Molecular characterization of the porcineSTAT4 and STAT6 genes

Jing Huang • Guojian Ma • Mengjin Zhu •

Jianzhi Pan • Wenchang Zhang • Shu-Hong Zhao

Received: 5 September 2011 / Accepted: 24 January 2012 / Published online: 7 February 2012

� Springer Science+Business Media B.V. 2012

Abstract Signal transducers and activators of transcrip-

tion (STATs) are members of a recently identified family

of transcription factors that activate gene transcription in

response to a number of different cytokines. STAT4 and

STAT6 were activated by interleukin (IL)-12 and IL-4

stimulation, which were important for the generation of

Th1 and Th2 cells. In this study, we cloned the cDNA

sequences and analyzed the genomic structure of porcine

STAT4 (poSTAT4) and STAT6 (poSTAT6) genes. Chro-

mosome localization assigned these two genes to SSC15

and SSC5, and they were most closely linked to maker

SWR1002 and DK. The RT-PCR revealed that both genes

were expressed in eight diverse tissues, with the highest

level in small intestine, followed by lung, kidney, muscle

and stomach, whereas expressions in heart, liver and spleen

were relatively weak. Transient transfection indicated that

poSTAT4 and poSTAT6 proteins distributed throughout

the whole porcine hip artery endothelial cell. A single

nucleotide polymorphism (A/G), which can be recognized

by restriction enzyme TaiI, was identified at the 30 untrans-

lated region of poSTAT6, and genotyping results showed

apparent variation in allele frequency between Chinese

indigenous and western breeds.

Keywords Pig � STAT4 � STAT6 � Genomic structure �Localization � SNP

Introduction

Signal transducers and activators of transcription (STATs)

are members of a recently identified family of transcription

factors that activate gene transcription in response to a

number of different cytokines [8]. The STATs are latent

cytoplasmic proteins that are promptly activated by tyro-

sine phosphorylation by the cytokine receptor associated

JAK (Janus) kinases after cytokine exposure [6]. The Janus

kinases-signal transducers and activators of transcription

(JAK-STAT) signaling pathway, which was identified from

the investigation into the transcriptional response to the

interferons, has been shown to be utilized by a large

number of cytokines, growth factors, and hormones [5]. Up

to now, seven mammalian STAT family members have

been identified, including STAT4 and STAT6 which were

focused on in this study. In particular, STAT4 was acti-

vated by interleukin (IL)-12 stimulation [2, 11], whereas

STAT6 was activated by IL-4 stimulation [14, 23]. It is

now well accepted that IL-12 and IL-4 are important for the

generation of Th1 and Th2 cells, respectively. CD4? Th

cells can be divided into at least two functionally distinct

subsets [18]. These two subsets are discriminated one from

another depending on the pattern of cytokines they secrete.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-012-1523-1) contains supplementarymaterial, which is available to authorized users.

J. Huang � G. Ma � M. Zhu � S.-H. Zhao (&)

Key Laboratory of Agricultural Animal Genetics, Breeding,

and Reproduction of Ministry of Education & Key Laboratory

of Swine Genetics and Breeding of Ministry of Agriculture,

Huazhong Agricultural University, Wuhan 430070, Hubei,

People’s Republic of China

e-mail: shzhao@mail.hzau.edu.cn

J. Huang � J. Pan

Institute of Animal Husbandry and Veterinary,

Zhejiang Academy of Agricultural Sciences,

Hangzhou 310021, Zhejiang, People’s Republic of China

W. Zhang

Department of Animals Science, Fujian Agriculture and Forestry

University, Fuzhou 350002, People’s Republic of China

123

Mol Biol Rep (2012) 39:6959–6965

DOI 10.1007/s11033-012-1523-1

Th1 cells are defined by their ability to produce IL-12 and

interferon-c (IFN-c) on stimulation, whereas Th2 cells

secrete a different panel of cytokines, such as IL-4, IL-5

and IL-13 [10]. One mechanism by which cytokines such

as IL-12 and IL-4 elicit biological responses is through

activation of the JAK-STAT signaling pathway [20]. Some

studies on Stat4 and Stat6-deficient mice showed that Stat6

appeared to be critical for the differentiation pathway of

Th2 cells, through the activation of STAT6 by IL-4 stim-

ulation of the IL-4 receptor. Similarly, Stat4 play an

extremely important role in the differentiation of Th1 cells

[12]. As known publicly, Th1 cells are important in cell-

mediated immunity, and Th2 cells are important regulators

of the humoral immune response.

Taken together, the critical roles of STAT4 and STAT6

genes in cytokine signaling pathways and immunity have

been clearly demonstrated through generation and analysis

of gene targeted mice. Due to the similarities between pig

and human, pig has been considered as an important model

organism used of human health research. However, little

information on STATs is known in pigs. In this study, we

focused on the genomic structures and cDNA sequences of

porcine STAT4 and STAT6 genes. In addition, their

chromosome assignments, tissue expression patterns, sub-

cellular cell localizations, polymorphisms and allele fre-

quencies were analyzed. The knowledge of STAT4 and

STAT6 genes gained in this study will contribute to

understand the function of the genes in the pig.

Materials and methods

Isolation of full-length coding regions of poSTAT4

and poSTAT6 genes

Human mRNA sequence of STAT6 (hSTAT6) (GenBank

accession nos. NM_003153.3) was compared with all

sequences available in the expressed sequence tags (EST)

and genome databases by using BLAST algorithm (http://

www.ncbi.nlm.nih.gov/blast/). We selected the porcine

ESTs that shared more than 80% sequence identity to the

corresponding human cDNA to assemble the porcine genes

using SeqMan (DNASTAR, Inc., Madison, WI, USA). The

primer pair (CDS-6F, CDS-6R for poSTAT6, Table S1) in

both 50 and 30 untranslated region (UTR) flanking the entire

coding sequence in cDNAs of STAT6 was designed from the

pig ESTs. Since we can not get the EST including the full-

length coding region of porcine STAT4 gene using the

method above, both human and murine mRNA sequences

were compared through BLAST algorithm in order to

assemble the presumed sequence covering the coding region

and then the primer pair (CDS-4F, CDS-4R for poSTAT4,

Table S1) was designed.

Total RNA was extracted from spleen tissue of the adult

Chinese indigenous Tongcheng pig (Hubei Province, China)

with TRIZOL reagent (Invitrogen, Carlsbad, CA, USA).

Reverse transcriptase-polymerase chain reaction (RT-PCR)

was performed using Taq polymerase (Fermentas, Vilnius,

Lithuania), as well as M-MLV reverse transcriptase (Pro-

mega, Madison, WI, USA). The predominant PCR product

was gel purified and subsequently cloned into the pEGM-

T-Easy vector (Promega) prior to sequencing.

Gene expression patterns of poSTAT4 and poSTAT6

Tissue distribution of these two genes was analyzed by

semi-quantitative PCR and real-time PCR. The primer

pairs (exp-4F, exp-4R for poSTAT4 and exp-6F, exp-6R

for poSTAT6, Table S1) were designed between the exon

21 and exon 22 of poSTAT4 and exon 9 and exon 10 of

poSTAT6 respectively. Total RNAs were extracted from

nine different tissues of heart, liver, spleen, lung, kidney,

myeloid cells, thymus and testis of the mature Tongcheng

pig. Real-time RT-PCR analysis was performed using

SYBR Green Real-time PCR Master Mix (TOYOBO) in

the Roche LightCycler 480. The relative quantities of po-

STAT4 and poSTAT6 mRNAs were assessed by the

comparative cycle threshold method and normalized with

porcine beta-anctin mRNA level as an endogenous control

with specific primers (ACTIN-F, ACTIN-R, Table S1). The

PCR program was 95�C for 4 min, followed by 30 s at

94�C, 30 s at 66�C for STAT4, and 60�C for STAT6, and

then 30 s at 72�C for 27 cycles and a final extension at

72�C for 5 min.

Somatic cell hybrid and radiation hybrid mapping

The porcine somatic cell hybrid panel (SCHP) [30] was

used for determining the chromosomal regional location

and the INRA-University of Minnesota porcine radiation

hybrid (IMpRH) [31] panel was applied for more precise

mapping of the genes.

The primer pairs (map-4F, map-4R designed in the

intron 24 for poSTAT4 and map-6F, map-6R designed in

intron 19 for poSTAT6, Table S1) were employed both for

the mapping by SCHP and IMpRH panel. PCR reactions

were performed in a volume of 10 lL of 19 PCR buffer

(Promega), containing 20 ng DNA from each cell hybrid

line, 0.2 lM of each primer, 100 lM of each dNTP,

1.5 mM MgCl2 and 2.0 units Taq DNA Polymerase (Pro-

mega). The PCR progress was 3 min at 94�C followed by

35 cycles of 30 s at 94�C, 30 s at 60�C for STAT4, and

64�C for STAT6, then 30 s at 72�C, and a final extension

of 5 min at 72�C. The PCR results were analyzed

on http://www.toulouse.inra.fr/lgc/pig/hybrid.htm [3] and

6960 Mol Biol Rep (2012) 39:6959–6965

123

http://www.toulouse.inra.fr/lgc/pig/RH/IMpRH.htm [17]

for SCHP and RH mapping, respectively.

Transient expression of poSTAT4 and poSTAT6

in porcine hip artery endothelial cells (PIEC)

The open reading frames (ORF), encoding poSTAT4 and

poSTAT6, were amplified from their cDNA clones and

subcloned into the XhoI–SalI and Eco47III–EcoRI sites of

the pEGFP-N1 vector (Clontech, Palo Alto, CA) to yield

their mammalian expression plasmids pGFP-STAT4 and

pGFP-STAT6. The primers (pEGFP-4F, pEGFP-4R for

poSTAT4 and pEGFP-6F, pEGFP-6R for poSTAT6) were

listed in Table S1. Both plasmids were sequenced to verify

the correct sequences were cloned.

PIEC were utilized to investigate the cellular localiza-

tions of the poSTAT4 and poSTAT6 proteins. The cells

were cultured in DMEM containing 10% (v/v) fetal bovine

serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin

under humidified air containing 5% CO2 at 37�C and

seeded onto glass cover slips in 6-well plates. Transient

transfections were performed using LipofectamineTM

2000

(Invitrogen, Carlsbad, CA, USA) when the cells reached

80% confluence, according to the manufacturer’s protocol.

At 24 h after transfection, cells were washed three times

with phosphate-buffered saline (PBS), and then fixed in 4%

formaldehyde for 15 min at room temperature. After the

final washing steps and incubation with 10 lM Hoe-

chst33342 for 10 min, the slides were mounted and sealed,

and analyzed by confocal microscopy (TCS-SP2). Leica

confocal software (Leica IM500) was used to generate

images of individual fluorescent markers as well as overlay

pictures to demonstrate the relative distribution of the

fusion protein.

Single nucleotide polymorphism (SNP) identification

and allele frequencies analysis

SNPs in the two genes were identified by sequencing PCR

products from two pig breeds Erhualian and Landrace. The

PCR restriction fragment length polymorphism (PCR-

RFLP) method was employed to genotype the polymorphic

sites. A total of 189 DNA samples from unrelated animals

representing eight breeds (Landrance, Yorkshire, Duroc

and five Chinese indigenous pig breeds, Erhualian, Da-

huabai, Wuzhishan, Little meishan and Zang pigs) were

genotyped and allele frequencies were determined about

the polymorphic site of poSTAT6. PCR reactions were

performed using the following specific primer pairs: SNP-

4F, SNP-4R for poSTAT4 and SNP-6F, SNP-6R for po-

STAT6 (Table S1).

Results and discussion

Molecular cloning and sequence analysis of poSTAT4

and poSTAT6

Analysis of the cDNA sequences of the porcine STAT4 and

STAT6 revealed the following results: (1) The deduced

full-length cDNA of poSTAT4 is 2,541 bp containing an

ORF of 2,247 bp encoding a protein of 748 amino acid

residues with a calculated molecular mass of 85.9 kDa and

an isoelectric point (pI) of 6.14. The poSTAT4 is 91

and 86% identical to the human (GenBank accession no.

NM_003151) and mouse homologues genes (GenBank

accession no. NM_011487). (2) The deduced poSTAT6

mRNA is 3,890 bp which contains a 2,547 bp ORF flanked

by a 145-bp 50-UTR and a 1198-bp 30-UTR, with a putative

polyadenylation signal AATAAA located at 3,827–3,832.

The poSTAT6 gene is predicted to encode a polypeptide of

848 amino acids with a molecular mass of 94.4 kDa and a pI

of 5.95. The poSTAT6 exhibits 86 and 85% sequence iden-

tity with human (GenBank accession no. NM_003153), and

mouse (GenBank accession no. NM_009284). The sequen-

ces of porcine STAT4 and STAT6 were deposited in

GenBank (GenBank accession no. EU1214100 and

EU1214128).

Similar to their orthologous genes in human (Fig. S1),

porcine STAT4 and STAT6 showed the highest homology

toward their amino terminus. STAT4 and STAT6 share a

number of the functional domain characteristics of STAT

proteins, including a DNA-binding domain in the middle

portion, a conserved SH2 domain mediating docking to the

receptor and STAT dimerization, and a C-terminal trans-

activation domain [15, 25]. In human, the NH2-terminal

130 amino acids are conserved among STAT family

members and this region is essential for tetramerization of

dimerized STAT molecules, which enables cooperative

DNA binding on the promoters containing multiple poten-

tial STAT recognition sites [28, 29]. In addition, a con-

served tyrosine residue exists near the carboxy-terminus,

and through phosphorylation of this residue, STAT proteins

are activated by Jak in order to form homo- or hetero-

dimers, then translocate into the nucleus working as intra-

nuclear transcription factors [9, 24].

After alignment of the poSTAT4 and poSTAT6 mRNA

sequences with genomic DNA sequences of the Sus scrofa

chromosome 15 clone (GenBank accession no. CU550692)

and chromosome 5 clone (GenBank accession no. CU633

428), the putative exon and intron regions were identified

(Table S2). Porcine STAT4 spans approximately 110 Kb

on the genome, it may contain 24 exons and 23 introns,

though we only can make sure about the 23 exons and

22 introns except the first ones (Table S2). Pocine

STAT6 spans approximately 15.2 Kb on the genome, and it

Mol Biol Rep (2012) 39:6959–6965 6961

123

contains 22 exons and 21 introns (Table S3). The gene

structure of these two genes is conserved among human,

mouse and pig. Further comparisons revealed the exon/

intron junctions of them; each of the 50-donor and 30-acceptor splice sites conformed to the expected consensus

sequences for eukaryotic splice junctions, namely, GT-AG

rule and the length of exonic and intronic sequences was

determined.

Tissue expression of poSTAT4 and poSTAT6 genes

RT-PCR and real-time PCR analysis of total RNA both

showed that poSTAT4 and poSTAT6 have similar

expression patterns (Figs. 1, 2). Both genes were expressed

with the highest level in thymus, followed by lung, kidney,

myeloid cells and testis, whereas expressions in heart, liver

and spleen were relatively weak in case of poSTAT4 gene.

Having compared this data with the expression patterns of

human and mouse STAT4 and STAT6 on NCBI, we con-

vinced that the expression pattern of porcine STAT4 and

STAT6 were in agreement with the human and mouse

genes, although STAT4 is only expressed in myeloid cells,

thymus and testis in human [32].

Chromosome location of the poSTAT4 and poSTAT6

genes

The localization by IMpRH panel revealed that poSTAT4

was mapped to chromosome 15, and was most closely

linked to the microsatellite marker SWR1002 (distance =

51cR, LOD = 7.73) and SW1945 (distance = 61cR,

LOD = 6.18), poSTAT6 was mapped to chromosome 5,

and was tightly linked to markers DK (distance = 31cR,

LOD = 11.67) and SW332 (distance = 48cR, LOD =

5.81) (Fig. 3).

The hSTAT4 and hSTAT6 genes have been mapped to

chromosome 2q32.2-q32.3 and 12q13 (http://www.ncbi.

nlm.nih.gov/mapview/). The mapping information of these

two genes is in consistent with comparative mapping data,

as porcine chromosome 15 and 5 regions have been sepa-

rately shown to share homology with human chromosome

2q and 12q [7]. Furthermore, mouse STAT4 was mapped to

chromosome 1 and STAT6 was mapped to the chromo-

some 10, which also showed homology with human chro-

mosome 2 and 12 [4].

After searching in the pig QTLdatabase, we found

that there is immune QTL named Lymphocyte number

(LYMPH) in SSC15, with the QTL center location 92.8 cM,

which included the location of porcine STAT4 gene

(http://www.animalgenome.org/cgi-bin/QTLdb/SS/draw_

chromap?chromos=15&optqtl=LYMPH); Similarly, we

also found immune QTL named inference-gamma level

(IFNG) in SSC5, with the QTL center location 2 cM, which

included the location of porcine STAT6 gene (http://

www.animalgenome.org/cgi-bin/QTLdb/SS/draw_chromap?

chromos=5&optqtl=IFNG). These results suggested that

porcine STAT4 and STAT6 genes may play important roles in

pig immune system.

Cellular localization of poSTAT4 and poSTAT6

in PIEC

The cellular localizations of poSTAT4 and poSTAT6 were

determined by fluorescence and confocal analysis in PIEC

transiently transfected with pEGFP-STAT4 and pEGFP-

STAT6, respectively. After labeling nuclei by staining with

Fig. 1 Tissue expression analysis of porcine STAT4 and STAT6

genes by RT-PCR. 1 DL2000 DNA marker, 2–9 heart, liver, spleen,

lung, kidney, myeloid cells, thymus and testis

0102030405060708090

100

hear

t

liver

sple

en

lung

kidn

ey

mye

loid

cells

thym

us

test

is

Rel

ativ

e m

RN

A e

xpre

ssio

n

STAT4

STAT6

Fig. 2 Tissue expression analysis of porcine STAT4 and STAT6

genes by real-time PCR

Fig. 3 Multiple point analysis result for porcine STAT4 and STAT6

genes RH mapping

6962 Mol Biol Rep (2012) 39:6959–6965

123

Hoechst33342, poSTAT4 and poSTAT6 fusion proteins

were found in distribution throughout the whole cell

(Fig. 4). In mouse cells, these two genes are primarily

localized in the cytoplasm [21]. This difference may be due

to the different cell lines and species, and another reason is

that cells in this experiment were activated in some case,

which resulting STAT gene transfer into the nucleus from

the cytoplasm.

In mouse, STAT family proteins were initially present in

inactive forms in the cytoplasm, while Jak kinase was

constitutively associated with the cytoplasmic membrane-

proximal regions of various receptors [19]. Upon ligand

binding, Jak kinases become catalytically activated, and

tyrosine residues in the cytoplasmic domain of the receptor

become phosphorylated at the same time. This phosphor-

ylation leads to the recruitment of STAT proteins via rec-

ognition of the receptor phosphotyrosines by the STAT

SH2 domains. The activated Jak kinases then phosphorylate

STAT proteins at their tyrosine residues. Thereafter, the

phosphorylated STAT proteins detach from the receptor,

become homodimerized or heterodimerized, and translocate

to the nucleus to activate transcription by interaction with

specific DNA sequences [27].

SNP detection and allele frequency distribution

of poSTAT6

Analysis of the cDNA sequences of poSTAT6 revealed a

SNP at the 30-UTR, which is at position 3417(A/G) (for this

polymorphism, haplotypes were detected in several pig

breeds through sequencing of several animals). The SNP

position was defined according to the coding sequence of

poSTAT6. The 276-bp PCR amplicon containing poSTAT6

SNP was detectable by digestion with TaiI, resulting in

allele A (276 bp) and allele G (225 and 51 bp, Fig. 5a).

Genotyping results showed apparent variation in allele fre-

quency between Chinese indigenous and western breeds but

did not show any difference within the five Chinese breeds

and within the three Western breeds (Fig. 5b). The reason

may due to the intense selection of western breeds and no

selection of the Chinese local breeds. STAT6 polymor-

phisms were recently associated with elevated total IgE

levels in a genome-wide association study [22]. Some SNPs

in STAT4 and STAT6 of the Janus kinase-signal transducer

and activator of transcription (Jak-Stat) signal transduction

pathway were examined in resent investigations. Their

results showed that hSTAT4 and hSTAT6 are significant

Fig. 4 Cellular localization of STAT4-pEGFP-N1 and STAT6-

pEGFP-N1 fusion protein in PIEC. The fusion proteins were both

mainly distributed in the cytoplasm (excited at 488 nm; a, A), nulclei

were stained with Hoechst33342 (excited at 360 nm; b, B). The

fluorescent signals were analyzed by confocal microscopy. The

overlay image was produced by merging two signals together (c, C)

Mol Biol Rep (2012) 39:6959–6965 6963

123

association with different diseases, such as inflammatory

bowel disease, minimal change nephrotic syndrome, asthma,

urinary schistosomiasis and so on [1, 13, 16, 26]. Due to the

bias distribution of the genotypes in the pure breed Landrace

pigs, we did not analyze the association between this SNP site

and the blood parameters and immune response traits in this

study.

Acknowledgments The authors thank Dr Martine Yerle and the

Laboratoire de Genetique Cellulaire, INRA, for providing SCHP and

RH DNA samples. This project was supported by key project of

National Natural Science Foundation of China (U0631005), National

High Science and Technology Foundation of China ‘‘863’’

(2010AA10A104), the creative team project of the Chinese Ministry

of Education (IRT0831), and the Fundamental Research Funds for the

Central Universities (2009PY001).

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