25.2-q25.3. It spans 217 460 bp， contains 19 exons， 18 introns， and has 15 transcription variants. TCF7L2 variant 1 encodes the longest variant， with the mRNA accession number： NM_001146274.1， and the corresponding nucleotide sequence is located at hg38 chr10： 112 950 407 to 113 167 493. The 2 000 bp DNA sequence upstream of chr10： 112 950 407 was selected for the prediction and analysis of promoters， transcription factor binding sites， CpG islands， and SNPs.
Methods
Acquisition of the 2 000 bp sequence of the 5’ regulatory region of the human TCF7L2 gene
In the UCSC （http：//genome.ucsc.edu/） website， the site of the TCF7L2 gene in the human genome （GRCh38/hg38） was searched. With the transcription initiation site as +1， a 2 000 bp DNA sequence upstream of the transcription start site was obtained， and used as a candidate promoter.
Acquisition of the 2 000 bp sequence of the 5’ regulatory region of the mouse homologous TCF7L2 gene
With NM_001146274.1 as a probe， the Blast tool （https：//blast.ncbi.nlm.nih.gov/Blast.cgi） was used to search the mouse RefSeqRNA database to obtain the mouse homologous sequence NM_001331147.1. NM_001331147.1 is the mRNA of mouse TCF7L2 gene variant 22. On the UCSC website， the site of NM_001331147.1 in the mouse genome （GRCm38/mm10） was searched， and the 2 000 bp sequence upstream of the 5’ transcription start site was obtained. Promoter region prediction and analysis
The Neural Network Promoter Prediction （http：//www.fruitfly.org/seq_tools/promoter.html）， Promoter2.0 （http：//www.cbs.dtu.dk/services/Promoter/）， TSSG （http：//linux1.softberry.com/berry.phtml？topic = tssg&group = programs&subgroup = promoter） and Proscan （https：//www-bimas.cit.nih.gov/molbio/proscan/） online software were used to predict the potential promoter region in the 5’ regulatory region of the human TCF7L2 gene， and perform comparative analysis. Parameter setting： Neural Network Promoter Prediction promoter threshold was set to be 0.8， and the default values of Promoter 2.0， TSSG and Proscan were used.
Motif recognition of TATA box， GC box and CAAT box
The motif recognition of TATA box， GC box and CAAT box could be realized by whether the corresponding characteristic sequence is found in the 5’ regulatory region sequence of TCF7L2 gene. The TATA box sequence format is TATAWAW （W stands for A or T）; the GC box sequence format is GGGCGG; and the CAAT box sequence format is CCAAT.
Prediction and analysis of transcription factor binding sites in promoter region
The online software AliBaba2.1 （http：//gene-regulation.com/pub/programs/alibaba2/index.html）， PROMO （http：//alggen.lsi.upc.es/） and JASPAR （http：//jaspar.genereg.net/） were used to predict the transcription factor binding sites in the 5’ regulatory region of the human TCF7L2 gene， and perform statistical analysis to screen out common transcription factors. AliBaba2.1 parameter setting： Min mat. Conservation was set to be 75%， and other parameters were default values. PROMO parameter setting： Considering factors were selected as the Only human factors， the Considering sites were selected as the Only human sites， and other parameters were default values. JASPAR parameter setting： Homo sapiens was selected for JASPAR matrix models species， the Relative profile score threshold was set to be 85%， and other parameters were set as default values.
Prediction of transcription factor binding sites in conserved regions of the TCF7L2 gene promoter in human and mouse
The online software CONREAL （http：//conreal.niob.knaw.nl/） was used to predict the binding sites of transcription factors in the conserved regions of the TCF7L2 gene promoter in human and mouse. The 2 000 bp sequences upstream of the 5’ transcription start sites of human and mouse TCF7L2 gene were input into the CONREAL program. Then， the threshold for PWMs was set to be 85%， the threshold for homology was set to be 75%， and three homology tools were selected to obtain the conserved regions in the two; and the two vertebrate transcription factor binding site databases JASPAR and Transfac v.8.2 were searched to obtain transcription factor binding sites that are located at the same position of the conserved regions of the sequence. Prediction of methylated CpG islands in the promoter region of the human TCF7L2 gene
EMBOSS （http：//www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/） and MethPrimer （http：//www.urogene.org/cgi-bin/methprimer/methprimer.cgi） online software were used to predict human TCF7L2 CpG islands in the promoter region of the gene. Parameter setting： CpG detection content/expected content （Obs/Exp）>0.60， C+ G content percentage>50%， CpG island length>200 bp.
SNP Screening in promoter region and its potential function prediction
The SNP screening tool： F-SNP software （http：//compbio.cs.queensu.ca/F-SNP/）. SNP function analysis software： SNP Function Prediction （http：//snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm）.
Results and Analysis
Prediction and analysis of the promoter region of the human TCF7L2 gene
The -1--2 000 bp sequence upstream of the 5’ regulatory region of the human TCF7L2 gene was obtained from the UCSC database， and the potential promoter regions of the 2 000 bp sequence was analyzed using 4 different online softwares. It was found by querying the UCSC database that the TCF7L2 gene is located on the "+" strand， so the potential promoters whose predicted region was on the positive-sense strand were selected. The results are shown in Table 1. The 2 000 bp sequence upstream of the 5’ regulatory region of the human TCF7L2 gene was subjected to full sequence alignment with the human TCF7L2 gene promoter sequence HPRM36386 （product number） queried on the genecopoeia website （http：//www.genecopoeia.com/） using the BLAST tool， and it was found that identity of the two reached 81%. HPRM36386 has a total length of 1 628 bp， and the transcription start site （TSS） is located at the A base of 1 470 bp. The 375-2 000 bp of the 2 000 bp sequence upstream of the 5’ regulatory region of the TCF7L2 gene was exactly the same as the HPRM36386 sequence， and the A base of 1 844 bp corresponded to the 1 470 bp A base of the HPRM36386 sequence. It was speculated that the TCF7L2 gene promoter was located within the range of the 1 700 bp sequence upstream of the 5’ regulatory region.
Motif recognition of TATA box， GC box and CAAT box
By searching the corresponding characteristic sequences of TATA box， GC box and CAAT box， we found that there was a TATA box in the 5’ regulatory region of the human TCF7L2 gene， located between -1 698 and -1 692 bp， and no GC box and CAAT box were found.
Prediction and analysis of transcription factor binding sites in the promoter region of human TCF7L2 gene In order to improve the accuracy of transcription factor binding site prediction， three softwares， AliBaba2.1， PROMO and JASPAR， were used to predict the transcription factor binding sites in the promoter region of the human TCF7L2 gene. The three softwares predicted 241， 944， and 1，035 （sense strand） transcription factor binding sites within 1 to 2 000 bp upstream of the 5’ regulatory region， involving 75， 76， and 208 transcription factors， respectively. There were 18 transcription factors that predicted by two softwares at the same time with the same binding site position （Table 2）.
Analysis of transcription factor binding sites in the conserved regions of the TCF7L2 gene promoter in human and mouse
According to the evolutionary footprint analysis， the CONREAL program was used to determine the conserved regions of the TCF7L2 gene promoter in human and mouse. The transcription factor binding sites in the conserved regions obtained by the three prediction methods （CONREAL， LAGAN， MAVID） were mostly the same. The CONREAL method obtained 207 transcription factor binding sites in the conserved regions （Table 3）， involving 66 kinds of transcription factors such as Sp1， CREB， c-Jun， GATA-1， AP-2alphaA， CdxA， FREAC-4， FREAC-3， cap， and HNF-3beta， among which Sp1， CREB， c-Jun， GATA-1， and AP-2alphaA were predicted by AliBaba2.1 and PROMO at the same time.
Analysis of methylated CpG islands in the promoter region of the human TCF7L2 gene
The online software EMBOSS and MethPrimer were used to predict methylated CpG islands in the 2 000 bp sequence upstream of the 5’ regulatory region of the human TCF7L2 gene. The prediction results of the EMBOSS software showed that there was a CpG island with a length of 499 bp， located at 1 443 to 1 941 bp of the predicted sequence （Fig. 1）. The prediction results of MethPrimer software showed a CpG island with a size of 450 bp located at 1 443 to 1 892 bp（Fig. 2）， which was consistent with the prediction result of the EMBOSS software.
Prediction and analysis of SNPs in the promoter region of the human TCF7L2 gene
The chromosomal loci （Chr 10： 112 948 407-112 950 406） at 1-2 000 bp upstream of the 5’ regulatory region of the human TCF7L2 gene were input in the F-SNP software， and 2 SNPs： rs11195470 and rs7083546 were searched （as shown in Table 4）. The SNP Function Prediction software was used to perform function prediction and race-specific allele frequency query. The results showed that rs11195470 and rs7083546 had extremely low conservativeness. There were racial differences in the rs11195470 allele frequency between the four populations. The rs11195470 allele frequency was the highest in Americans of Nordic and Western European descent （CEU）， and the same for the Japanese and Nigerian （Table 5）. In order to explore the influence of rs11195470 and rs7083546 polymorphisms on transcription factor binding， the AliBaba2.1 software was used to predict the transcription factor binding sites of the sequence where the SNPs are located. The results are shown in Table 6. Discussion
TCF7L2 belongs to the transcription factors of the TCF/LEF family and is involved in human diseases including cancer， type 2 diabetes and bipolar affective disorder[15]. The TCF7L2 gene is encoded by 19 exons， and the selection of the transcription start site and alternative splicing produce hundreds of potential protein variants[15-16]. TCF7L2 gene has tissue-specific splicing， suggesting that TCF7L2 may have different functions in different cells[15]. A variety of genetic diseases， mainly metabolic and cardiovascular diseases， are largely due to an extreme upstream TCF7L2 binding element driving the transcription of metabolism-related genes[17]. In the Wnt signaling pathway， TCF7L2 protein binds with β-catenin to form an active nuclear complex， activates the proglucagon gene， and promotes the secretion of glucagon-like polypeptide-1 （GLP-1） from small intestinal L cells to maintain Steady state of blood glucose[18].
Wu et al.[17] showed that TCF7L2 binds tightly to the specific binding region of the PIK3R1 promoter， and significantly controls the transcription of the encoded protein p85， thereby affecting the activation of the PI3K/AKT signaling pathway and insulin secretion. Microarray research also found consistent sequences of proprotein convertase （PC） 1 and 2 genes binding to TCF7L2， suggesting that TCF7L2 plays a role in insulin processing[16]. Because the TCF7L2 target gene has tissue specificity， studies on the binding mode of TCF7L2 genomic DNA show that TCF7L2 specifically binds to multiple genes that regulate liver cell sugar metabolism， including Pck1， Fbp1， Irs1， Irs2， Akt2， Adipor1， Pdk4， and Cpt1a[19]. Lavanya Miyer et al.[20] also identified a series of validated TCF7L2 cardiac-specific target genes， such as Hand2， Tbx20， Rock2 and Dstn. However， the binding mode of TCF7L2 and its candidate target genes involved in pancreatic gluconeogenesis remains unclear. In T2D， the TCF7L2 gene rs7903146 （C/T） polymorphism showed a significant association in almost all groups and races[21]. Shanmugapriya Chandrasekaran et al.[22] pointed out that TCF7L2 rs7903146 T+ genotype caused a decrease in GLP-1 level， and GLP-1 level increased the risk of type 2 diabetes by reducing postprandial insulin levels and increasing insulin resistance. Andreas Leiherer et al.[23] studied the molecular mechanism of the TCF7L2 polymorphism rs7903146 and the risk of T2D and found that： serotonin was the most important metabolite in carriers of dangerous alleles， and serotonin was significantly related to the rs7903146 genotype after being fully adjusted. This finding suggests for the first time that the influence of TCF7L2 polymorphism on the risk of type 2 diabetes may involve serotonin-dependent pathway. Recent studies have shown that TCF7L2 has a regulatory effect on β cell quality. TCF7L2 may play an important role in the pathogenesis of diabetes by participating in regulating the proliferation and apoptosis of pancreatic β-cells[24]. Kathleen A. Bailey et al.[25] showed that the decreased expression of TCF7L2 in β cells led to impaired insulin secretion， and further pointed out that β cells with low TCF7L2 expression occupied less area of the pancreas， confirming that the expression of TCF7L2 is of great significance to the survival and proliferation of β cells. The silencing of mammalian pancreatic islets or biological β-cell line TCF7L2 leads to decreased expression of proinsulin gene， decreased glucose-stimulated insulin secretion， exocytosis defect of insulin-containing particles， and decreased insulin secretion[26]. In August 2014， a team from Lund University published a report stating that TCF7L2 can positively regulate the expression of transcription factors such as MAFA， PDX-1 and NKX6.1 by targeting insulin gene enhancer binding protein-1 （ISL1）[27]. This finding provides evidence for suggesting the role of TCF7L2 in β cell regeneration. Yao et al.[28] proposed a model to explain the new mechanism of geniposide to promote the regeneration and survival of β cells. In this model， β-catenin/TCF7L2 is the core component required for the function of geniposide. Geniposide can activate the β-catenin/TCF7L2 transcription complex and induce βcell regeneration by stimulating βcell proliferation and differentiation. Meanwhile， geniposide promotes the survival of β cells by inhibiting the level of c-casp3 to inhibit β-cell apoptosis， and supports β-catenin/TCF7L2 as a possible target for diabetes treatment to promote the formation of new β cells. The promoter is located near the transcription start point and is a DNA sequence that RNA polymerase binds and initiates transcription. Four promoter analysis softwares with different principles and algorithms were used to analyze the 2 000 bp sequence upstream of the 5’ regulatory region of the human TCF7L2 gene， and it was found that there were at least 5 potential promoter regions on the positive-sense strand of the gene， located at -167 to -117 bp， -242 to -192 bp， -600 to -308 bp， -853 to -801 bp， and -1 700 to -1 501 bp， respectively. Combining with results of the sequence alignment with HPRM36386， it could be basically confirmed that the human TCF7L2 gene promoter was located within the range of 1 700 bp upstream of the 5’ regulatory region， the core promoter regions was located at -242--192 bp and -853--803 bp， and the transcription start site was located at the A base of 1 844 bp in the 2 000 bp sequence of the 5’ regulatory region. In the gene expression regulatory network， the binding of transcription factors and cis-acting elements can initiate the expression of a set of specific genes， or turn off the expression of a set of genes. Three online softwares， AliBaba2.1， PROMO and JASPAR predicted hundreds of transcription factor binding sites in the promoter region of the TCF7L2 gene， and there were 18 transcription factors predicted by two softwares at the same time with the same binding position. These transcription factors have a relatively high probability of existence， and they can be verified first in the subsequent experimental verification of important transcription factors. According to the evolutionary footprint method， 207 common transcription factor binding sites was obtained by the CONREAL prediction method in the conserved regions of the TCF7L2 gene promoter in human and mouse， and a total of 66 transcription factors were involved after processing and reduction. These transcription factors are involved in cell proliferation， differentiation， apoptosis， tumorigenesis， inflammation， immune response， Wnt signaling pathway， insulin signaling pathway， β cell development regulation， lipid biosynthesis， etc. These predictions， on the other hand， provide evidence for the known functions of TCF7L2， and suggest at the same time that the expression of TCF7L2 is regulated by a variety of transcription factors and is in a complex metabolic network， that is， TCF7L2 has many important physiological functions. CpG island methylation can inhibit the normal transcription process of the promoter， thereby reducing gene expression. The analysis results of CpG islands in the promoter region of TCF7L2 gene by EMBOSS and MethPrimer were basically the same. There was a CpG island in the promoter region of the human TCF7L2， which was located between 1 443 to 1 941 bp of the 2 000 bp sequence of the 5’ regulatory region， close to the first exon， conforming to the distribution characteristics of CpG islands. SNP is a third-generation genetic marker， and it has been confirmed in various racial studies that multiple SNPs in TCF7L2 are associated with T2D. This study found that there were two SNPs in the promoter region of the human TCF7L2 gene： rs11195470 and rs7083546. The transcription factor binding sites at the SNPs disappeared after allele mutations， indicating that rs11195470 and rs7083546 polymorphisms have an impact on the transcriptional regulation of TCF7L2 gene. Its relevance to T2D needs to be further studied in specific populations. TCF7L2 gene rs7903146 C/T polymorphism is associated with T2D in the Han population in the eastern part of Heilongjiang Province， and the rs7903146 C/T variant can lead to insulin resistance and decreased insulin sensitivity[29]. TCF7L2 gene SNPs can increase the susceptibility to T2D， which may be related to inhibiting the proliferation and differentiation of pancreatic β cells， affecting the processing of proinsulin， and affecting the function of pancreatic islet cells[30].
Conclusions
Bioinformatics softwares were used to predict and analyze the 2 000 bp sequence of the 5’ regulatory region of the human TCF7L2 gene. It could be basically determined that the promoter was located in the range of 1 700 bp upstream of the 5’ regulatory region， with the core promoter region located at -242 to -192 bp， -853 to -803 bp， and the transcription start site was located at the A base of 1 844 bp of the 2 000 bp sequence. There was a CpG island between 1 443 and 1 941 bp of the 2 000 bp sequence， and a TATA box at 303-309 bp. AliBaba2.1， PROMO and JASPAR softwares respectively predicted 241， 944 and 1 035 （positive-sense strand） transcription factor binding sites. 207 common transcription factor binding sites were predicted in the conserved regions of the TCF7L2 gene promoter in human and mouse， involving 66 transcription factors. Two SNPs were predicted in the promoter region： rs11195470 and rs7083546.
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Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
Key words TCF7L2; Promoter; Transcription factor; Transcription factor binding sites; Bioinformatics
Received： July 3， 2020 Accepted： September 9， 2020
Supported by the Diabetes Special Fund Project of Hubei University of Science and Technology （2016-18XZ12）.
Siyuan BAO （1976-）， male， P. R. China， senior experimentalist， master， devoted to research about molecular biology.
*Corresponding author. E-mail： baosyuan@163.com.
Type 2 diabetes is a slowly progressive disease， mostly in developing countries. At present， the number of diabetic patients in China is about 121 million， of which type 2 diabetes （T2D） patients account for about 90%[1]. The pathogenesis of type 2 diabetes is very complicated， and genetic factors and environmental factors together determine the occurrence of type 2 diabetes[2]. Searching for diabetes susceptibility factors and genes and preventing and treating diabetes at the molecular level are the current priorities. TCF7L2 is currently one of the genes with the highest degree of association with T2D found in the genome-wide association study （GWAS）. TCF7L2， also known as T cell transcription factor 4 （TCF4）， is located on human chromosome 10q25.3. It is abundantly expressed in human pancreatic β cells and adipose tissue. Its expression product is a key protein of the Wnt signaling pathway and has a regulatory effect on the function of pancreatic β cells. In studies on different races， it has been confirmed that single nucleotide polymorphisms （SNPs） at multiple sites are associated with T2D. Several recent studies have confirmed that TCF7L2 rs7903146 C>T is related to the development of T2D[3-7]. Mari Cassol Ferreira et al.[8] showed that the existence of TCF7L2 rs7903146 T allele in T2D patients was related to the increase in postprandial insulin， proinsulin and C-peptide secretion; and after exenatide acetate treatment， the plasma insulin and C peptide peaks of T allele carriers after meals were significantly lower than those of patients who did not carry the T allele. Li et al.[9] found that individuals with the T allele of the TCF7L2 rs12255372G/T gene polymorphism may be more susceptible to T2D in the Chinese population. Li et al.[10] found that the TCF7L2 rs290481 T>C polymorphism was significantly related to the occurrence of T2D in the Han population in eastern China， and the T→C mutation of rs290481 promoted the increase of FPG level. Xia et al.[11] used genome editing technology to reveal that the rs7903146 region of TCF7L2 seems to be the interaction region between the ACSL5 promoter and TCF7L2， and the deletion of this region in two human colon cell lines significantly reduced the expression of ACSL5; and given that ACSL5 is involved in lipid biosynthesis， it is speculated that inhibiting ACSL5 enzyme activity can significantly improve insulin sensitivity. Dan et al.[12] pointed out that miR-181a-5p regulated TGFβ/Smad and Wnt signaling pathways by directly targeting Smad7 and TCF7L2， and promoted 3T3-L1 preadipocyte differentiation and adipogenesis. The expression level of TCF7L2 is closely related to the dedifferentiation of β cells. Down-regulation of TCF7L2 expression can increase the dedifferentiation of β cells induced by high glucose， and overexpression of TCF7L2 can reduce the dedifferentiation of β cells induced by high glucose[13]. TCF7L2 is involved in the regulation of glucose metabolism and may become a potential therapeutic target for patients with prediabetes[14]. Although TCF7L2 has been deeply and meticulously explored， the exact mechanism by which TCF7L2 and their respective SNPs change the metabolic process and affect the susceptibility to type 2 diabetes remains to be determined. It has not yet reached a consensus on how TCF7L2 increases the prevalence of T2D. To clarify these problems， it is necessary to understand the expression regulation mechanism of the TCF7L2 gene itself， and study it from the perspective of network regulation. The human TCF7L2 gene promoter sequence has not been recorded in the NCBI database， and the bioinformatics analysis of the TCF7L2 promoter has not been reported. In order to obtain the promoter sequence and characteristics of the human TCF7L2 gene， we used bioinformatics research methods to screen out potential promoter sequences from the genome database， and analyzed transcription factors and their binding sites， CpG islands， SNPs， etc.， laying a theoretical foundation for the study on the expression regulation of TCF7L2 gene and its mechanism of participating in the pathogenesis of T2D.
Materials and Methods
Materials
The human TCF7L2 gene is located at 10q25.2-q25.3. It spans 217 460 bp， contains 19 exons， 18 introns， and has 15 transcription variants. TCF7L2 variant 1 encodes the longest variant， with the mRNA accession number： NM_001146274.1， and the corresponding nucleotide sequence is located at hg38 chr10： 112 950 407 to 113 167 493. The 2 000 bp DNA sequence upstream of chr10： 112 950 407 was selected for the prediction and analysis of promoters， transcription factor binding sites， CpG islands， and SNPs.
Methods
Acquisition of the 2 000 bp sequence of the 5’ regulatory region of the human TCF7L2 gene
In the UCSC （http：//genome.ucsc.edu/） website， the site of the TCF7L2 gene in the human genome （GRCh38/hg38） was searched. With the transcription initiation site as +1， a 2 000 bp DNA sequence upstream of the transcription start site was obtained， and used as a candidate promoter.
Acquisition of the 2 000 bp sequence of the 5’ regulatory region of the mouse homologous TCF7L2 gene
With NM_001146274.1 as a probe， the Blast tool （https：//blast.ncbi.nlm.nih.gov/Blast.cgi） was used to search the mouse RefSeqRNA database to obtain the mouse homologous sequence NM_001331147.1. NM_001331147.1 is the mRNA of mouse TCF7L2 gene variant 22. On the UCSC website， the site of NM_001331147.1 in the mouse genome （GRCm38/mm10） was searched， and the 2 000 bp sequence upstream of the 5’ transcription start site was obtained. Promoter region prediction and analysis
The Neural Network Promoter Prediction （http：//www.fruitfly.org/seq_tools/promoter.html）， Promoter2.0 （http：//www.cbs.dtu.dk/services/Promoter/）， TSSG （http：//linux1.softberry.com/berry.phtml？topic = tssg&group = programs&subgroup = promoter） and Proscan （https：//www-bimas.cit.nih.gov/molbio/proscan/） online software were used to predict the potential promoter region in the 5’ regulatory region of the human TCF7L2 gene， and perform comparative analysis. Parameter setting： Neural Network Promoter Prediction promoter threshold was set to be 0.8， and the default values of Promoter 2.0， TSSG and Proscan were used.
Motif recognition of TATA box， GC box and CAAT box
The motif recognition of TATA box， GC box and CAAT box could be realized by whether the corresponding characteristic sequence is found in the 5’ regulatory region sequence of TCF7L2 gene. The TATA box sequence format is TATAWAW （W stands for A or T）; the GC box sequence format is GGGCGG; and the CAAT box sequence format is CCAAT.
Prediction and analysis of transcription factor binding sites in promoter region
The online software AliBaba2.1 （http：//gene-regulation.com/pub/programs/alibaba2/index.html）， PROMO （http：//alggen.lsi.upc.es/） and JASPAR （http：//jaspar.genereg.net/） were used to predict the transcription factor binding sites in the 5’ regulatory region of the human TCF7L2 gene， and perform statistical analysis to screen out common transcription factors. AliBaba2.1 parameter setting： Min mat. Conservation was set to be 75%， and other parameters were default values. PROMO parameter setting： Considering factors were selected as the Only human factors， the Considering sites were selected as the Only human sites， and other parameters were default values. JASPAR parameter setting： Homo sapiens was selected for JASPAR matrix models species， the Relative profile score threshold was set to be 85%， and other parameters were set as default values.
Prediction of transcription factor binding sites in conserved regions of the TCF7L2 gene promoter in human and mouse
The online software CONREAL （http：//conreal.niob.knaw.nl/） was used to predict the binding sites of transcription factors in the conserved regions of the TCF7L2 gene promoter in human and mouse. The 2 000 bp sequences upstream of the 5’ transcription start sites of human and mouse TCF7L2 gene were input into the CONREAL program. Then， the threshold for PWMs was set to be 85%， the threshold for homology was set to be 75%， and three homology tools were selected to obtain the conserved regions in the two; and the two vertebrate transcription factor binding site databases JASPAR and Transfac v.8.2 were searched to obtain transcription factor binding sites that are located at the same position of the conserved regions of the sequence. Prediction of methylated CpG islands in the promoter region of the human TCF7L2 gene
EMBOSS （http：//www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/） and MethPrimer （http：//www.urogene.org/cgi-bin/methprimer/methprimer.cgi） online software were used to predict human TCF7L2 CpG islands in the promoter region of the gene. Parameter setting： CpG detection content/expected content （Obs/Exp）>0.60， C+ G content percentage>50%， CpG island length>200 bp.
SNP Screening in promoter region and its potential function prediction
The SNP screening tool： F-SNP software （http：//compbio.cs.queensu.ca/F-SNP/）. SNP function analysis software： SNP Function Prediction （http：//snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm）.
Results and Analysis
Prediction and analysis of the promoter region of the human TCF7L2 gene
The -1--2 000 bp sequence upstream of the 5’ regulatory region of the human TCF7L2 gene was obtained from the UCSC database， and the potential promoter regions of the 2 000 bp sequence was analyzed using 4 different online softwares. It was found by querying the UCSC database that the TCF7L2 gene is located on the "+" strand， so the potential promoters whose predicted region was on the positive-sense strand were selected. The results are shown in Table 1. The 2 000 bp sequence upstream of the 5’ regulatory region of the human TCF7L2 gene was subjected to full sequence alignment with the human TCF7L2 gene promoter sequence HPRM36386 （product number） queried on the genecopoeia website （http：//www.genecopoeia.com/） using the BLAST tool， and it was found that identity of the two reached 81%. HPRM36386 has a total length of 1 628 bp， and the transcription start site （TSS） is located at the A base of 1 470 bp. The 375-2 000 bp of the 2 000 bp sequence upstream of the 5’ regulatory region of the TCF7L2 gene was exactly the same as the HPRM36386 sequence， and the A base of 1 844 bp corresponded to the 1 470 bp A base of the HPRM36386 sequence. It was speculated that the TCF7L2 gene promoter was located within the range of the 1 700 bp sequence upstream of the 5’ regulatory region.
Motif recognition of TATA box， GC box and CAAT box
By searching the corresponding characteristic sequences of TATA box， GC box and CAAT box， we found that there was a TATA box in the 5’ regulatory region of the human TCF7L2 gene， located between -1 698 and -1 692 bp， and no GC box and CAAT box were found.
Prediction and analysis of transcription factor binding sites in the promoter region of human TCF7L2 gene In order to improve the accuracy of transcription factor binding site prediction， three softwares， AliBaba2.1， PROMO and JASPAR， were used to predict the transcription factor binding sites in the promoter region of the human TCF7L2 gene. The three softwares predicted 241， 944， and 1，035 （sense strand） transcription factor binding sites within 1 to 2 000 bp upstream of the 5’ regulatory region， involving 75， 76， and 208 transcription factors， respectively. There were 18 transcription factors that predicted by two softwares at the same time with the same binding site position （Table 2）.
Analysis of transcription factor binding sites in the conserved regions of the TCF7L2 gene promoter in human and mouse
According to the evolutionary footprint analysis， the CONREAL program was used to determine the conserved regions of the TCF7L2 gene promoter in human and mouse. The transcription factor binding sites in the conserved regions obtained by the three prediction methods （CONREAL， LAGAN， MAVID） were mostly the same. The CONREAL method obtained 207 transcription factor binding sites in the conserved regions （Table 3）， involving 66 kinds of transcription factors such as Sp1， CREB， c-Jun， GATA-1， AP-2alphaA， CdxA， FREAC-4， FREAC-3， cap， and HNF-3beta， among which Sp1， CREB， c-Jun， GATA-1， and AP-2alphaA were predicted by AliBaba2.1 and PROMO at the same time.
Analysis of methylated CpG islands in the promoter region of the human TCF7L2 gene
The online software EMBOSS and MethPrimer were used to predict methylated CpG islands in the 2 000 bp sequence upstream of the 5’ regulatory region of the human TCF7L2 gene. The prediction results of the EMBOSS software showed that there was a CpG island with a length of 499 bp， located at 1 443 to 1 941 bp of the predicted sequence （Fig. 1）. The prediction results of MethPrimer software showed a CpG island with a size of 450 bp located at 1 443 to 1 892 bp（Fig. 2）， which was consistent with the prediction result of the EMBOSS software.
Prediction and analysis of SNPs in the promoter region of the human TCF7L2 gene
The chromosomal loci （Chr 10： 112 948 407-112 950 406） at 1-2 000 bp upstream of the 5’ regulatory region of the human TCF7L2 gene were input in the F-SNP software， and 2 SNPs： rs11195470 and rs7083546 were searched （as shown in Table 4）. The SNP Function Prediction software was used to perform function prediction and race-specific allele frequency query. The results showed that rs11195470 and rs7083546 had extremely low conservativeness. There were racial differences in the rs11195470 allele frequency between the four populations. The rs11195470 allele frequency was the highest in Americans of Nordic and Western European descent （CEU）， and the same for the Japanese and Nigerian （Table 5）. In order to explore the influence of rs11195470 and rs7083546 polymorphisms on transcription factor binding， the AliBaba2.1 software was used to predict the transcription factor binding sites of the sequence where the SNPs are located. The results are shown in Table 6. Discussion
TCF7L2 belongs to the transcription factors of the TCF/LEF family and is involved in human diseases including cancer， type 2 diabetes and bipolar affective disorder[15]. The TCF7L2 gene is encoded by 19 exons， and the selection of the transcription start site and alternative splicing produce hundreds of potential protein variants[15-16]. TCF7L2 gene has tissue-specific splicing， suggesting that TCF7L2 may have different functions in different cells[15]. A variety of genetic diseases， mainly metabolic and cardiovascular diseases， are largely due to an extreme upstream TCF7L2 binding element driving the transcription of metabolism-related genes[17]. In the Wnt signaling pathway， TCF7L2 protein binds with β-catenin to form an active nuclear complex， activates the proglucagon gene， and promotes the secretion of glucagon-like polypeptide-1 （GLP-1） from small intestinal L cells to maintain Steady state of blood glucose[18].
Wu et al.[17] showed that TCF7L2 binds tightly to the specific binding region of the PIK3R1 promoter， and significantly controls the transcription of the encoded protein p85， thereby affecting the activation of the PI3K/AKT signaling pathway and insulin secretion. Microarray research also found consistent sequences of proprotein convertase （PC） 1 and 2 genes binding to TCF7L2， suggesting that TCF7L2 plays a role in insulin processing[16]. Because the TCF7L2 target gene has tissue specificity， studies on the binding mode of TCF7L2 genomic DNA show that TCF7L2 specifically binds to multiple genes that regulate liver cell sugar metabolism， including Pck1， Fbp1， Irs1， Irs2， Akt2， Adipor1， Pdk4， and Cpt1a[19]. Lavanya Miyer et al.[20] also identified a series of validated TCF7L2 cardiac-specific target genes， such as Hand2， Tbx20， Rock2 and Dstn. However， the binding mode of TCF7L2 and its candidate target genes involved in pancreatic gluconeogenesis remains unclear. In T2D， the TCF7L2 gene rs7903146 （C/T） polymorphism showed a significant association in almost all groups and races[21]. Shanmugapriya Chandrasekaran et al.[22] pointed out that TCF7L2 rs7903146 T+ genotype caused a decrease in GLP-1 level， and GLP-1 level increased the risk of type 2 diabetes by reducing postprandial insulin levels and increasing insulin resistance. Andreas Leiherer et al.[23] studied the molecular mechanism of the TCF7L2 polymorphism rs7903146 and the risk of T2D and found that： serotonin was the most important metabolite in carriers of dangerous alleles， and serotonin was significantly related to the rs7903146 genotype after being fully adjusted. This finding suggests for the first time that the influence of TCF7L2 polymorphism on the risk of type 2 diabetes may involve serotonin-dependent pathway. Recent studies have shown that TCF7L2 has a regulatory effect on β cell quality. TCF7L2 may play an important role in the pathogenesis of diabetes by participating in regulating the proliferation and apoptosis of pancreatic β-cells[24]. Kathleen A. Bailey et al.[25] showed that the decreased expression of TCF7L2 in β cells led to impaired insulin secretion， and further pointed out that β cells with low TCF7L2 expression occupied less area of the pancreas， confirming that the expression of TCF7L2 is of great significance to the survival and proliferation of β cells. The silencing of mammalian pancreatic islets or biological β-cell line TCF7L2 leads to decreased expression of proinsulin gene， decreased glucose-stimulated insulin secretion， exocytosis defect of insulin-containing particles， and decreased insulin secretion[26]. In August 2014， a team from Lund University published a report stating that TCF7L2 can positively regulate the expression of transcription factors such as MAFA， PDX-1 and NKX6.1 by targeting insulin gene enhancer binding protein-1 （ISL1）[27]. This finding provides evidence for suggesting the role of TCF7L2 in β cell regeneration. Yao et al.[28] proposed a model to explain the new mechanism of geniposide to promote the regeneration and survival of β cells. In this model， β-catenin/TCF7L2 is the core component required for the function of geniposide. Geniposide can activate the β-catenin/TCF7L2 transcription complex and induce βcell regeneration by stimulating βcell proliferation and differentiation. Meanwhile， geniposide promotes the survival of β cells by inhibiting the level of c-casp3 to inhibit β-cell apoptosis， and supports β-catenin/TCF7L2 as a possible target for diabetes treatment to promote the formation of new β cells. The promoter is located near the transcription start point and is a DNA sequence that RNA polymerase binds and initiates transcription. Four promoter analysis softwares with different principles and algorithms were used to analyze the 2 000 bp sequence upstream of the 5’ regulatory region of the human TCF7L2 gene， and it was found that there were at least 5 potential promoter regions on the positive-sense strand of the gene， located at -167 to -117 bp， -242 to -192 bp， -600 to -308 bp， -853 to -801 bp， and -1 700 to -1 501 bp， respectively. Combining with results of the sequence alignment with HPRM36386， it could be basically confirmed that the human TCF7L2 gene promoter was located within the range of 1 700 bp upstream of the 5’ regulatory region， the core promoter regions was located at -242--192 bp and -853--803 bp， and the transcription start site was located at the A base of 1 844 bp in the 2 000 bp sequence of the 5’ regulatory region. In the gene expression regulatory network， the binding of transcription factors and cis-acting elements can initiate the expression of a set of specific genes， or turn off the expression of a set of genes. Three online softwares， AliBaba2.1， PROMO and JASPAR predicted hundreds of transcription factor binding sites in the promoter region of the TCF7L2 gene， and there were 18 transcription factors predicted by two softwares at the same time with the same binding position. These transcription factors have a relatively high probability of existence， and they can be verified first in the subsequent experimental verification of important transcription factors. According to the evolutionary footprint method， 207 common transcription factor binding sites was obtained by the CONREAL prediction method in the conserved regions of the TCF7L2 gene promoter in human and mouse， and a total of 66 transcription factors were involved after processing and reduction. These transcription factors are involved in cell proliferation， differentiation， apoptosis， tumorigenesis， inflammation， immune response， Wnt signaling pathway， insulin signaling pathway， β cell development regulation， lipid biosynthesis， etc. These predictions， on the other hand， provide evidence for the known functions of TCF7L2， and suggest at the same time that the expression of TCF7L2 is regulated by a variety of transcription factors and is in a complex metabolic network， that is， TCF7L2 has many important physiological functions. CpG island methylation can inhibit the normal transcription process of the promoter， thereby reducing gene expression. The analysis results of CpG islands in the promoter region of TCF7L2 gene by EMBOSS and MethPrimer were basically the same. There was a CpG island in the promoter region of the human TCF7L2， which was located between 1 443 to 1 941 bp of the 2 000 bp sequence of the 5’ regulatory region， close to the first exon， conforming to the distribution characteristics of CpG islands. SNP is a third-generation genetic marker， and it has been confirmed in various racial studies that multiple SNPs in TCF7L2 are associated with T2D. This study found that there were two SNPs in the promoter region of the human TCF7L2 gene： rs11195470 and rs7083546. The transcription factor binding sites at the SNPs disappeared after allele mutations， indicating that rs11195470 and rs7083546 polymorphisms have an impact on the transcriptional regulation of TCF7L2 gene. Its relevance to T2D needs to be further studied in specific populations. TCF7L2 gene rs7903146 C/T polymorphism is associated with T2D in the Han population in the eastern part of Heilongjiang Province， and the rs7903146 C/T variant can lead to insulin resistance and decreased insulin sensitivity[29]. TCF7L2 gene SNPs can increase the susceptibility to T2D， which may be related to inhibiting the proliferation and differentiation of pancreatic β cells， affecting the processing of proinsulin， and affecting the function of pancreatic islet cells[30].
Conclusions
Bioinformatics softwares were used to predict and analyze the 2 000 bp sequence of the 5’ regulatory region of the human TCF7L2 gene. It could be basically determined that the promoter was located in the range of 1 700 bp upstream of the 5’ regulatory region， with the core promoter region located at -242 to -192 bp， -853 to -803 bp， and the transcription start site was located at the A base of 1 844 bp of the 2 000 bp sequence. There was a CpG island between 1 443 and 1 941 bp of the 2 000 bp sequence， and a TATA box at 303-309 bp. AliBaba2.1， PROMO and JASPAR softwares respectively predicted 241， 944 and 1 035 （positive-sense strand） transcription factor binding sites. 207 common transcription factor binding sites were predicted in the conserved regions of the TCF7L2 gene promoter in human and mouse， involving 66 transcription factors. Two SNPs were predicted in the promoter region： rs11195470 and rs7083546.
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