Computational STAT4 rSNP Analysis, Transcriptional Factor Binding Sites and Disease

The Janus Kinase-Signal Transducers and Activators of Transcription (JAK-STAT) pathways play a critical role in immune, neuronal, hematopoietic and hepatic systems ¹. JAK-STAT is a principal signal transduction pathway in cytokine and growth factor signaling as well as regulating various cellular processes such as cell proliferation, differentiation migration and survival ². JAK-STAT provides the principle intracellular signaling mechanism required for a wide array of cytokines ^{3, 4}. The STAT portion of the signaling cascade has seven mammalian family members which are STAT1, 2, 3, 4, 5a, 5b and 6 ^{3, 4}. These STATs bind thousands of transcriptional factor binding sites (TFBS) in the genome and regulate the transcription of many protein-coding genes, miRNAs and long noncoding RNAs ⁴. The STAT 4 gene which is important for signaling by interleukins (IL-12 and IL-23) and type 1 interferons ⁴ has been found to have several simple nucleotide polymorphisms (SNPs) associated with human disease ^{5, 6, 7, 8, 9, 10, 11, 12}. STAT4 transduces IL-12, IL-23 and type 1 interferon-mediated signals into helper T (Th) cells (Th1 and Th17) differentiation, monocyte activation, and interferon-gamma production ^{12, 13}. The STAT4-dependent cytokine regulation is found in the pathogenesis of autoimmune disease ^{14, 15} such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) ^{16, 17}.

The STAT4 gene maps to human chromosome 2q32.3 and is about 143 kb in size. The coding region consists of 22 exons with a large 70 kb intron between exons 2 and 3. Several SNPs in the gene have been significantly association with Behcet’s Disease ¹⁸, diabetes risk ¹¹, hepatitis B virus-related hepatocellular carcinoma ^{6, 10, 19, 20}, inflammatory bowel disease ²¹, juvenile idiopathic arthritis ²², primary biliary cirrhosis and Crohn's disease ²³, severe renal insufficiency in lupus nephritis ⁸, systemic lupus erythematosus ⁵ and ulcerative colitis ²⁴ (Table 1). The rs7574865 STAT4 SNP has been found to be significantly associated with diabetes ¹¹, hepatitis B virus-related hepatocellular carcinoma ^{6, 10, 19, 20}, inflammatory bowel disease ²¹, juvenile idiopathic arthritis ²², primary biliary cirrhosis and Crohn's disease ²³, severe renal insufficiency in lupus nephritis ⁸, systemic lupus erythematosus ⁵ and ulcerative colitis ²⁴. The rs11889341 STAT4 SNP has been found to be significantly associated with diabetes ¹¹, hepatitis B virus (HBV) infection, HBV-related cirrgisus and hepatocellular carcinoma ²³, severe renal insufficiency in lupus nephritis ⁸, and systemic lupus erythematosus ⁵. The rs8179673 STAT4 SNP has been found to be significantly associated with diabetes ¹¹, hepatitis B virus (HBV) infection, HBV-related cirrgisus and hepatocellular carcinoma ²³ and systemic lupus erythematosus ⁵. The rs7582694 STAT4 SNP has been found to be significantly associated with hepatitis B virus (HBV) infection, HBV-related cirrgisus and hepatocellular carcinoma ²³ and severe renal insufficiency in lupus nephritis ⁸. The rs7574070 and rs7572482 STAT4 SNPs have been found to be significantly associated with Behcet’s disease ¹⁸. The rs7572482 STAT4 SNP is located in the promoter region while the remaining SNPs are located in the large 70 kb intron between exon 2 and 3. The reports listed above indicate that these SNPs are in strong linkage disequilibrium (LD) with each other.

Single nucleotide changes that affect gene expression by impacting gene regulatory sequences such as promoters, enhances, and silencers are known as regulatory SNPs (rSNPs) ^{25, 26, 27, 28}. A rSNPs within a transcriptional factor binding site (TFBS) can change a transcriptional factor’s (TF) ability to bind its TFBS ^{29, 30, 31, 32} in which case the TF would be unable to effectively regulate its target gene ^{33, 34, 35, 36, 37}. This concept is examined for the above STAT4 rSNPs and their allelic association with TFBS, where computation analyses ^{38, 39, 40, 41} was used to identify TFBS alterations created by the STAT4 rSNPs. Recent reports have also introduced the concept of modeling of epigenetic modifications to transcriptional factor binding sites in the control of gene expression ^{42, 43}. In this report, the rSNP associations with changes in potential TFBS are discussed with their possible relationship to these diseases in humans.

The JASPAR CORE database ^{44, 45} and ConSite ⁴⁶ were used to identify the potential STAT4 TFBS in this study. JASPAR is a database of transcription factor DNA-binding preferences used for scanning genomic sequences where ConSite is a web-based tool for finding cis-regulatory elements in genomic sequences. The TFBS and rSNP location within the binding sites have previously been discussed ⁴⁷. The Vector NTI Advance 11.5 computer program (Invitrogen, Life Technologies) was used to locate theTFBS in theSTAT4 gene (NCBI Ref Seq NM_003151) from 4 kb upstream of the transcriptional start site to 8.3 kb past the 3’UTR which represents a total of 130.9 kb. The JASPAR CORE database was also used to calculate each nucleotide occurrence (%) within the TFBS, where upper case lettering indicate that the nucleotide occurs 90% or greater and lower case less than 90%. The occurrence of each SNP allele in the TFBS is also computed from the database (Table 2 & Appendix).

Genome-wide association studies (GWAS) over the last decade have identified nearly 6,500 disease or trait-predisposing SNPs where only 7% of these are located in protein-coding regions of the genome ^{48, 49} and the remaining 93% are located within non-coding areas ^{50, 51} such as regulatory or intergenic regions. SNPs which occur in the putative regulatory region of a gene where a single base change in the DNA sequence of a potential TFBS may affect the process of gene expression are drawing more attention ^{25, 27, 52}. A SNP in a TFBS can have multiple consequences. Often the SNP does not change the TFBS interaction nor does it alter gene expression since a transcriptional factor (TF) will usually recognize a number of different binding sites in the gene. In some cases the SNP may increase or decrease the TF binding which results in allele-specific gene expression. In rare cases, a SNP may eliminate the natural binding site or generate a new binding site. In which cases the gene is no longer regulated by the original TF. Therefore, functional rSNPs in TFBS may result in differences in gene expression, phenotypes and susceptibility to environmental exposure ⁵². Examples of rSNPs associated with disease susceptibility are numerous and several reviews have been published.^{52, 53, 54, 55}.

The rs7574865 rSNP STAT4-G allele ^{G (+ strand) or C (} located in the unique MAX and ZNF354C TFBS have a 100% occurrence in humans while the unique FOXL1 TFBS has a 17% occurrence (Table 2). Since these binding sites (BS) occur multiple times in the gene, the rSNP G allele should not have much of an impact gene regulation (Table 2). The minor rs7574865 rSNP STAT4-T allele ^{T (+ strand) or A (} located in the unique ARID3A, FOXQ1 and PDX1 TFBS have a 100% occurrence in humans while the NR1HE::RXRa TFBS has a 44% occurrence (Figure 1, Table 2). Since all the unique BS for this allele occur multiple times in this gene, it would not be expected that these TFBS would have much of an effect on STAT4 regulation except for the NR1HE:: RXRa BS which occurs only once in the gene (Table 2) and is a key regulator of macrophage function, controlling transcriptional programs involved in lipid homeostasis and inflammation (Appendix). Since NR1HE:: RXRa protein duplex is part of the NR1 subfamily of the retinoid nuclear receptor superfamily, the presence of its TFBS created by the minor T allele could in part be responsible for the diseases listed in Table 1, that are significantly associated with this rSNP.

The rs11889341 rSNP STAT4-C allele [C (+ strand) or G (- strand) located in the unique AR TFBS has a 100% occurrence in humans and occurs only once in the gene (Figure 2, Table 2). The androgen receptor is a steroid-hormone activated transcription factor which stimulates transcription of androgen responsive genes that are expressed in bone marrow, mammary gland, prostate, testicular and muscle tissues. The absence of this TFBS created by the minor STAT4-T allele should have a major effect relating to the diseases listed in Table 1. The minor rs11889341 rSNP STAT4-T allele ^{T (+ strand) or A (} located in the unique CDX2, FOXOD1, FOXI1, FOXO1, FOXP1, FOXP2 and GATA3 TFBS have a 100% occurrence in humans. These TFBS occur multiple times in the gene so they would not be expected to have much impact on the regulation of the gene, except for the FOXP1 TFBS which occurs only once in the gene (Table 2). Although FOXP1 is a member of the subfamily P of the forkhead box (FOX) transcription factors which play important roles in the regulation of tissue- and cell type-specific gene transcription during both development and adulthood, it is doubtful that the presence of this TFBS only with the minor T allele would have much impact on the regulation of the gene since there are other family members TFBS represented with the minor allele (Table 2). The SNP T allele is also located in the two unique MEF2C TFBS which have a 95 and 97% occurrence in humans and each occurs only once in the gene (Table 2). The MEF2C TF controls cardiac morphogenesis and myogenesis, and is also involved in vascular development and consequently the presence or absence of this TFBS should have an impact on the diseases listed in Table 1.

The rs8179673 rSNP STAT4-T allele ^{T (+ strand) or A (} located in the unique ARID3A, GATA3 and NKX3-1 TFBSs have a 100% occurrence in humans while the LHX3 and NFIL3 TFBS have a 95 and 96% occurrence (Table 2). Since these TFBS occur more than once in the gene, it is doubtful that these BS would have much impact on the regulation of the gene. The minor rs8179673 rSNP STAT4-C allele ^{C (+ strand) or G (} located in the unique FOXH1, FOXO1, FOXQ1, SOX2 and SOX6 TFBS have a 100% occurrence in humans while the HNF1a and HNF4g TFBS have a 67 and 93% occurrence, respectively (Table 2). All of these TFBS occur only once in the gene (Table 2). However, the FOXH1, FOXO1, FOXQ1, SOX2 and SOX6 TFBS are involved with transcription machinery and are represented in families consisting of multiple members. Therefore, it is unlikely that the presence or absence of one family member would have much impact on gene regulation especially since there are TFBS for multiple family members represented by the minor C allele (Table 2, Appendix). The unique HNF1a and HNF4g TFBS which also occur only once in the gene are BS for the HNF1a and HNF4g transcriptional activators that regulates the tissue specific expression of multiple genes, especially in pancreatic islet cells and in liver (Table 2, Appendix).

The rs7574070 rSNP STAT4-A allele ^{A (+ strand) or T (} located in the unique CEBPb and ZNF354C TFBS have a 100% occurrence in humans while the RFX1 and RFX5 TFBS have a 84 and 78% occurrence, respectively (Table 2). The CEBPb, RFX1 and RFX5 TFBS occur only once in the gene while the ZNF354C TFBS occurs 87 times in the gene (Table 2); consequently, only the CEBPb, RFX1 and RFX5 TFBS might have an impact on gene regulation. The CEBPb TF is an important transcriptional activator regulating the expression of genes involved in immune and inflammatory responses; consequently, the loss of the BS with the minor C allele could have an impact on Behcet’s disease (Table 1). The RFX1 & 5 TFs are important regulatory factors essential for MHC class II gene expression and the loss of these BS with the presence of the minor C allele could also have an impact on Behcet’s disease (Table 1). The rs7574070 rSNP STAT4-C allele ^{C (+ strand) or G (} located in the unique STAT4 and THAP2C TFBS have a 43 and 99% occurrence in humans and occur only once in the gene (Table 2). The STAT4 TF is important in regulating genes associated with systemic lupus erythematosus and rheumatoid arthritis (Appendix); consequently, the occurrence of this TFBS only with the minor C allele could have an impact on Behcet’s disease (Table 1). The THAP2C TF is involved with a large spectrum of important biological functions (Appendix) and also contribute to Behcet’s disease when the TFBS is only represented in the minor C allele (Table 2, Appendix).

The rs7572482 rSNP STAT4-A allele ^{A (+ strand) or T (} located in the unique ARID3A, FOS, NFE2L1::MAFG and POU5F1::SOX2 TFBS have a 100% occurrence in humans except for the POU5F1::SOX2 TFBS for which it has a 90% occurrence (Table 2). The ARID3A and NFE2L1::MAFG TFBS occur multiple times in the gene while the FOS and POU5F1::SOX2 TFBS only occurs once (Table 2). The FOS TF is a regulator of cell proliferation, differentiation and transformation while the POU5F1::SOX2 TFs play a key role in embryonic development and stem cell pluripotency (Appendix) which could have an impact on Behcet’s disease (Table 1). The rs7572482 rSNP STAT4-G allele ^{G (+ strand) or C (} located in the unique ARNT, ARNT::AHR and ZBTB33 TFBS have a 100% occurrence in humans except for the ARNT::AHR TFBS for which it has a 96% occurrence (Table 2). The ARNT and ARNT::AHR TFBS occur 14 times in the STAT4 gene while the ZBTB33 TFBS only occurs once. The ZBTB33 TF is a transcriptional regulator involved with zinc finger motifs and may contribute to the repression of target genes of the Wnt signaling pathway (Appendix). Similar logic can be used to evaluate the potential TFBS within the other STAT4 rSNPs found in the Table 1 & Table 2.

Human diseases or conditions can be associated with rSNPs of the STAT4 gene as illustrated above. What a change in the rSNP alleles can do, is to alter the DNA landscape around the SNP for potential TFs to attach and regulate a gene. As an example, the potential TFBS associated with the rs7574865 rSNP STAT4-T allele from Table 2 are illustrated in Figure 1 as well as the rs11889341 common rSNP STAT4-C allele illustrated in Figure 2. As can be seen in Table 2, these potential TFBS change when an individual carries the alternate allele. The importance of this has been illustrated in Figure 2 with the AR TFBS where the common C allele has this function and the minor T allele does not. The AR TF is a steroid-hormone activated transcription factor which stimulates transcription of androgen responsive genes. A second example would be the HNF1a and HNF4g TFBS where the minor rs8179673 T allele has this function while the common allele does not. This TF regulates the tissue specific expression of multiple genes, especially in pancreatic islet and liver cells. A third example found with the rs7574070 common rSNP STAT4-A allele and not for the minor C allele is for the RFX1 & 5 TFBS whose TFs are important regulatory factors essential for MHC class II gene expression. Other examples can be found in Table 2.

SNPs that alter the TFBS are not only found in the promoter regions but in the introns, exons and the UTRs of a gene. The nucleus of the cell is where epigenetic alterations occur and TFs operate to convert chromosomes into single stranded DNA for mRNA transcription while it is the cytoplasm where mRNA is processed by separating exons and introns for protein translation. Consequently, it doesn’t matter where TFs bind the DNA in the nucleus because it is only there that TFs function. The SNPs outlined in this report should be considered as rSNPs since they change the DNA landscape for TF binding and have been associated with disease. In this report, examples have been described to illustrate that a change in rSNP alleles in the STAT4 gene can provide different TFBS which in turn are also associated with disease in humans. The potential alterations in TFBS obtained by computational analyses need to be verified by future protein/DNA electrophoretic mobility gel shift assays and gene expression studies.

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