Computational EPAS 1 rSNP Analysis , Transcriptional Factor Binding Sites and High Altitude Sickness or Adaptation

Purpose – The endothetal Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) gene which encodes hypoxiainducible-factor-2 alpha (HIF2a) is a transcription factor that is involved in the response to hypoxia. EPAS1 has been found to have four (rs56721780, rs6756667, rs7589621, rs1868092) simple nucleotide polymorphisms (SNPs) associated with human disease. These SNPs were computationally examined with respect to changes in potential transcriptional factor binding sites (TFBS) and these changes were discussed in relation to disease and alterations in high altitude adaptation in humans. Methods -The JASPAR CORE and ConSite databases were instrumental in identifying the TFBS. The Vector NTI Advance 11.5 computer program was employed in locating all the TFBS in the EPAS1 gene from 1.6 kb upstream of the transcriptional start site to 539 bps past the 3’UTR. The JASPAR CORE database was also involved in computing each nucleotide occurrence (%) within the TFBS. Results – The EPAS1 SNPs in the promoter, intron two and the 3’UTR regions have previously been found to be significantly associated with disease and different levels of high-altitude hypoxia among native Tibetans. The SNP alleles were found to alter the DNA landscape for potential transcriptional factors (TFs) to attach resulting in changes in TFBS and thereby, alter which transcriptional factors potentially regulate the EPAS1 gene such as for the glucocorticoid and mineralocorticoid nuclear receptor binding sites created by the rs7589621 rSNP EPAS1-G allele. These receptors regulate carbohydrate, protein and fat metabolism. Also the minor rs7589621 rSNP EPAS1-A creates a punitive TFBS for the FOXC TF which is an important regulator of cell viability and resistance to oxidative stress. These EPAS1 SNPs should be considered as regulatory (r) SNPs. Conclusion -The alleles of each rSNP were found to generate unique TFBS resulting in potential changes in TF EPAS1 regulation. The punitive changes in TFBS created by the four rSNPs could very well influence the significant cline in allele frequencies seen in Tibetans with increasing altitude or the haplotype association with high altitude polycythemia in male Han Chinese. These regulatory changes were discussed with respect to changes in human health that result in disease and sickness. DOI : 10.14302/issn.2326-0793.jpgr-15-889 Freely Available Online www.openaccesspub.org | JPGR CC-license DOI : 10.14302/issn.2326-0793.jpgr-15-889 Vol-1 Issue 4 Pg. no.32 Introduction: The endothetal Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) gene which encodes hypoxia-inducible -factor-2 alpha (HIF2a) is a transcription factor that is involved in the response to hypoxia. Hypoxia is a major geographical condition associated with high-altitude environments [1]. Hypoxia-inducible-factors (HIFs) are heterodimers consisting of an oxygen-labile HIFa subunit and a stable HIFb subunit [2]. During hypoxia conditions, three isoforms either HIF1a, HIF2a or HIF3a and HIF1b are activated and function as transcriptional regulators of genes involved with the hypoxia response [3-5]. Genome wide association studies (GWAS) on high -altitude adaptation have implicated several single nucleotide polymorphisms (SNPs) in the regulatory region of the EPAS1 gene which are responsible for the genetic adaptation of high-altitude hypoxia in Tibetans [6-8]. Genetic variation in the regulatory region of the EPAS1 gene may influence gene expression and contribute to changes in biological functions [9]. EPAS1 is expressed in organs that are involved in oxygen transport and metabolism, such the lung, placenta and vascular endothelium [10], and is associated with many biological processes and diseases related to metabolism [11], angiogenesis [12, 13], inflammation [14, 15] and cancer [16-18]. The EPAS1 gene maps to human chromosome 2p21 and is about 120 kb in size with a coding region consisting of 15 exons [19]. Four HIF2a SNPs (rs56721780, rs6756667, rs7589621 and rs1868092) have been significantly associated with different levels of high-altitude hypoxia among native Tibetans [20]. The rs56721780 SNP in the HIF2a promoter region has also been significantly associated with high-altitude adaptation of Tibetans [9] while the rs6756667 SNP from intron two has been significantly associated with susceptibility to acute mountain sickness in individuals unaccustomed to high altitude environments [21]. The rs1868092 SNP near the HIF2a 3’UTR has been associated with high altitude polycythemia in male Han Chinese at the Qinghai-Tibetan plateau [22]. Single nucleotide changes that affect gene expression by impacting gene regulatory sequences such as promoters, enhances, and silencers are known as regulatory SNPs (rSNPs) [23-26]. A rSNPs within a transcriptional factor binding site (TFBS) can change a transcriptional factor’s (TF) ability to bind its TFBS [2730] in which case the TF would be unable to effectively regulate its target gene [31-35]. This concept is examined for the above four HIF2a rSNPs and their allelic association with TFBS, where computation analyses [36-39] is used to identify TFBS alterations created by the HIF2a rSNPs. In this report, the rSNP associations with changes in potential TFBS are discussed with their possible relationship to disease or sickness in humans.


Introduction:
The endothetal Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) gene which encodes hypoxia-inducible -factor-2 alpha (HIF2a) is a transcription factor that is involved in the response to hypoxia.Hypoxia is a major geographical condition associated with high-altitude environments [1].Hypoxia-inducible-factors (HIFs) are heterodimers consisting of an oxygen-labile HIFa subunit and a stable HIFb subunit [2].During hypoxia conditions, three isoforms either HIF1a, HIF2a or HIF3a and HIF1b are activated and function as transcriptional regulators of genes involved with the hypoxia response [3][4][5].Genome wide association studies (GWAS) on high -altitude adaptation have implicated several single nucleotide polymorphisms (SNPs) in the regulatory region of the EPAS1 gene which are responsible for the genetic adaptation of high-altitude hypoxia in Tibetans [6][7][8].Genetic variation in the regulatory region of the EPAS1 gene may influence gene expression and contribute to changes in biological functions [9].EPAS1 is expressed in organs that are involved in oxygen transport and metabolism, such the lung, placenta and vascular endothelium [10], and is associated with many biological processes and diseases related to metabolism [11], angiogenesis [12,13], inflammation [14,15] and cancer [16][17][18].
The EPAS1 gene maps to human chromosome 2p21 and is about 120 kb in size with a coding region consisting of 15 exons [19].Four HIF2a SNPs (rs56721780, rs6756667, rs7589621 and rs1868092) have been significantly associated with different levels of high-altitude hypoxia among native Tibetans [20].The rs56721780 SNP in the HIF2a promoter region has also been significantly associated with high-altitude adaptation of Tibetans [9] while the rs6756667 SNP from intron two has been significantly associated with susceptibility to acute mountain sickness in individuals unaccustomed to high altitude environments [21].The rs1868092 SNP near the HIF2a 3'UTR has been associated with high altitude polycythemia in male Han Chinese at the Qinghai-Tibetan plateau [22].
This concept is examined for the above four HIF2a rSNPs and their allelic association with TFBS, where computation analyses [36][37][38][39] is used to identify TFBS alterations created by the HIF2a rSNPs.In this report, the rSNP associations with changes in potential TFBS are discussed with their possible relationship to disease or sickness in humans.

Methods
The JASPAR CORE database [40,41] and ConSite [42] were used to identify the potential STAT4 Vol-1 Issue 4 Pg.no.-33 sequences.The TFBS and rSNP location within the binding sites have previously been discussed [43].The Vector NTI Advance 11.5 computer program (Invitrogen, Life Technologies) was used to locate the TFBS in the EPAS1 gene (NCBI Ref Seq NM_001430) from 1.6 kb upstream of the transcriptional start site to 539 bps past the 3'UTR which represents a total of 91 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 3).

EPAS1 rSNPs and TFBS
The allele frequencies of four EPAS1 SNPs (rs56721780, rs6756667, rs7589621 and rs1868092) significantly associated with different levels of highaltitude hypoxia among native Tibetans [20] are presented in Table 1 along with low altitude Han Chinese and Japanese populations.The common rs56721780 SNP EPAS1 -G allele creates nine unique punitive TFBS for the REL, RELA, RUNX1,TFAP2A, TFAP1 (var.2),TFAP2B, TFAP2B(var.2),TFAP2C and TFAP2C (var.2) TFs, which are involved with inflammation, immunity, differentiation, cell growth, tumorigenesis, apoptosis, hematopoiesis, transcriptional activation and repression, respectively (Tables 2 & 3).The minor EPAS1 -C allele creates two unique punitive TFBS for the FOXP3 and HOXA5 TFs which are involved with the homeostasis of the immune system and specific identities on the anterior-posterior axis during development, respectively (Tables 2 & 3).There are also four conserved TBFS for the HLTF, HNF4G, NFAT5 and SOX10 TFs which are involved altering chromatin structure, transcription, regulation of osmoprotective and inflammatory genes and embryonic development, respectively (Table 2, Table 3).
There are also twenty-one conserved TFBS for the ATF4,  The encoded protein is a homeobox transcription factor that is related to the protein encoded by the Drosophila even-skipped (eve) gene, a member of the pair-rule class of segmentation genes.

FIGLA Folliculogenesis Specific Basic Helix-Loop-Helix
The protein is a basic helix-loop-helix transcription factor that regulates multiple oocyte-specific genes, including genes involved in folliculogenesis and those that encode the zona pellucida.

FOS Jun Proto-Oncogene
The Fos gene family consists of 4 members: FOS, FOSB, FOSL1, and FOSL2.These genes encode leucine zipper proteins that can dimerize with proteins of the JUN family, thereby forming the transcription factor complex AP-1.The FOS proteins have been implicated as regulators of cell proliferation, differentiation, and transformation.Controls osteoclast survival and size.As a dimer with JUN, activates LIF transcription.This gene is a member of KDWK gene family.The product of this gene associates with GMEB1 protein, and the complex is essential for parvovirus DNA replication.

GSX1
GS Homeobox 1 Activates the transcription of the GHRH gene.Plays an important role in pituitary development.Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis.

HOXA5 Hoxa5
Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis.

HOXB2
Homeobox B2 Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis.

HOXB3 Homeobox B3
The encoded protein functions as a sequence-specific transcription factor that is involved in development.

INSM1 Insulinoma-Associated 1
This gene is a sensitive marker for neuroendocrine differentiation of human lung tumors.

ISL2 ISL LIM Homeobox 2
Transcriptional factor that defines subclasses of motoneurons that segregate into columns in the spinal cord and select distinct axon pathways.Transcriptional regulator which is important for the differentiation and maintenance of meso-diencephalic dopaminergic (mdDA) neurons during development.This gene encodes a member of the SOX (SRY-related HMG-box) family of transcription factors involved in the regulation of embryonic development and in the determination of the cell fate.

SREBF1 Sterol regulatory element binding transcription factor 1
Transcriptional activator required for lipid homeostasis.Regulates transcription of the LDL receptor gene as well as the fatty acid and to a lesser degree the cholesterol synthesis pathway.

SREBF2 Sterol regulatory element binding transcription factor 2
This gene encodes a member of the a ubiquitously expressed transcription factor that controls cholesterol homeostasis by regulating transcription of sterol-regulated genes.The encoded protein contains a basic helix-loop-helix-leucine zipper (bHLH-Zip) domain and binds the sterol regulatory element 1 motif.

SRY SRY (sex determining region Y)box 10
Transcriptional regulator that controls a genetic switch in male development.It is necessary and sufficient for initiating male sex determination by directing the development of supporting cell precursors TBP TATA Box Binding Protein General transcription factor that functions at the core of the DNA-binding multiprotein factor TFIID. Binding of TFIID to the TATA box is the initial transcriptional step of the pre-initiation complex (PIC), playing a role in the activation of eukaryotic genes transcribed by RNA polymerase II.

TBX4 T-Box 4
Involved in the transcriptional regulation of genes required for mesoderm differentiation.

TBX5 T-Box 5
This gene is a member of a phylogenetically conserved family of genes that share a common DNA-binding domain, the T-box.T-box genes encode transcription factors involved in the regulation of developmental processes.

TEAD1 TEA Domain Family Member 1
This gene encodes a ubiquitous transcriptional enhancer factor that is a member of the TEA/ATTS domain family.This protein directs the transactivation of a wide variety of genes and, in placental cells, also acts as a transcriptional repressor.

TEAD3 TEA Domain Family Member 3
This gene product is a member of the transcriptional enhancer factor (TEF) family of transcription factors, which contain the TEA/ATTS DNA-binding domain.It is predominantly expressed in the placenta and is involved in the transactivation of the chorionic somatomammotropin-B gene enhancer.

TEAD4 TEA Domain Family Member 4
It is preferentially expressed in the skeletal muscle, and binds to the M-CAT regulatory element found in promoters of muscle-specific genes to direct their gene expression.The protein encoded by this gene is a transcription factor that binds the consensus sequence 5'-GCCNNNGGC-3' and activates the transcription of some genes while inhibiting the transcription of others.

USF2 Upstream Transcription Factor 2, C-Fos Interacting
Transcription factor that binds to a symmetrical DNA sequence (E-boxes) (5-CACGTG-3) that is found in a variety of viral and cellular promoters.

YY1 YY1 Transcription Factor
Multifunctional transcription factor that exhibits positive and negative control on a large number of cellular and viral genes by binding to sites overlapping the transcription start site

YY2 YY2 Transcription Factor
The protein encoded by this gene is a transcription factor that includes several Kruppel-like zinc fingers in its C-terminal region.It possesses both activation and repression domains, and it can therefore have both positive and negative effects on the transcription of target genes.

ZNF354C Zinc finger protein 354C
May function as a transcription repressor.
Table 3.The EPAS1 (HIF2a) SNPs that were examined in this study where the minor allele is in red.Also listed are the transcriptional factors (TF), their potential binding sites (TFBS) containing these SNPs and DNA strand orientation.TFs in red differ between the SNP alleles.Where upper case nucleotide designates the 90% conserved BS region and red is the SNP location of the alleles in the TFBS.Below the TFBS is the nucleotide occurrence (%) obtained from the Jaspar Core database.Also listed are the number (#) of binding sites in the gene for the given TF.Note: TFs can bind to more than one nucleotide sequence.2 & 3 2 & 3).

Discussion:
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 [44,45] and the remaining 93% are located within non-coding areas [46,47] such as regulatory or intergenic regions.-100% occurrence, respectively in humans (Table 3).
Since these binding sites (BS) occur only once in the gene except for the RUNX1 TFBS which occurs twice, the rSNP G allele should have a tremendous impact on gene regulation by these TFs (Table 3).TFBSs have a 100% occurrence in humans and are found only once in the EPAS1 gene (Table 3).
Consequently, the corresponding glucocorticoid and mineralocorticoid nuclear receptors which bind their respective BS should have a major impact on the regulation of the EPAS1 gene (Tables 2 & 3).The minor rs7589621 rSNP EPAS1-A allele [A (+ strand) or T (strand)] located in the unique LIN54, POU3F1-4, TEAD1, 3, 4 TFBS has a 100, 95, 95, 90, 99, 92, 100 and 94% occurrence, respectively in humans (Table 3).However, only the POU3F1-3 TFBS occur once in the EPAS1 gene, consequently, their corresponding TFs should have an impact on the EPAS1 gene regulation.The remaining TFBS occur multiple times in the gene and would not be expected to have much impact on gene regulation (Figure 2, Tables 2 & 3).
The rs1868092 rSNP EPAS1-A allele [A (+ strand) or T (-strand)] located in the unique NR2C2 and YY1 TFBS have a 81 and 94%% occurrence, respectively in humans and only occur once in the EPAS1 gene (Tables 2 & 3).The NR2C2 orphan nuclear receptor which can occur as a repressor of activator of transcription and the YY1 TF which exhibits both positive and negative control of transcript should have an impact on the regulation of the EPAS1 gene (Table 2).
The minor rs1868092 rSNP EPAS1-G allele [G (+ strand) or C (-strand)] located in the unique HIC2, KLF5, NFIA, and TEAD1 TFBS have a 99, 100, 100 and 95% occurrence, respectively in humans and occur only once in the EPAS1 gene (Table 3).Since the corresponding TFs function as activators, enhancers and repressors, the occurrence of these TFBS should impact the regulation of the EPAS1 gene (Tables 2 & 3).
Human diseases or conditions can be associated with rSNPs of the EPAS1 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 punitive TFBS associated with the rs56721780 common rSNP EPAS1-G allele from Table 3 as illustrated in Figure 1 as well as the rs7589621 common rSNP STAT4-G allele as illustrated in Figure 2. As can be seen in Table 3  Table 3).The rs56721780 rSNP common EPAS1-G allele is found in each of these TFBS.As shown, this rSNP is located in the promoter region of the EPAS1 gene.
Also included with the potential TFBS is their % sequence homology to the duplex.3).The rs7589621 rSNP common EPAS1-G allele is found in each of these TFBS.As shown, this rSNP is located in intron two of the EPAS1 gene.Also included with the potential TFBS is their % sequence homology to the duplex.

FOXH1
Forkhead Box H1 Transcriptional activator.Recognizes and binds to the DNA sequence 5-TGT[GT][GT]ATT-3.Required for induction of the goosecoid (GSC) promoter by TGF-beta or activin signaling.FOXP3 Forkhead Box P3 Transcriptional regulator which is crucial for the development and inhibitory function of regulatory T-cells (Treg).Plays an essential role in maintaining homeostasis of the immune system by allowing the acquisition of full conventional T-cells.Suppressive function and stability of the Treg lineage, and by directly modulating the expansion and function of conventional T-cells.GBX1 Gastrulation Brain Homeobox 1 Sequence-specific DNA binding transcription factor activity and sequence-specific DNA binding.An important paralog of this gene is DLX5.GBX2 Gastrulation Brain Homeobox 2 May act as a transcription factor for cell pluripotency and differentiation in the embryo GMEB2 Glucocorticoid Modulatory Element Binding Protein 2

Hypermethylated In Cancer 1
This gene functions as a growth regulatory and tumor repressor gene.HIC2 Hypermethylated In Cancer 2 Transcriptional repressor HLF Hepatic Leukemia Factor The encoded protein forms homodimers or heterodimers with other PAR family members and binds sequence-specific promoter elements to activate transcription.HLTF Helicase-like transcription factor This gene encodes a member of the SWI/SNF family.Members of this family have helicase and ATPase activities and are thought to regulate transcription of certain genes by altering the chromatin structure around those genes.HMBOX1 Homeobox Containing 1 Transcription factor.Isoform 1 acts as a transcriptional repressor.HNF4g Hepatocyte Nuclear Factor 4, Gamma Transcription factor.Has a lower transcription activation potential than HNF4-alpha HOXA2 Homeobox A2 Intestine-Specific Homeobox Transcription factor that regulates gene expression in intestine.May participate in vitamin A metabolism most likely by regulating BCO1 expression in the intestine.JDP(var.2) Jun Dimerization Protein 2Component of the AP-1 transcription factor that represses transactivation mediated by the Jun family of proteins.Involved in a variety of transcriptional responses associated with AP-1 such as UV-induced apoptosis, cell differentiation, tumorigenesis and antitumogeneris.JUN Jun Proto-OncogeneTranscription factor that recognizes and binds to the enhancer heptamer motif 5-TGA[CG] TCA-3.signaling pathway stimulation.Promotes activity of NR5A1 when phosphorylated by HIPK3 leading to increased steroidogenic gene expression upon cAMP signaling pathway Neural Retina Leucine Zipper This gene encodes a basic motif-leucine zipper transcription factor of the Maf subfamily.The encoded protein is conserved among vertebrates and is a critical intrinsic regulator of photoceptor development and function.insulin, somatostatin, glucokinase, islet amyloid polypeptide and glucose transporter type 2 gene transcription.Particularly involved in glucose-dependent regulation of insulin gene transcription.PHOX2A Paired-Like Homeobox 2a May be involved in regulating the specificity of expression of the catecholamine biosynthetic genes.Acts as a transcription activator/factor.POU2F1 POU Class 2 Homeobox 1 Transcription factor that binds to the octamer motif (5-ATTTGCAT-3) and activates the promoters of the genes for some small nuclear RNAs (snRNA) and of genes such as those for histone H2B and immunoglobulins.Modulates transcriptiontransactivation by NR3C1, AR and PGR POU3F1 POU Class 3 Homeobox 1 Transcription factor that binds to the octamer motif (5-ATTTGCAT-3).Thought to be involved in early embryogenesis and neurogenesis POU3F2 POU Class 3 Homeobox 2 This gene encodes a member of the POU-III class of neural transcription factors.The encoded protein is involved in neuronal differentiation and enhances the activation of corticotropin-releasing hormone regulated genes.POU3F3 POU Class 3 Homeobox 3 This gene encodes a POU-domain containing protein that functions as a transcription factor.The encoded protein recognizes an octamer sequence in the DNA of target genes.This protein may play a role in development of the nervous system.POU3F4 POU Class 3 Homeobox 4 This gene encodes a member of the POU-III class of neural transcription factors.This family member plays a role in inner ear development.The protein is thought to be involved in the mediation of epigenetic signals which induce striatal neuron-precursor differentiation.POU5F1 POU Class 5 Homeobox 1B This gene encodes a transcription factor containing a POU homeodomain that plays a key role in embryonic development and stem cell pluripotency.Aberrant expression of this gene in adult tissues is associated with tumorigenesis.Forms a trimeric complex with SOX2 on DNA and controls the expression of a number of genes involved in embryonic development such as YES1, FGF4, UTF1 and ZFP206.CC-license Vol-1 Issue 4 Pg.no.-39 ).The minor rs7589621 SNP EPAS1-A allele creates eleven unique punitive TFBS for the FOXC1, HNF4G, LIN54, NEUROD2, POU3F1-4, TEAD1, and TEAD3 & 4, TFs which are involved with cell viability and resistance to oxidative stress, transcriptional repression, regulation of cell cycle genes, neuronal determination, early embryogenesis and neurogenesis, and enhancer for transcription, respectively (Tables 2 & 3).There are also thirty-one conserved TFBS for the BARX1, BSX, DBP, DLX6, EN2, ESX1, EVX1 & 2, GMEB2, GSX1, HLF, HLTF, HMBOX1, HOXA2, HOXB2, HOXB3, ISL2, ISX, LBX2, NFATC3, NKX2-3, RORA AND TBP TFs which are involved with craniofacial development, transcriptional activation, circadian rhythm, roles in forebrain, central nervous system, placental development, specification of neuronal cell types, DNA replication, pituitary development, transcription activation, altering chromatin structure, transcriptional repressor, regulates development, axon pathways, regulates gene expression in the intestine, induces expression of cytokine genes in T-cells, homeodomain, nuclear hormone receptors, and the pre-initiation complex, respectively (Tables 2 & 3).The common rs1868092 SNP EPAS1-A allele creates four unique TFBS for the HOXA5, NR2C2, YY1 & 2 TFs which are involved with the development regulatory system, repression or activation of transcription, and positive and negative control of transcription at the transcription start site, respectively (Tables 2 & 3).The minor EPAS1-G allele creates seven unique TFBS for the HIC2, KLF5, MZF1, NFIA, NFIC, NFIX and TEAD1 TFs which are involved with transcriptional repression, transcription, hemopoietic development, transcription and replication, and enhancer of transcription, respectively (Tables 2 & 3).There are also seven conserved punitive TFBS for the E2F6, HLTF, HNF4G, NKX2-3, NKX2-8, SOX10 and THAP1 TFs which are involved in tumor suppression, chromatin structure, transcriptional activation, homeodomain, regulatory, and regulates endothelial cell proliferation, respectively (Tables 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[23,25,48].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 [48].Examples of rSNPs associated with disease susceptibility are numerous and several reviews have been published [48-51].The rs56721780 rSNP EPAS1-G allele [G (+ strand) or C (-strand)] located in the unique RELA, RUNX1, TFAP2A, B & C TFBS have a 100%, 92% and 94 The minor rs56721780 rSNP EPAS1-C allele [C (+ strand) or G (-Vol-1 Issue 4 Pg.no.-53 strand)] located in the unique FOXP3 and HOXA5 TFBS have a 65% and 31% occurrence, respectively in humans.Since these TFBS have a low occurrence in humans and occur more than once in the gene, the respective TF would not be expected to have much impact on the regulation of the EPAS1 gene (Figure 1, Table 3).The rs6756667 rSNP EPAS1-A allele [A (+ strand) or T (-strand) located in the unique CEBPa, FOS::JUN, HIC 1 & 2, HOXA2, NFIA and NRL TFBS has a 78, 94, 95, 98, 90, 100, 86 and 100% occurrence, respectively in humans (Figure 2, Table 3); however, only the CEBPa, NFIA and NRL TFBS occur only once in the gene.Consequently the corresponding three TFs should impact the regulation of the EPAS1 gene.The minor rs6756667 rSNP EPAS1-G allele [G (+ strand) or T (-strand)] located in the unique ATF7, GMEB2, JDP2, and MGA TFBS have a 99, 100, 99, 100% occurrence, respectively in humans and all only occur once in the EPAS1 gene.Consequently, the TFs for these TFBS could have some impact on the regulation of the EPAS1 gene (Tables 2 & 3).The rs7589621 rSNP EPAS1-G allele [G (+ strand) or C (-strand)] located in the unique NR3C1 & 2 , these potential TFBS change when an individual carries the alternate allele.The importance of this has been illustrated in Figure 1 with the punitive TFAP2A, B & C TFBS where the common G allele has binding sites for these TFs and the minor C allele does not.The TFAP2A, B & C TFs act as activators and repressors and are involved in a large spectrum of biological functions such as proper eye, face, body wall, limb and neural tube development (cf.Table 2).Another example would be the punitive NR3C1 & 2 TFBS where the common

Figure 1 .
Figure 1.Double stranded DNA from the EPAS1 gene showing the potential TFBS for twenty different TFs which can bind their respective DNA sequence either above (+) or below (-) the duplex (cf.

Figure 2 .
Figure 2. Double stranded DNA from the EPAS1 gene showing the potential TFBS for forty eight different TFs which can bind their respective DNA sequence either above (+) or below (-) the duplex (cf.Table3).The

Table 2 (
Continuation) JUND(var.2) Jun D Proto-OncogeneTranscription factor that recognizes and binds to the enhancer heptamer motif 5'-TGA[CG] TCA-3'.The encoded protein may play a role in the regulation of vertebrate limb myogenesis.Mutations in the related mouse protein may be associated with craniofacial and/or skeletal abnormalities, in addition to neurovascular dysfunction observed in Alzheimer's disease.MGA MGA, MAX Dimerization Protein Functions as a dual-specificity transcription factor, regulating the expression of both MAX-network and T-box family target genes.Functions as a repressor or an activator.Acts as a regulator of transcriptional activation.Plays a role in the inducible expression of cytokine genes in T-cells, especially in the induction of the IL-2.Recognizes and binds the palindromic sequence 5'-TTGGCNNNNNGCCAA-3' present in viral and cellular promoters and in the origin of replication of adenovirus type 2. These proteins are individually capable of activating transcription and replication.NFIL3 Nuclear factor, interleukin 3 regulated Expression of interleukin-3 (IL3; MIM 147740) is restricted to activated T cells, natural killer (NK) cells, and mast cell lines.www.openaccesspub.org| JPGR CC-license Vol-1 Issue 4 Pg.no.-38

Table 2 (
Continuation)Sequence-specific DNA binding transcription factor activity and RNA polymerase II distal enhancer sequence-specific DNA binding transcription factor activity.Transcription factor which binds to specific A/T-rich DNA sequences in the promoter regions of a number of genes.Involved in transcriptional regulation in islet beta cells.Binds to the insulin promoter and is involved in regulation of the insulin gene.Orphan nuclear receptor that can act as a repressor or activator of transcription.An important repressor of nuclear receptor signaling pathways such as retinoic acid receptor, retinoid X, vitamin D3 receptor, thyroid hormone receptor and estrogen receptor pathways.Glucocorticoids regulate carbohydrate, protein and fat metabolism, modulate immune responses through supression of chemokine and cytokine production and have critical roles in constitutive activity of the CNS, digestive, hematopoietic, renal and reproductive systems.The protein encoded by this gene plays a role in protecting cells from oxidative stress and damage induced by ionizing radiation.

Table 2 (
Continuation) Homeobox 1B This intronless gene was thought to be a transcribed pseudogene of POU class 5 homeobox 1; however, it has been reported that this gene can encode a functional protein.The protein has been shown to be a weak transcriptional activator and may play a role in carcinogenesis and eye development.
This gene encodes a member of the runt domain-containing family of transcription factors.Found in a number of enhancers and promoters, and can either activate or suppress transcription.It also interacts with other transcription factors.It functions as a tumor suppressor, and the gene is frequently deleted or transcriptionally silenced in cancer.SOX10SRY (sex determining region Y)box 10

Table 3 (
Continuation) Vol-1 Issue 4 Pg.no.-52 regulation of carbohydrate, protein and fat metabolism, mediates aldosterone actions on salt and water balance, and catecholamine biosynthetic genes, respectively (Tables