Identifying changes in punitive transcriptional factor binding sites from regulatory single nucleotide polymorphisms that are significantly associated with disease or sickness

Abstract

AIM

To identify punitive transcriptional factor binding sites (TFBS) from regulatory single nucleotide polymorphisms (rSNPs) that are significantly associated with disease.

METHODS

The genome-wide association studies have provided us with nearly 6500 disease or trait-predisposing SNPs where 93% are located within non-coding regions such as gene regulatory or intergenic areas of the genome. In the regulatory region of a gene, a SNP can change the DNA sequence of a transcriptional factor (TF) motif and in turn may affect the process of gene regulation. SNP changes that affect gene expression and impact gene regulatory sequences such as promoters, enhancers, and silencers are known as rSNPs. Computational tools can be used to identify unique punitive TFBS created by rSNPs that are associated with disease or sickness. Computational analysis was used to identify punitive TFBS generated by the alleles of these rSNPs.

RESULTS

rSNPs within nine genes that have been significantly associated with disease or sickness were used to illustrate the tremendous diversity of punitive unique TFBS that can be generated by their alleles. The genes studied are the adrenergic, beta, receptor kinase 1, the v-akt murine thymoma viral oncogene homolog 3, the activating transcription factor 3, the type 2 demodkinase gene, the endothetal Per-Arnt-Sim domain protein 1, the lysosomal acid lipase A, the signal Transducer and Activator of Transcription 4, the thromboxane A2 receptor and the vascular endothelial growth factor A. From this sampling of SNPs among the nine genes, there are 73 potential unique TFBS generated by the common alleles compared to 124 generated by the minor alleles indicating the tremendous diversity of potential TFs that are capable of regulating these genes.

CONCLUSION

From the diversity of unique punitive binding sites for TFs, it was found that some TFs play a role in the disease or sickness being studied.

Key Words: Regulatory single nucleotide polymorphisms, Alleles, Transcriptional factors, Transcriptional factor binding sites, Linkage disequilibrium, Disease or sickness

Core tip: Disease or trait-predisposing single nucleotide polymorphisms (SNPs) in or near genes can alter the transcriptional factor binding sites (TFBS) for the TFs regulating the gene; thereby affecting the health of an individual. In this report, the disease or sickness associated regulatory SNPs (rSNPs) within a sampling of nine human genes were studied with respect to the alterations in TFBS. From this sampling there were 73 punitive unique TFBS generated by the common rSNP alleles compared to 124 generated by the minor alleles indicating the tremendous diversity of potential TFs that are capable of affecting the health of person.

INTRODUCTION

The genome-wide association studies (GWAS) have over the past decade provided us with nearly 6500 disease or trait-predisposing single nucleotide polymorphisms (SNPs). Only seven percent of these SNPs are located in protein-coding regions of the genome[1,2] while the remaining 93% are located within non-coding regions[3,4] such as gene regulatory or intergenic areas of the genome. Much attention has been drawn to SNPs that occur in the putative regulatory region of a gene where a single nucleotide change in the DNA sequence of a potential transcriptional factor (TF) motif may affect the process of gene regulation[5-7]. A nucleotide change in a transcriptional factor binding site (TFBS) can have multiple consequences. Since a TF can usually recognize a number of different binding motifs in a gene, the SNP may not change the TFBS interaction with the TF and consequently not alter the process of gene expression. In other cases the nucleotide change may increase or decrease the TF’s ability to bind DNA which would result in allele-specific gene expression. In some cases a nucleotide change may eliminate the natural binding motif or generate a new binding site (BS) as a result the gene is no longer regulated by the original TF[8,9]. Single nucleotide changes that affect gene expression by impacting gene regulatory sequences such as promoters, enhancers, and silencers are known as regulatory SNPs (rSNPs)[5,6,10,11]. 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[7,12-16]. Advances in understanding the functional relevance of SNPs in non-coding regions of the human genome using epi-genomics and genome engineering have been recently reviewed[17]. Computational tools can be used to identify punitive TFBS created by rSNPs which are associated with disease or sickness[18]. To this end, computational analysis has been used to identify punitive or potentially unique TFBS generated by the alleles of rSNPs[19] where unique TFBS occur with only one of the two rSNP alleles.

In this report, rSNPs within a sample of nine human genes (Table 1) which have been significantly associated with disease or sickness were selected to illustrate the tremendous diversity of unique punitive TFBS that can be generated by SNP alleles (Table 2)[8,9,20-27]. The SNP alleles from these reports were found to share common TFBS between alleles but each SNP allele can also create unique TFBS only for that allele (Table 2). As an example in Table 2, the rs948988 ADRBK1-G allele creates two potential unique TFBS for the Kruppel-like factors 1 and 4 (KLF1,4) TFs that do not occur with the alternate ADRBK1-A allele while the ADRBK1-A allele creates ten other punitive unique TFBS not found with ADRBK1-G allele. Many of the rSNPs have been reported to be in linkage disequilibrium (LD) (Table 1), where LD is considered to be the non-random association of SNP alleles within a gene. LD between SNPs in the regulatory region of a gene can indicate strong associations of certain haplotypes and TFBS with sickness or disease[28].

Table 1 Genes and their single nucleotide polymorphisms that have been found to be associated with disease or sickness.

Protein and gene symbol	Chromosome	SNP	SNP location	Mutation	LD	Ref.
Adrenergic, beta, receptor kinase 1	11q13.1	rs948988	intron 2	c.190 + 653G > A	Yes	[9,19]
		rs4370946	3'UTR	c.*217C > T	Yes
v-akt murine thymoma viral oncogene homolog 3	1q44	rs4590656	intron 1	c.46 + 3654C > T	Yes	[8,19]
		rs10157763	intron 1	c.46 + 11386C > T	Yes
		rs2125230	intron 1	c.47-26830G > A	Yes
Activating transcription factor 3	1q32.3	rs3125289	promoter	c.-5 + 9322T > C	Unknown	[19,20]
		rs11119982	promoter	c.-4-23516C > T	Unknown
Type 2 demodkinase gene	14q24.3	rs225015	3'UTR	c.*1453G > A	Yes	[19,21]
		rs225011	intron 1	c. 330 + 366C > T	Yes
		rs12885300	5'UTR	c.-451C > T	Yes
Endothetal Per-Arnt-Sim domain protein 1	2p21	rs6756667	intron 2	c.218-3881A > G	No	[22]
		rs1868092	3'UTR	c.*2403G > A	No
Lysosomal acid lipase A	10q23.31	rs1412444	intron 2	c.229 + 2506C > T	n/a	[23]
Signal transducer and activator of transcription 4	2q32.3	rs8179673	intron 2	c.274-28290T > C	Yes	[24]
		rs10181656	intron 2	c.274-28828C > G	Yes
Thromboxane A2 receptor	19p13.3	rs2238631	intron 1	c.-84 + 2229G > A	Yes	[19,25]
		rs2238632	intron 1	c.-84 + 2030C > T	Yes
		rs2238634	intron 1	c.-84 + 1799G > T	Yes
Vascular endothelial growth factor A	6p21.1	rs34357231	promoter	c. -2550-2568D > I	Yes	[19,26,28]
		rs1570360	promoter	c.-614A > G	Yes
		rs3025039	3'UTR	c.*237C > T	Yes

Also listed is the gene chromosome location, single nucleotide polymorphism location in the gene and the resulting genetic mutation as well as the occurrence of linkage disequilibrium between single nucleotide polymorphisms found within each gene. SNP: Single nucleotide polymorphism; LD: Linkage disequilibrium; n/a: Not available.

Table 2 Genes whose single nucleotide polymorphisms are significantly associated with human disease or sickness.

Gene symbol
ADRBK1	Ethnic group	B		B
	disease or sickness	12		12
	SNP	rs948988 (G/A)		rs4370946 (C/T)
	alleles (MAF)	G	A (0.29)	C	T (0.2)
	potential unique TFBS	KLF1, 4	BATF:JUN	E2F1,3,4,6	ARNT:AHR
			ESR2	EGR1	ATOH1
			FOS	INSM1	ELF1
			FOSL2	KLF4	ESR2
			JUND	NFKB1	NR3C1
			JUN:FOS	NRF1
			MYB	SP1, 2
			NFE2L1:MAF
			NR3C1
			SOX17
AKT3	Ethnic group	G		C		C
	disease or sickness	1		14		14
	SNP	rs4590656 (C/T)		rs10157763 (C/T)		rs2125230 (G/A)
	alleles (MAF)	C	T (0.41)	C	T (0.33)	G	A (0.2)
	potential unique TFBS	ARNT:AHR	GFI	ELF5	CTCF	ARNT:AHR	GATA1
		HIF1a:ARNT	HNF4A	ELK1	NFATC2	FEV	HNF4a
			PAX2	MYCN	SOX17	HIF1a:ARNT	HOXA5
			SPIB	SPIB	ZNF354C	SPI1	IRF1
				SPI1			NR2F1
				TFAP2A			SOX17
ATF3	Ethnic group	C		C
	disease or sickness	15		15
	SNP	rs3125289 (C/T)		rs11119982 (C/T)
	alleles (MAF)	C	T (0.10)	C	T (0.36)
	potential unique TFBS	ARNT	FOXA1, 2	HLTF	ARID3A
		ARNT:AHR	FOXL1		MAX
		GABPa	FOXO3		MYB
		MYC	HLTF		USF1
		MYCN	SOX10		ZEB1
		MZF1	SOX17
		SPIB	SRY
		USF1
DIO2	Ethnic group	F		F		C
	disease or sickness	2		2		17
	SNP	rs225015 (G/A)		rs225011 (C/T)		rs12885300 (C/T)
	alleles (MAF)	G	A (0.4)	C	T (0.42)	C	T (0.23)
	potential unique TFBS	EBF1	ELF1	CRX	FOXL1		ARID3A
		ESRRA	ELK1	RXRa	MEF2A		BATF:JUN
		PPARg:RXRa	ERG		PDX1		IRF1
		RFX5	ETS1				JUN:FOS
		THAP1	FLI1				PAX2
			RUNX1				SOX6
			SOX9
			SPI1
			TCF7L2
EPAS1	Ethnic group	G		G
	disease or sickness	1		1
	SNP	rs6756667 (A/G)		rs1868093 (A/G)
	alleles (MAF)	A	G (0.20)	A	G (0.25)
	potential unique TFBS	CEBPa	ATF7	NR2C2	HIC2
		NFIA	GMEB2	NFIA	KLF5
		NRL	JDP2	YY1	MGA
					TEAD1
					USF1
LIPA	Ethnic group	C, D, E
	disease or sickness	20, 21
	SNP	rs1412444 (C/T)
	alleles (MAF)	C	T (0.32)
	potential unique TFBS	ELF1	FOXA1
		ETS1	FOXL1
		GABPa	FOXO3
		HOXA5	HNF1B
		SPI1	MEF2A
			NFKB1
			NFIC
			PAX2
			SOX6
			SOX9
			SRY
			THAP1
STAT4	Ethnic group	A, C		A, C
	disease or sickness	2,6,10		2,10
	SNP	rs8179673 (T/C)		rs10181656 (C/G)
	alleles (MAF)	T	C (0.26)	C	G (0.26)
	potential unique TFBS	EN1	FOXA2	AR	HNF4g
		NFIL3	FOXH1	E2F6	STAT3
			FOXO1	NR1H3:RXRa
			FOXP1	ZNF263
			FOXQ1
			HNF1a
			HNF4g
TBXA2R	Ethnic group	A		A		A
	disease or sickness	22		22		22
	SNP	rs2238631 (G/A)		rs2238632 (C/T)		rs2238634 (G/T)
	alleles (MAF)	G	A (0.2)	C	T (0.21)	G	T (0.22)
	potential unique TFBS	FOXC1	ELK1		ARNT		HLTF
		TFAP2a	ELK4		CREB1		HNF4a
			ETS1		HIF1a:ARNT		NR2F1
			GATA2		MAX		NR2E3
			HAND1:TCFE2a		USF1		NR4A2
			SPZ1
VEGFA	Ethnic group	G		G		G
	disease or sickness	1		1		1
	SNP	rs34357231 (I/D)		rs1570360 (G/A)		rs3025039 (C/T)
	alleles (MAF)	D	I (0.28)	G	A (0.13)	C	T (0.09)
	potential unique TFBS	HNF4a	AR	EGR1	EGR2	BRCA1	NFE2::MAF
		HNF4g	EGR1,2	MZF1	EHF	ESR2	RFX5
		JUN	KLF5	SP2	FOXH1	HIF1A:ARNT	YY1
		MYB	MZF1_1-4		MAFK	NFE2L1:MAFG
		NFIC	NFYB		SPIB
		NR2C2	NFATC2		THAP1
		NR4A2	NKX2-5
		PAX2	NKX3-2
		RFX5	SP1, 2
			STAT5A:STAT5B
Ethnic Group	Disease		Disease			Disease
A. Asian	1. Chronic mountain sickness		8. Juvenile idiopathic arthritis			15. Hypospadias
B. Black	2. Diabetes		9. Primary biliary cirrhosis and Crohn's disease			16. Mental retardation
C. Caucasian	3. Hepatitis B virus- related hepatocellular		10. Lupus			17. Osteoarthritis
D. Chinese	4. Hepatitis B virus infection		11. Ulcerative colitis			18. Insulin resistance
E. Hispanic	5. HBV viral clearance		12. Cardiovascular disease			19. Hepatic glucose output
F. Pima Indians	6. Hepatocellular carcinoma		13. Renal cell carcinoma risk			20. Coronary artery disease
G. Tibetan	7. Inflammatory bowel disease		14. Aggressive prostate cancer			21. Myocardial infarction
						22. Asthma

Also listed are the SNP alleles and frequencies within the ethnic group as well as the potential unique transcriptional factor binding site created with each SNP allele. For a complete list of significant gene SNPs see references (Table 1). SNP: Single nucleotide polymorphisms; ADRBK1: Adrenergic, beta, receptor kinase 1; AKT3: V-akt murine thymoma viral oncogene homolog 3; ATF3: Activating transcription factor 3; DIO2: Type 2 demodkinase gene; EPAS1: Endothetal Per-Arnt-Sim domain protein 1; STAT4: Signal transducer and activator of transcription 4; TBXA2R: Thromboxane A2 receptor; VEGFA: Vascular endothelial growth factor A; LIPA: Lysosomal acid lipase A.

MATERIALS AND METHODS

Identifying TFBS

The JASPAR CORE database[29,30] and ConSite[31] were used to identify the TFBS in this study. JASPAR is a collection 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 Vector NTI Advance 11 computer program (Invitrogen, Life Technologies) was used to locate SNPs and TFBS within all genes listed in Table 1.

RESULTS

The protein and gene symbol, chromosome position of the gene, SNP number and location within the gene and nucleotide (mutation) change are listed in Table 1. Also listed is whether or not linkage disequilibrium occurs between the SNPs within each gene (Table 1). Nine genes, ethnic groups, disease or sickness, SNPs and alleles as well as potential unique TFBS per allele that have been reported are found in Table 2. Not all of the SNPs for each gene are listed in the tables but can be found in the accompanying references (Table 1). From Table 2, it can be seen that there occur incidences when the SNP common allele does not have any unique punitive TFBS while the minor allele provide several (e.g., rs12885300 in DIO2; rs2238632 and rs2238634 in TBXA2R). There are other incidences where the SNP common allele provides one or two unique punitive TFBS while the minor alleles again provide several (e.g., rs948988 in ADRBK1; rs4590656 in AKT3; rs11119982 in ATF3; rs8179673 in STAT4 and rs2238631 in TBXA2R). A near balance between SNP alleles in unique punitive TFBS can also be found in the table (e.g., rs4370946 in ADRBK1; rs10157763 and rs2125230 in AKT3; rs3125289 in ATF3; rs6756667 in EPAS1; rs34357231 and rs3025039 in VEGFA). The minor allele of the SNP usually generates more punitive unique TFBS than the common allele (e.g., rs948988 in ADRBK1; rs1412444 in LIPA and rs8179673 in STAT4). In fact, from this sampling of SNPs among the nine genes, there are 73 potential unique TFBS generated by the common alleles compared to 124 by the minor alleles (Table 2).

DISCUSSION

The possible relationship of these punitive unique TFBS to disease and sickness has previously been discussed for each gene in the accompanying references (Table 1). The use of rSNPs that are in LD within a gene to identify punitive TFBS can be illustrated with a few SNPs from these nine genes. The ADRBK1 gene, which transcribes the GRK2 kinase, is an important regulator of beta-adrenergic signaling and plays a central role in heart failure (HF) pathology[32-34]. Two rSNPs in LD within the ADRBK1 gene are rs948988 and rs4370946 whose minor alleles create punitive unique TFBS for ESR2 that is a binding site for the beta estrogen receptor which is expressed in blood monocytes and pulmonary epithelial cells (Tables 1-3). The ESR2 TFBS is not found with the common (rs948988 and rs4370946) alleles of the gene and may be related to HF. The NR3C1 TFBS for the glucocorticoid receptor which regulates carbohydrate, protein and fat metabolism is also only found with the minor alleles of these rSNPs (Tables 2 and 3) and should have an impact on HF. Other TFBS generated by the rs948988 minor allele of interest in HF might be the MYB and NFE2L.1:MAF TFs which are involved with hematopoietic progenitor cells and cell differentiation of erythrocytes as well as the rs4370946 common allele for the NRF1 TF which is involved with heme biosynthesis and mitochondrial DNA transcription and replication (Tables 2 and 3).

Table 3 Transcriptional factors, protein name and their description or function.

TF	Protein name	TF description/function
AR	Androgen receptor	The protein functions as a steroid-hormone activated transcription factor
		Upon binding the hormone ligand, the receptor dissociates from accessory proteins, translocates into the nucleus, dimerizes, and then stimulates transcription of androgen responsive genes. They are expressed in bone marrow, mammary gland, prostate, testicular and muscle tissues where they exist as dimers coupled to Hsp90 and HMGB proteins
ARID3A	AT rich interactive domain 3A (BRIGHT-like)	This gene encodes a member of the ARID (AT-rich interaction domain) family of DNA binding proteins
ARNT	Aryl hydrocarbon receptor nuclear translocator	Involved in the induction of several enzymes that participate in xenobiotic metabolism
ARNT:AHR	Hypoxia-inducible factor 1:Aryl hydrocarbon	The dimer alters transcription of target genes. Involved in the induction
	receptor nuclear translocator	of several enzymes that participate in xenobiotic metabolism
ATF7	Activating Transcription Factor 7	Plays important functions in early cell signaling
		Has no intrinsic transcriptional activity, but activates transcription on formation of JUN or FOS heterodimers
ATOH1	Atonal homolog 1	Transcriptional regulator. Activates E box-dependent transcription in collaboration with TCF3/E47
BATF::JUN	Basic leucine zipper transcription factor, ATF-like Jun proto-oncogene	The protein encoded by this gene is a nuclear basic leucine zipper protein that belongs to the AP-1/ATF superfamily of transcription factors. The leucine zipper of this protein mediates dimerization with members of the Jun family of proteins. This protein is thought to be a negative regulator of AP-1/ATF transcriptional events
BRCA1	Breast cancer 1, early onset	This gene encodes a nuclear phosphoprotein that plays a role in maintaining genomic stability, and it also acts as a tumor suppressor
CEBPA	CCAAT/enhancer binding protein, alpha	CCAAT/enhancer binding protein is a DNA-binding protein that recognizes two different motifs: the CCAAT homology common to many promoters and the enhanced core homology common to many enhancers
CREB1	cAMP responsive element binding protein 1	Phosphorylation-dependent transcription factor that stimulates transcription upon binding to the DNA cAMP response element, a sequence present in many viral and cellular promoters
CRX	Cone-rod homeobox	The protein encoded by this gene is a photoreceptor-specific transcription factor which plays a role in the differentiation of photoreceptor cells. This homeodomain protein is necessary for the maintenance of normal cone and rod function
CTCF	CCCTC-binding factor (zinc finger protein)	This gene is a member of the BORIS + CTCF gene family and encodes a transcriptional regulator protein with 11 highly conserved zinc finger domains. This nuclear protein is able to use different combinations of the zinc finger domains to bind different DNA target sequences and proteins
E2F1-6	E2F transcription factors 1-6	The protein encoded by this gene is a member of the E2F family of transcription factors. The E2F family plays a crucial role in the control of cell cycle and action of tumor suppressor proteins and is also a target of the transforming proteins of small DNA tumor viruses. The E2F proteins contain several evolutionally conserved domains found in most members of the family. These domains include a DNA binding domain, a dimerization domain which determines interaction with the differentiation regulated transcription factor proteins, a transactivation domain enriched in acidic amino acids, and a tumor suppressor protein association domain which is embedded within the transactivation domain
EBF1	Transcription factor COE1	EBF1 has been shown to interact with ZNF423 and CREB binding proteins
EGR1	Early growth response 1	The protein encoded by this gene belongs to the EGR family of C2H2-type zinc-finger proteins. It is a nuclear protein and functions as a transcriptional regulator. The products of target genes it activates are required for differentiation and mitogenesis
EGR2	Early growth response 2	The protein encoded by this gene is a transcription factor with three tandem C2H2-type zinc fingers
		Sequence-specific DNA-binding transcription factor
EHF	Ets homologous factor	This gene encodes a protein that belongs to an erythroblast transformation-specific transcription factor subfamily characterized by epithelial-specific expression. The encoded protein acts as a transcriptional repressor and may be involved in epithelial differentiation and carcinogenesis
ELF1	E74-like factor 1 (ets domain transcription factor)	The encoded protein is primarily expressed in lymphoid cells and acts as both an enhancer and a repressor to regulate transcription of various genes
ELF5	E74-like factor 5	A member of an epithelium-specific subclass of the Ets Transcription factor family
ELK1	ELK1, member of ETS oncogene family	This gene is a member of the Ets family of transcription factors and of the ternary complex factor subfamily. The protein encoded by this gene is a nuclear target for the ras-raf-MAPK signaling cascade
ELK4	ELK4, ETS-domain protein (SRF accessory protein 1)	This gene is a member of the Ets family of transcription factors and of the ternary complex factor subfamily. Proteins of the ternary complex factor subfamily form a ternary complex by binding to the serum response factor and the serum response element in the promoter of the c-fos proto-oncogene
EN1	Engrailed homeobox 1	Homeobox-containing genes are thought to have a role in controlling development
ERG	v-ets avian erythroblastosis virus E26 oncogene homolog	This gene encodes a member of the erythroblast transformation-specific family of transcriptions factors. All members of this family are key regulators of embryonic development, cell proliferation, differentiation, angiogenesis, inflammation, and apoptosis
ESR2	Estrogen receptor beta	Estrogen receptor β is a member of the family of estrogen receptors and the superfamily of nuclear receptor transcription factors and is expressed by many tissues including blood monocytes and tissue macrophages, colonic and pulmonary epithelial cells
ESRRA	Estrogen-related receptor alpha	This nuclear receptor acts as a site-specific transcription regulator and has been also shown to interact with estrogen and the transcripton factor TFIIB by direct protein-protein contact. The binding and regulatory activities of this protein have been demonstrated in the regulation of a variety of genes including lactoferrin, osteopontin, medium-chain acyl coenzyme A dehydrogenase and thyroid hormone receptor genes
ETS1	Protein C-ets-1	The protein encoded by this gene belongs to the erythroblast transformation-specific family of transcription factors and has been shown to interact with TTRAP, UBE2I and Death associated protein
FEV	ETS oncogene family	It functions as a transcriptional repressor
FLI1	Fli-1 proto-oncogene, ETS transcription factor	Sequence-specific transcriptional activator
FOS	FBJ murine osteosarcoma viral oncogene homolog	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. As such, the FOS proteins have been implicated as regulators of cell proliferation, differentiation, and transformation. In some cases, expression of the FOS gene has also been associated with apoptotic cell death
FOSL1 and 2	FOS-like antigen 1 and 2	GO annotations related to this gene include RNA polymerase II regulatory region sequence-specific DNA binding and sequence-specific DNA binding transcription factor activity
FOXA1	Forkhead box A1	Transcription factor that is involved in embryonic development, establishment of tissue-specific gene expression and regulation of gene expression in differentiated tissues. Is thought to act as a "pioneer" factor opening the compacted chromatin for other proteins through interactions with nucleosomal core histones and thereby replacing linker histones at target enhancer and/or promoter sites
		Involved in the development of multiple endoderm-derived organ systems such as liver, pancreas, lung and prostate. Modulates the transcriptional activity of nuclear hormone receptors
FOXA2	Forkhead box A2	Involved in embryonic development, establishment of tissue-specific gene expression and regulation of gene expression in differentiated tissues
FOXC1	Forkhead box C1	An important regulator of cell viability and resistance to oxidative stress in the eye
FOXL1	Forkhead box L1	Transcription factor required for proper proliferation and differentiation in the gastrointestinal epithelium. Target gene of the hedgehog signaling pathway
FOXO1	Forkhead Box O1	Transcription factor that is the main target of insulin signaling and regulates metabolic homeostasis in response to oxidative stress
FOXO3	Forkhead Box O3	This gene belongs to the forkhead family of transcription factors which are characterized by a distinct forkhead domain. This gene likely functions as a trigger for apoptosis through expression of genes necessary for cell death
FOXP1	Forkhead box P1	This gene belongs to subfamily P of the forkhead box transcription factor family. Forkhead box transcription factors play important roles in the regulation of tissue- and cell type-specific gene transcription during both development and adulthood. Transcriptional repressor. It plays an important role in the specification and differentiation of lung epithelium
FOXQ1	Forkhead box Q1	This gene belongs to the forkhead family of transcription factors which is characterized by a distinct DNA-binding forkhead domain. Plays a role in hair follicle differentiation
GABPA	GA-binding protein alpha chain	One of three GA-binding protein transcription factor subunits which functions as a DNA-binding subunit which shares identity with a subunit encoding the nuclear respiratory factor 2 gene and is likely involved in activation of cytochrome oxidase expression and nuclear control of mitochondrial function
GATA1	GATA binding protein 1	The protein plays an important role in erythroid development by regulating the switch of fetal hemoglobin to adult hemoglobin
GATA2	GATA binding protein 2	A member of the GATA family of zinc-finger transcription factors that are named for the consensus nucleotide sequence they bind in the promoter regions of target genes and play an essential role in regulating transcription of genes involved in the development and proliferation of hematopoietic and endocrine cell lineages
GATA3	GATA binding protein 3	Plays an important role in endothelial cell biology
GFI	Growth factor independent 1 transcription repressor	This gene encodes a nuclear zinc finger protein that functions as a transcriptional repressor. This protein plays a role in diverse developmental contexts, including hematopoiesis and oncogenesis. It functions as part of a complex along with other cofactors to control histone modifications that lead to silencing of the target gene promoters
GMEB2	Glucocorticoid modulatory element binding protein 1	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
HAND1:TCFE2α	Heart- and neural-crest derivatives-expressed protein 1: Transcription factor E2A	Hand1 belongs to the basic helix-loop-helix family of transcription factors
		The Tcfe2a gene encodes the transcription factor E2A, a member of the “class I” a family of basic helix-loop-helix transcription factors (also known simply as "E-proteins"). The transcription factor E2A controls the initiation of B lymphopoiesis
HIC1	Hypermethylated in cancer 1	This gene functions as a growth regulatory and tumor repressor gene
HIF1A:ARNT HLTF	Hypoxia-inducible factor 1: Aryl hydrocarbon receptor nuclear translocator Helicase-like transcription factor	HIF1 is a homodimeric basic helix-loop-helix structure composed of HIF1a, the alpha subunit, and the aryl hydrocarbon receptor nuclear translocator (Arnt), the beta subunit. The protein encoded by HIF1 is a Per-Arnt-Sim transcription factor found in mammalian cells growing at low oxygen concentrations. It plays an essential role in cellular and systemic responses to hypoxia Member of the SWItch/Sucrose Non Fermentable family which have helicase and ATPase activities and are thought to regulate transcription of certain genes by altering the chromatin
HNF1A	Hepatocyte nuclear factor 1 homeobox A	Transcriptional activator that regulates the tissue specific expression of multiple genes, especially in pancreatic islet cells and in liver
HNF1B	HNF1 homeobox B	This gene encodes a member of the homeodomain-containing superfamily of transcription factors. The protein binds to DNA as either a homodimer, or a heterodimer with the related protein hepatocyte nuclear factor 1-alpha. The gene has been shown to function in nephron development, and regulates development of the embryonic pancreas
HNF4α	Hepatocyte nuclear factor 4, alpha	The protein encoded by this gene is a nuclear transcription factor which binds DNA as a homodimer. The encoded protein controls the expression of several genes, including hepatocyte nuclear factor 1 alpha, a transcription factor which regulates the expression of several hepatic genes. This gene may play a role in development of the liver, kidney, and intestines
HNF4γ	Hepatocyte nuclear factor 4, gamma	Steroid hormone receptor activity and sequence-specific DNA binding transcription factor activity. An important paralog of this gene is RXRA
HOXA5	Homeobox protein Hox-A5	DNA-binding transcription factor which may regulate gene expression, morphogenesis, and differentiation
ISNM1	Insulinoma-associated 1	Insulinoma-associated 1 gene is intronless and encodes a protein containing both a zinc finger DNA-binding domain and a putative prohormone domain. This gene is a sensitive marker for neuroendocrine differentiation of human lung tumors
IRF1,2	Interferon regulatory factor	Members of the interferon regulatory transcription factor family that contain a conserved
		N-terminal region of about 120 amino acids, which folds into a structure that binds specifically to the interferon consensus sequence
JDP2	Jun dimerization protein 2	Component 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-Oncogene	This gene is the putative transforming gene of avian sarcoma virus 17. It encodes a protein which is highly similar to the viral protein, and which interacts directly with specific target DNA sequences to regulate gene expression
JUND	Jun D proto-oncogene	The protein encoded by this intronless gene is a member of the JUN family, and a functional component of the AP1 transcription factor complex. This protein has been proposed to protect cells from p53-dependent senescence and apoptosis
JUN::FOS	Jun proto-oncogene FBJ murine osteosarcoma viral oncogene homolog	Promotes activity of NR5A1 when phosphorylated by HIPK3 leading to increased steroidogenic gene expression upon cAMP signaling pathway stimulation Has a critical function in regulating the development of cells destined to form and maintain the skeleton. It is thought to have an important role in signal transduction, cell proliferation and differentiation
KLF1	Kruppel-like factor 1 (erythroid)	Transcription regulator of erythrocyte development that probably serves as a general switch factor during erythropoiesis. Is a dual regulator of fetal-to-adult globin switching
KLF4	Krueppel-like factor 4	Transcription factor that can act both as activator and as repressor. Regulates the expression of key transcription factors during embryonic development
KLF5	Krueppel-like factor 5	This gene encodes a member of the Kruppel-like factor subfamily of zinc finger proteins. The encoded protein is a transcriptional activator that binds directly to a specific recognition motif in the promoters of target genes. This protein acts downstream of multiple different signaling pathways and is regulated by post-translational modification. It may participate in both promoting and suppressing cell proliferation. Expression of this gene may be changed in a variety of different cancers and in cardiovascular disease. Alternative splicing results in multiple transcript variants
MAX	MYC associated factor X	The protein encoded by this gene is a member of the basic helix-loop-helix leucine zipper family of transcription factors
MAFK	v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog K	Since they lack a putative transactivation domain, the small Mafs behave as transcriptional repressors when they dimerize among themselves. However, they seem to serve as transcriptional activators by dimerizing with other (usually larger) basic-zipper proteins and recruiting them to specific DNA-binding sites
MEF2A	Myocyte enhancer factor 2A	The protein encoded by this gene is a DNA-binding transcription factor that activates many muscle-specific, growth factor-induced, and stress-induced genes. Mediates cellular functions not only in skeletal and cardiac muscle development, but also in neuronal differentiation and survival
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
MYB	Myb proto-oncogene protein	This gene encodes a transcription factor that is a member of the MYB family of transcription factor genes. Transcriptional activator and plays an important role in the control of proliferation and differentiation of hematopoietic progenitor cells
MYC	v-myc myelocytomatosis viral oncogene homolog	The protein encoded by this gene is a multifunctional, nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis and cellular transformation
MYCN	v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)	This gene is a member of the MYC family and encodes a protein with a basic helix-loop-helix domain. Amplification of this gene is associated with a variety of tumors, most notably neuroblastomas
MZF1_1-4	Myeloid zinc finger 1	Binds to target promoter DNA and functions as transcription regulator. May be one regulator of transcriptional events during hemopoietic development. Isoforms of this protein have been shown to exist at protein level
NFATC2	Nuclear factor of activated T-cells, cytoplasmic 2	This protein is present in the cytosol and only translocates to the nucleus upon T cell receptor stimulation, where it becomes a member of the nuclear factors of activated T cells transcription complex
NFIA	Nuclear Factor I/A	Recognizes and binds the palindromic sequence 5-TTGGCNNNNNGCCAA-3 present in viral and cellular promoters transcription and replication and in the origin of replication of adenovirus type 2. These proteins are individually capable of activating transcription and replication
NFIC	Nuclear factor 1 C-type	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
NFE2::MAF	Nuclear factor, erythroid 2 V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog	Regulates erythroid and megakaryocytic maturation and differentiation. Plays a role in all aspects of hemoglobin production from globin and heme synthesis to procurement of iron. When overexpressed, represses anti-oxidant response element-mediated transcription
NFE2L1:MAFG	Nuclear factor erythroid 2-related factor 1 Transcription factor MafG	Nuclear factor erythroid 2-related factor coordinates the up-regulation of cytoprotective genes via the antioxidant response element. MafG is a ubiquitously expressed small maf protein that is involved in cell differentiation of erythrocytes. It dimerizes with P45 NF-E2 protein and activates expression of a and b-globin
NFIL3	Nuclear factor, interleukin 3 regulated	Expression of interleukin-3 (MIM 147740) is restricted to activated T cells, natural killer cells, and mast cell lines
NFKB1	Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1	natural killer-kappa-B is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis
NFYB	Nuclear transcription factor Y, beta	The protein encoded by this gene is one subunit of a trimeric complex, forming a highly conserved transcription factor that binds with high specificity to CCAAT motifs in the promoter regions in a variety of genes. This gene product, subunit B, forms a tight dimer with the C subunit, a prerequisite for subunit A association. The resulting trimer binds to DNA with high specificity and affinity. Subunits B and C each contain a histone-like motif
NHLH1	Nescient helix loop helix 1	The helix-loop-helix proteins are a family of putative transcription factors, some of which have been shown to play an important role in growth and development of a wide variety of tissues and species
NKX2-5	Natural killer 3 homeobox 2	This gene encodes a member of the NK family of homeobox-containing proteins
		Transcriptional repressor that acts as a negative regulator of chondrocyte maturation
NKX3-2	Natural killer 3 homeobox 2	This gene encodes a member of the NK family of homeobox-containing proteins Transcriptional repressor that acts as a negative regulator of chondrocyte maturation
NR1H3:RXRa	Nuclear Receptor Subfamily 1, Group H, Member 3 Retinoid X receptor, alpha	The protein encoded by this gene belongs to the NR1 subfamily of the nuclear receptor superfamily The NR1 family members are key regulators of macrophage function, controlling transcriptional programs involved in lipid homeostasis and inflammation. This protein is highly expressed in visceral organs, including liver, kidney and intestine. It forms a heterodimer with retinoid X receptor, and regulates expression of target genes containing retinoid response elements Studies in mice lacking this gene suggest that it may play an important role in the regulation of cholesterol homeostasis
NR2C2	Nuclear receptor subfamily 2, group C, member 2	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
NR2E3	Nuclear receptor subfamily 2, group E, member 3	This protein is part of a large family of nuclear receptor transcription factors involved in signaling pathways
NR2F1 (COUP)	Nuclear receptor subfamily 2, group F, member 1	Binds to the ovalbumin promoter and, in conjunction with another protein (S300-II) stimulates initiation of transcription. Binds to both direct repeats and palindromes of the 5'-AGGTCA-3' motif. An important paralog of this gene is RXRA
NR3C1	Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)	Glucocorticoids regulate carbohydrate, protein and fat metabolism, modulate immune responses through suppression of chemokine and cytokine production and have critical roles in constitutive activity of the CNS, digestive, hematopoietic, renal and reproductive systems
NR4A2	Nuclear receptor subfamily 4, group A, member 2	Transcriptional regulator which is important for the differentiation and maintenance of meso-diencephalic dopaminergic neurons during development
NRF1	Nuclear respiratory factor 1	This gene encodes a protein that homodimerizes and functions as a transcription factor which activates the expression of some key metabolic genes regulating cellular growth and nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication
NRL	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
PAX2	Paired box gene 2	Probable transcription factor that may have a role in kidney cell differentiation
PDX1	Pancreatic and duodenal homeobox 1	Activates insulin, somatostatin, glucokinase, islet amyloid polypeptide and glucose transporter type 2 gene transcription. Particularly involved in glucose-dependent regulation of insulin gene transcription
PPARγ:RXRα	Peroxisome proliferator-activated receptor gamma Retinoid X receptor, alpha	Peroxisome proliferator-activated receptor gamma is a member of the nuclear receptor family of ligand-activated transcription factors that heterodimerize with the retinoic X receptor to regulate gene expression. Peroxisome proliferator-activated receptor gamma is located primarily in the adipose tissue, lymphoid tissue, colon, liver and heart and is thought to regulate adipocyte differentiation and glucose homeostasis
RXRa	Retinoid X receptor, alpha	Retinoid X receptors and retinoic acid receptors, are nuclear receptors that mediate the biological effects of retinoids by their involvement in retinoic acid-mediated gene activation
RFX5	Regulatory factor X, 5	Activates transcription from class II MHC promoters. Recognizes X-boxes. Mediates cooperative binding between RFX and natural killer-Y. Regulatory factor X binds the X1 box of MHC-II promoters
RUNX1	Runt-related transcription factor 1	Heterodimeric transcription factor that binds to the core element of many enhancers and promoters. The protein encoded by this gene represents the alpha subunit of core binding factor and is thought to be involved in the development of normal hematopoiesis
SOX6	SRY (sex determining region Y)-box 6	The encoded protein is a transcriptional activator that is required for normal development of the central nervous system, chondrogenesis and maintenance of cardiac and skeletal muscle cells
SOX9	SRY (sex determining region Y)-box 9	The protein encoded by this gene recognizes the sequence CCTTGAG along with other members of the involved in chondrogenesis by acting as a transcription factor for these genes
SOX10	SRY (sex determining region Y)-box 10	This gene encodes a member of the SRY-related HMG-box family of transcription factors involved in the regulation of embryonic development and in the determination of the cell fate
SOX17	SRY (sex determining region Y)-box 17	Acts as transcription regulator that binds target promoter DNA and bends the DNA
SP1	Specificity Protein 1	Can activate or repress transcription in response to physiological and pathological stimuli. Regulates the expression of a large number of genes involved in a variety of processes such as cell growth, apoptosis, differentiation and immune responses
SP2	Specificity Protein 2	This gene encodes a member of the Sp subfamily of Sp/XKLF transcription factors. Sp family proteins are sequence-specific DNA-binding proteins characterized by an amino-terminal trans-activation domain and three carboxy-terminal zinc finger motifs. This protein contains the least conserved DNA-binding domain within the Sp subfamily of proteins, and its DNA sequence specificity differs from the other Sp proteins. It localizes primarily within subnuclear foci associated with the nuclear matrix, and can activate or in some cases repress expression from different promoters
SPIB	Transcription factor Spi-B	SPI1 and SPIB are members of a subfamily of erythroblast transformation-specific transcription factors; erythroblast transformation-specific proteins share a conserved erythroblast transformation-specific domain that mediates specific DNA binding SPIB and SPI1 bind to a purine-rich sequence, the PU box (5-prime-GAGGAA-3-)
SPI1	Spleen focus forming virus proviral integration oncogene spi1	This gene encodes an erythroblast transformation-specific-domain transcription factor that activates gene expression during myeloid and B-lymphoid cell development
SPZ1		This gene encodes a basic helix-loop-helix-zip transcription factor which functions in the mitogen-activate protein kinase signaling pathway
SRY	Sex determining region Y	Transcriptional regulator that controls a genetic switch in male development
STAT3	Signal transducer and activator of transcription 3 (acute-phase response factor)	Signal transducer and transcription activator that mediates cellular responses to interleukins, KITLG/SCF and other growth factors
STAT5A:STAT5B	Signal transducer and activator of transcription 5A and transcription 5B	Carries out a dual function: signal transduction and activation of transcription Regulates the expression of milk proteins during lactation
TCF7L2	Transcription factor 7-like 2 (T-cell specific, HMG-box)	This gene encodes a high mobility group box-containing transcription factor that plays a key role in the Wnt signaling pathway. The protein has been implicated in blood glucose homeostasis
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
TFAP2a	Activator protein 2	The AP2a protein acts as a sequence specific DNA-binding transcription factor recognizing and binding to the specific DNA sequence and recruiting transcription machinery
THAP1	THAP domain containing, apoptosis associated protein 1	DNA-binding transcription regulator that regulates endothelial cell proliferation and G1/S cell-cycle progression
USF1	Upstream transcription factor 1	This gene encodes a member of the basic helix-loop-helix leucine zipper family, and can function as a cellular transcription factor. The encoded protein can activate transcription through pyrimidine-rich initiator (Inr) elements and E-box motifs
YY1	YY1 transcription factor	YY1 is a ubiquitously distributed transcription factor belonging to the GLI-Kruppel class of zinc finger proteins. The protein is involved in repressing and activating a diverse number of promoters. YY1 may direct histone deacetylases and histone acetyltransferases to a promoter in order to activate or repress the promoter, thus implicating histone modification in the function of YY1
ZEB1	Zinc finger E-box-binding homeobox 1	A member of the delta-EF1 (TCF8)/Zfh1 family of 2-handed zinc finger/homeodomain proteins and interacts SMADs with receptor-mediated, activated full-length activated full-length SMADs
ZNF263	Zinc finger protein 263	Might play an important role in basic cellular processes as a transcriptional repressor. An important paralog to ZNF496
ZNF354C	Zinc finger protein 354C	May function as a transcription repressor

The type 2 demodkinase 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[35-38]. Two rSNPs in LD within the DIO2 gene are rs225015 and rs225011 whose major alleles create unique punitive TFBS for TFs that are involved with energy balance and glucose metabolism (Tables 1-3). The ESRRα an alpha estrogen-related receptor that is involved with regulating thyroid hormone receptor genes while PPARα:RXRα and RXRα are involved with the regulation of adipocyte differentiation and glucose homeostasis (Tables 2 and 3). The minor allele of the rs225015 rSNP creates a unique punitive TFBS for the TCF7L2 TF whose protein is implicated in blood glucose homeostasis (Tables 2 and 3). The minor allele of the rs225011 rSNP creates a unique punitive TFBS for the PDX1 TF whose protein activates insulin, somatostatin, glucokinase, islet amyloid polypeptide and glucose transporter type 2 gene transcription (Tables 2 and 3).

The thromboxane A2 receptor (TBXA2R) gene is a member of the seven-transmembrane G-protein-coupled receptor super family, which interacts with intracellular G proteins, regulates different downstream signaling cascades, and induces many cellular responses including the intracellular calcium influx, cell migration and proliferation as well as apoptosis[39]. Two rSNPs in LD within the TBXA2R gene are rs2238631 and rs2238634 whose minor alleles create unique punitive TFBS for TFs that are involved in signaling cascades and apoptosis (Tables 1-3). The ELK1 and SPZ1 TFs are involved with the ras-raf-MAPK signaling cascade while the ETS1 TF is involved with cell death (Tables 2 and 3). NR2E3 is part of a large family of nuclear receptor TFs involved in signaling pathways (Tables 2 and 3).

The other six genes can be analyzed in the same manner to identify punitive TFBS created by the rSNP alleles of these genes (Tables 2 and 3). 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. This change in the DNA landscape can alter gene regulation which in turn can result in a change of a biological process or signaling pathway resulting in disease or illness. The process laid out in this report is a convenient way of identifying potential TFBS created by rSNP alleles that have been found to be significantly associated with disease or sickness. Any potential alterations in TFBS obtained by computational analyses need to be verified by protein/DNA electrophoretic mobility gel shift assays and gene expression studies[40]. CHIP-seq[41] experiments have become the standard method of validating TFBS and studying gene regulation[42-44].

In conclusion, SNPs in the regulatory region of a gene can alter the DNA landscape for TFs resulting in TFBS changes. Consequently, alterations in TF binding can affect gene regulation. Examples of this for nine genes are presented in this report where SNP alleles will either have no effect on TF binding or each allele will create unique punitive TFBS and alter a TFs ability to bind the DNA and regulate the gene.

COMMENTS

Background

Transcriptional factors (TFs) bind the DNA near a gene at transcriptional factor binding sites (TFBS) in order to regulate the gene. Single nucleotide polymorphisms (SNPs) that occur in the TFBS can alter the TFs ability to bind the DNA and thereby affect gene regulation. Such regulatory (r)SNPs have been associated with human disease and sickness. In this report, the alteration of TFBS created by rSNP alleles associated with disease has been documented for nine human genes. Sometimes the rSNP alleles will have no effect on the TFBS and not change the TF ability to bind the DNA. Other times each allele will create unique punitive TFBS that alter the TFs ability to regulate the gene.

Research frontiers

This article addresses an emerging concept in understanding how rSNPs which are significantly associated with disease can alter the TFBS for TFs that regulate a gene.

Innovations and breakthroughs

TFBS alteration by rSNPs is a newly emerging field of research and provides a different direction in examining changes in gene regulation resulting in human disease and sickness.

Applications

Given the great diversity of punitive unique TFBS generated by each allele of a rSNP, the author suspects that alterations in TFBS affect how well a gene is expressed. The outcome may result in disease or sickness. The methods outlined in the article should be applied to all rSNPs that are associated with disease or sickness of a regulatory nature.

Terminology

rSNP: A regulatory single nucleotide polymorphism that affects gene expression; TF: Transcriptional factor that is involved with regulating a gene; TFBS: Transcription factor DNA binding site in the regulatory region of a gene; Unique TFBS: A TFBS created by one rSNP allele and not the alternate allele.

Peer-review

This study is technically well performed and a very interesting result. The interpretation was also sound. The report applied a computational approach to predict functional rSNPs in TFBS, focusing on several genes published earlier. Computational modeling and analysis for functional prediction is one of the approaches recently developed, particularly to address GWAS findings.