Regulatory SNPs and transcriptional factor binding sites in ADRBK1, AKT3, ATF3, DIO2, TBXA2R and VEGFA

Abstract

Regulatory single nucleotide polymorphisms (rSNPs) which change the transcriptional factor binding sites (TFBS) for transcriptional factors (TFs) to bind DNA were reviewed for the ADRBK1 (GRK2), AKT3, ATF3, DIO2, TBXA2R and VEGFA genes. Changes in the TFBS where TFs attach to regulate these genes may result in human sickness and disease. The highlights of this previous work were reviewed for these genes.

Introduction

Genome-wide association studies (GWAS) over the last decade have identified nearly 6,500 disease or trait-predisposing simple nucleotide polymorphisms (SNPs) where only 7% of these are located in protein-coding regions of the genome ^{1, 2} and the remaining 93% are located within non-coding areas ^{3, 4} such as regulatory or inter-genic regions. SNPs which occur in the putative regulatory region of a gene where a single base change in the DNA sequence of a potential transcriptional factor binding site (TFBS) may affect the process of gene expression are drawing more attention. ^5–7 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 allele-specific gene expression cases, a TF's affinity for binding a response element may be altered where the SNP increases or decreases the TF binding ability. In rare cases, a SNP may eliminate the natural binding site or generate a new binding site. In such 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.⁷ Examples of rSNPs associated with disease susceptibility are numerous and several reviews have been published.^7–10 In this review, a rSNP will be considered as a SNP that alters the DNA landscape for TF binding resulting in different TFBS that may affect gene regulation.

In genetics, linkage disequilibrium (LD) is defined as the non-random association in a given population between the alleles of two or more loci.¹¹ LD between SNPs in the regulatory region of a gene can be used as a method of identifying associations of certain haplotypes with sickness or disease in a population.^{12, 13} This can be achieved when levels of LD between SNPs within haplotypes are seen to change substantially in a disease or sickness group of a population when compared to the normal baseline population. In such cases, the relationship between LD, SNPs and TFBS can be used to identify potential binding changes of TFs responsible for gene regulation which could result in disease or sickness.^14–21 In this report, LD is considered to be the non-random association of SNP alleles within the gene. The purpose of this review is to highlight some of these genetic associations for six genes with known human disease and sickness that have been recently studied.^{14, 16–19, 22, 23}

ADRBK1 (GRK2) rSNPs, TFBS and Heart Disease

G protein-coupled receptor kinases (GRKs) are a family of seven serine/threonine protein kinases with important and varied roles in regulating cellular signaling.^24–26 The GRK2 gene (ADRBK1), which is located at chromosome 11q13.1 and is about 20 kb in size, is an important regulator of beta-adrenergic signaling and plays a central role in heart failure (HF) pathology. ^27–29 Consequently, the inhibition of GRK2 has become an emerging treatment option of HF since the inhibition appears to be a powerful therapeutic approach and appears to provide complementation to β-blockade.²⁸ When the heart is injured or stressed there is activation of the sympathetic nervous system (SNS) and adrenergic overdrive via excessive catecholamines that leads to uncoupling desensitization of β-adrenergic receptors (βARs) in the heart, which is brought about by the up regulation of GRK2.^30–32 Lymphocyte GRK2 levels increase during acute myocardial infarction (MI) and are associated with worsening cardiac function ³³ enhanced GRK2 activity has been shown to contribute to cardiac decompensation.^{34, 35} Independent of its uncoupling properties on βARs, it appears to be a pro-death kinase especially in stressed myocytes.^{36, 37} Consequently, the inhibition of GRK2 or its genetic ablation has been show to improve cardiac function in myocardial injury.³⁸

rSNPs in the promoter region (rs12286664 and -703bp), introns (rs1894111, rs7128315 and rs948988) and 3’ UTR (rs4370946) of the ADRBK1 gene are in LD in the black population.²⁷ These rSNP alleles alter the DNA landscape for potential transcriptional factors (TFs) to attach resulting in changes in transcriptional factor binding sites (TFBS). The alleles of each rSNP were found to produce unique TFBS resulting in potential changes in TF ADRBK1 regulation.¹⁸ As an example, the rs7128315 rSNP common ADRBK1-G allele creates TFBS for the SREBF1 & 2 TFs which are involved with the cholesterol synthesis pathway. These TFBS occur only once in the gene and do not occur when the rs7128315 rSNP minor ADRBK1-A allele (0.32) is present.¹⁸ SREBF1 & 2 are master regulators of lipid homeostasis ^{39, 40} and have been associated with coronary heart disease in Han Chinese ⁴¹ and heart dysfunction.⁴²

AKT3 rSNPs, TFBS and High Altitude Sickness

The phosphatidylinositol 3-kinase (PI3K)/AKT pathway plays a key role in numerous cellular functions including proliferation, adhesion, migration, invasion, metabolism and survival⁴³ as well as angiogenesis. The PI3K/AKT pathway modulates the expression of angiogenic factors such as nitric oxide and angiopoietins.⁴⁴ The v-akt murine thymoma viral oncogene homolog 3 (AKT3), which is located at chromosome 1q44 and is about 362.8 kb in size. It is one of three isoforms of the AKTs which are major downstream targets of growth factor receptor tyrosine kinases that signal through PI3K.⁴⁵ The AKT3 single nucleotide polymorphism (rSNP, rs4590656) from intron one was recently found to be significantly associated with three physiological parameters (hemoglobin, hematocrit and red blood cell count) in chronic mountain sickness patients indicating the strong association of this gene with angiogenesis.⁴⁶ The rs4590656 rSNP common AKT3-C allele creates TFBS for the ARNT:AHR and HIF1α:ARNT TFs which are involved with xenobiotic metabolism and cellular and systemic responses to hypoxia, respectively; while these TFBS are not present with the rs4590656 rSNP minor AKT3-T allele (0.41).¹⁶ Other intron one AKT3 rSNPS (rs10157763, rs10927067 and rs2125230) have been significantly associated with aggressive prostate cancer (PCA). ⁴⁷ These rSNPs also alter the TFBS for TFs that regulate the AKT3 gene,¹⁶ where the rs2125230 rSNP minor AKT3-A allele (0.26) creates a unique TFBS for the IRF1 TF that occurs only once in the gene.¹⁶ The IRF1 (interferon regulatory factor 1) TF is a member of the interferon regulatory transcription factor (IRF) family which share several common effects which include antiviral agents that fight tumors. Other intron one rSNPs (rs4132509, rs12031994, rs2345994) have been found to be significantly associated with renal cell carcinoma risk (RCC).⁴⁸ The rs12031994 rSNP minor AKT3-A allele (0.13) creates a unique TFBS that occurs only once in the gene for the TAL1:GATA1 TF which has been implicated in the genesis of hemopoietic malignancies.⁴⁹ All seven AKT3 rSNP have been found to alter the DNA landscape resulting in changes in transcriptional factor binding sites (TFBS)¹⁶ for potential transcriptional factors (TFs) to attach.

ATF3 rSNPs, TFBS and Hypospadias

The activating transcription factor 3 (ATF3) gene, which is located at chromosome 1q32.3 and is about 55.4 kb in size. It is a member of the activating transcription factor/cAMP responsive element binding (CREB) protein family of transcription factors, which share the basic region-leucine zipper (bZip) DNA binding motif (TGACGTCA). This gene is induced by a variety of signals including many of those encountered by cancer cells, and is involved in the complex process of cellular stress response.^50–52 ATF3 has been viewed as a hub of the cellular adaptive response network which allows cells adapt to disturbances in homeostasis.⁵³ This gene has been shown to be up-regulated during sexual differentiation ⁵⁴ which indicates a potential role in hypospadias. Three unlinked ATF3 rSNPs (rs3125289, rs1877474 and rs11119982) which span a 16 kb region of intron one have been independently found to be significantly associated with the risk of hypospadias.⁵⁵ These rSNP alleles alter the DNA landscape for potential transcriptional factors (TFs) to attach resulting in changes in transcriptional factor binding sites (TFBS). These TFBS changes have been examined with respect to the human etiology of hypospadias which has been found to be significantly associated with the rSNPs .¹⁷

The rs3125289 rSNP AFT3-T allele (0.55) creates a unique TFBS for the SRY TF which is a transcriptional regulator that controls a genetic switch in male development. The TFBS for the SRY TF is only found once in the ATF3 gene and does not occur with the rs3125289 rSNP minor AFT3-C allele.¹⁷ The rs11119982 rSNP AFT3-T allele (0.51) creates a unique TFBS for the MYB TF which is plays an important role in the control of proliferation and differentiation of hematopoietic progenitor cells. The TFBS for the MYB TF is only found once in the ATF3 gene and does not occur with the rs11119982 rSNP minor AFT3-C allele.¹⁷

DIO2 rSNPs, TFBS and Type 2 Diabetes Mellitus

The type 2 deiodeinase gene (DIO2) encodes a deiodinase that coverts the thyroid prohormone, thyroxine (T4), to the biologically active triiodothyronine (T3) where T3 plays an important role in the regulation of energy balance and glucose metabolism.^56–59 DIO2 is found in the thyroid gland, cardiac and skeletal muscle, brown adipose tissue, placenta, pituitary, central nervous system (CNS) and at low levels in kidney and pancreases.^60–62 The DIO2 gene maps to human chromosome 14q24.3 and is about 15 kb in size. The coding region consists of two exons separated by an intron of approximately 7.4 kb.⁶³ Several SNPs have been found in the gene which have been studied is association with mental retardation (MR),⁶⁴ osteoarthritis ⁶⁵ and early-onset type 2 diabetes mellitus (T2DM).⁶⁶ Three of the common SNPs in the gene (rs225014, rs225012 and rs225010) have been found to be in strong LD with each other while the rs225012 and rs225010 SNPs have been shown to have a positive association with MR.⁶⁴ The haplotypes of two SNPs (rs225014 and rs12885300) have been shown to have a significant association with symptomatic osteoarthritis in Dutch women.⁶⁵ Three SNPs (rs225011, rs225014 and rs225015) which are in LD were found to be modestly associated with early-onset of T2DM in Pima Indians while these SNPs and rs6574549 were found to be nominally associated with hepatic glucose output.⁶⁶ The rs6574549 SNP was also found to be associated with fasting insulin, insulin action and energy expenditure.⁶⁶ These studies suggest that some DIO2 SNPs may be affecting the regulatory network for the gene expression in humans. An examination was made between DIO2 SNPs in LD and the transcription factor binding site (TFBS) changes resulting from the SNPs.²³ Regulatory SNPs (rSNPs) were found in the promoter region involving a novel SNP (-2035bp), 5’UTR (rs12885300), also in intron one (rs225010, rs225011 and rs225012), exon two [rs225014 (Thr92Ala)] and 3’ UTR (rs6574549 and rs225015) of the DIO2 gene which are all in LD.⁶⁶ The rs225012 rSNP common DIO2-G allele creates four unique TFBS for the E2F6, ELF1, EGR1 and SPI1 TFs which occur only once in the DIO2 gene and are involved with the control of the cell cycle and action of tumor suppressor proteins, transcription regulation, and gene expression during myeloid and B-lymphoid cell development, respectively.²³ The rs225012 rSNP minor DIO2-A allele (0.29) creates two unique TFBS for the HOXA5 and NKX3–2 TFs which occur only once in the DIO2 gene and are involved with the development regulatory system and negative regulation, respectively.²³ The rs225014 rSNP common DIO2-T allele (0.82) creates a unique TFBS for the FOXC1 TF which occurs only once in the DIO2 gene and is an important regulator of cell viability and resistance to oxidative stress. ²³ The rs6574549 rSNP common DIO2-G allele creates a unique TFBS for the LHX3 TF which occurs only once in the DIO2 gene and is required for pituitary development and motor neuron specification.²³ The rs6574549 rSNP rare DIO2-G allele (0.06) creates a unique TFBS for the POU2F2 TF which occurs only once in the DIO2 gene and regulates transcription in a number of tissues in addition to activating immunoglobulin gene expression.²³ The rs225015 rSNP common DIO2-G allele creates a unique TFBS for the EBF1 TF which occurs only once in the DIO2 gene and is a transcriptional activator.²³ The rs225015 rSNP minor DIO2-A allele (0.40) creates a unique TFBS for the TCF7L2 TF which occurs only once in the DIO2 gene and is implicated in blood glucose homeostasis.²³

TBXA2R rSNPs, TFBS and Asthma

The thromboxane A2 receptor (TBXA2R) gene which is located at chromosome 19p13.3 is a member of the seven-transmembrane G-protein-coupled receptor super family, which interacts with intracellular G proteins. It regulates different downstream signaling cascades, and induces many cellular responses including the intracellular calcium influx, cell migration and proliferation, and apoptosis.⁶⁷ This gene is abundantly expressed in tissues (at the mRNA and protein levels) targeted by the TBXA2R ligand thromboxane A2 (TXA2) that include erythroleukaemia cells, vascular and bronchial smooth muscle, uterus and placental tissue, endothelium, epithelium, trophoblasts, thymus, liver and small intestine.⁶⁸ The activation of TBXA2R in bronchial smooth muscle cells by its ligand results in intercellular calcium mobilization with subsequent bronchoconstriction. This contributes to bronchial smooth muscle hyperplasia and airway remodeling, which occurs in response to chronic airway inflammation in asthma.⁶⁹

Four linked TBXA2R rSNPs (rs2238631, rs2238632, rs2238633 and rs2238634) which span a 431bp region of intron one have been found to be in LD with two exon 3 SNPs [rs11318632, (c.795 T > C) and rs4523 (c.924 T > C)],⁷⁰ which are approximately 8.4 kb downstream from the intron one rSNPs. The rs11318632 and rs4523 rSNPs from exon 3 are synonymous changes and unlikely to influence the characteristics of the receptor protein. The exon 3 rSNPs have been associated with asthma and its related phenotypes in Asian populations. The rs4523 SNP has been found to be associated with adult asthma in a Japanese population⁷¹ and childhood atopic asthma in a Chinese population.⁷² The rs11318632 SNP has been found to be associated with atopic asthma in a Korean population.⁷³ Two haplotypes (H2 & H4) involving the four linked TBXA2R SNPs from intron one where found to influence TBXA2R transcriptional activity and were also associated with asthma-related phenotypes.⁷⁰

Four rSNPs (rs2238631, rs2238632, rs2238633 and rs2238634) in intron one, two rSNPs (rs1131882 and rs4523) in exon 3 and one rSNP (rs5756) in the 3’UTR of the thromboxane A2 receptor (TBXA2R) gene have been associated with childhood-onset asthma in Asians. These rSNP alleles alter the DNA landscape for potential transcriptional factors (TFs) to attach resulting in changes in transcriptional factor binding sites (TFBS). These TFBS changes were examined with respect to asthma which has been found to be significantly associated with the rSNPs.

The rs2238631 rSNP common TBXA2R-G allele creates a unique TFBS for the FOXC1 TF which occurs only once in the TBXA2R gene and is an important regulator of cell viability and resistance to oxidative stress.¹⁹ The rs2238631 rSNP minor TBXA2R-A allele (0.18) creates two unique TFBS for the ELK1 and ELK4 TFs which each occur only once in the TBXA2R gene and are involved with the ras-raf-MAPK signaling cascade, transcriptional activation and repression, respectively.¹⁹ The rs2238632 rSNP minor TBXA2R-T allele (0.49) creates two unique TFBS for the CREB1 and HIF1α:ARNT TFs which each occur only once in the gene and are involved with DNA binding, cellular and systemic responses to hypoxia, respectively.¹⁹ The rs2238634 rSNP minor TBXA2R-T allele (0.19) creates a unique TFBS for the HLTF TF which occurs only once in the TBXA2R gene and whose protein alters the chromatin structure around certain genes during transcription.¹⁹ The rs1131882 rSNP common TBXA2R-T allele (0.57) creates a unique TFBS for the SOX17 TF which occurs only once in the gene and is a transcription regulator that binds target promoter DNA and bends the DNA.¹⁹ The rs4523 rSNP minor TBXA2R-C allele (0.21) creates a unique TFBS for the AR TF which occurs only once in the gene and is a steroid hormone receptor that regulate eukaryotic gene expression and affects cellular proliferation and differentiation in target tissues.¹⁹

VEGFA rSNPs, TFBS and Hypoxia

The vascular endothelial growth factor (VEGF) is a family of key regulators in critical physiological and pathological angiogenesis⁷⁴ including tissue growth, wound healing, rheumatoid arthritis, proliferative retinopathies, cardiovascular disease and cancer⁷⁵ and is a growth factor activator for angiogenesis, vasculogenesis and endothelial cell growth. It has been shown that the VEGF is an important component of the pathogenesis of high altitude adaptation and sickness in most studies^76–81 but not all.⁸² Presently seven VEGF family members and 14 alternative splicing variants have been identified in humans.^83–85 Of the 14 splicing variants, 12 are VEGFA isoforms⁸⁵ with three (VEGFA-121, -165 & -189) being differentially expressed in humans visiting or living in high altitude environments and as well as in chronic mountain sickness (CMS) patients.^{77, 78} Among all family members, VEGFA, which is located at chromosome 6p12, is the most potent and best know angiogenic protein and exerts its biologic effect through interaction with cell-surface receptors which triggers a cascade of downstream dimerizations and phosphorylations.⁸⁶

Mountain sickness (MS) occurs among humans visiting or inhabiting high altitude environments. Genetic analyses of seven rSNPs in the promoter region of VEGFA gene were analyzed in lowland (Han) and highland (Tibetan) Chinese ¹⁴. The seven VEGFA rSNPs were evaluated in Han and Tibetan patients with acute and chronic mountain sickness, respectively. The rSNPs are rs699947, rs34357231, rs79469752, rs13207351, rs28357093, rs1570360 and rs2010963 which are found in the promoter ranging from -2578 bps to -634bps from the transcriptional start site (TSS), respectively. These rSNPs are found in TFBS and all alter these binding sites.¹⁴ Arterial oxygen saturation of hemoglobin (SaO₂) has been found to be significantly associated with the rs699947, rs34357231, rs13207351 and rs1570360 rSNPs in Han patients with AMS. ¹⁴ MS was found to be significantly associated with these rSNPs when compared to their Han and Tibetan control groups indicating these nucleotide substitutions results in TFBS changes which apparently have a physiological effect in the development of high altitude sickness.¹⁴ LD was found between rSNPs (rs13207351 and rs1570360) and between rSNPs (rs79469752 and rs28357093) among the control and sickness groups.^{14 87} The TFBS [HIF1α:ARNT] of the hypoxia inducible factor-1α and aryl hydrocarbon receptor nuclear translocator TFs is generated by the rs699947 common rSNP VEGFA-C allele but not with the minor VEGFA-A allele (0.23). This rSNP is not in LD with the other rSNPs in the VEGFA promoter.¹⁴ The HIF1α and ARNT TFs play an essential role in cellular and systemic responses to hypoxia and its TFBS occurs only in the promoter of the VEGFA gene,¹⁴ consequently, the rs699947 rSNP may have an impact on SaO₂ in patients with high altitude mountain sickness.

SNPs, TFBS and Ethnic Populations

A given rSNP allele frequency can vary between ethnic or racial groups due to historical population bottlenecks. This would affect the occurrence of TFBS and the TFs regulating genes and should impact the groups that are susceptible to disease or sickness. As an example, the rs225015 rSNP DIO2-A allele (above) creates a TFBS for the TCF7L2 TF, which is implicated in blood glucose homeostasis, and has an allele frequency of 0.40 in most ethnic and racial groups; however, the frequency is 0.81 in Pima Indians .⁶⁶ This rSNP is modestly associated with early-onset Type 2 Diabetes Mellitus and hepatic glucose output in the Pima Indians of Arizona⁶⁶ which may in part result from the higher occurrence of DIO2-A allele and TCF7L2 TFBS then in most other ethnic or racial groups.

Concluding Remarks

In summary, SNPs which occur in the non-coding regions of these genes have been found to be associated with human diseases or sicknesses. These non-coding regions host the binding sites for the transcription factors that regulate gene expression. The rs7128315 ADRBK1 rSNP which has been associated with coronary heart disease and dysfunction in Asians alters the SREBF1 & 2 TFBS which could interfere with lipid homeostasis. The rs2125230 AKT3 rSNP which has been associated with aggressive prostate cancer alters the IRF1 TFBS which could interfere with antiviral agents that fight tumors. The rs3125289 AFT3 rSNP which has been associated with the risk of hypospadias alters the SRY TFBS which could affect male development. The rs225015 DIO2 rSNP which is modestly associated with early-onset of T2DM in Pima Indians alters the TCF7L2 TFBS which is implicated in blood glucose homeostasis. The rs4523 TBXA2R rSNP which has been associated with asthma in Asians alters the AR TFBS which could affect cellular proliferation and differentiation. The rs699947 VEGFA rSNP which has been associated with arterial oxygen saturation of hemoglobin alters the HIF1α:ARNT TFBS which could affect the cellular and systemic responses to hypoxia. In conclusion regulatory SNPs underlying the allele-specific changes in transcriptional factor binding sites can alter the transcriptional factors that regulate these genes.