Evolution and Expression Patterns of Forkhead Box N1 in Pig

Liu Yang, Wang Liang, Li Ling, Liu Di, *, and Zhang Dong-jie

1 College of Animal Sciences and Technology, Northeast Agricultural University, Harbin 150030, China

2 Institute of Animal Husbandry, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China

Abstract: The thymus is essential for T-cell development. The transcription factor Foxn1 plays an important role in the development and function of thymic epithelial cells (TECs) in vertebrates. However, the transcriptional regulation and expression pattern of Foxn1 in pig is not known. Here, the complete sequence of pig Foxn1 was sequenced. Sequence analysis showed that the pig Foxn1 gene was 14 730 bp in length. Its cDNA full coding sequence (CDS) consisted of 1 941 bp nucleotides that encoded a 646-amino acid polypeptide. Its amino acid sequence comprised a conserved forkhead 3 domain spanning amino acids 269-365.Phylogenetic reconstruction of the Foxn1 nucleotide sequence of the pig and other species revealed that the pig Foxn1 gene was closely related to the sheep and cattle Foxn1 genes. Foxn1 gene was conserved in mammals. RT-PCR analysis showed that Foxn1 was only expressed in the thymus, skin and tongue, but not in the heart, liver, spleen, lung, kidney, adrenal gland, subcutaneous fat,longissimus dorsi, large intestine, small intestine, stomach, mesenteric lymph node, throat and ovary. These findings indicated that the expression pattern of Foxn1 was tissue-specific.

Key words: Sus scrofa, Foxn1, sequence, evolution, expression

Introduction

The thymus, which is the main site of T lymphocyte development, is one of the most important organs of the immune system. It is also one of the first organs to degenerate in normal healthy individuals. Thymic involution results in a reduction in production of native T cells and in turn, a decline in the function of the immune system (Bredenkamp et al., 2014).Transcription factor forkhead box N1 (Foxn1) is an epithelial cell-autonomous gene that predominantly regulates the development of thymic epithelial cells(TECs) and skin keratinocytes (Palamaro et al.,2014). It is initially named whn (winged-helix nude)and belongs to the forkhead box gene family (Schuff et al., 2006). The forkhead box gene family comprises a diverse group of "winged-helix" transcription factors that play an important role in a variety of biochemical and cellular processes, such as development, metabolism, aging and cancer. Their transcriptional properties rely notably on the presence of two specific domains,a transcriptional regulatory domain and a DNA-binding domain (DBD), which are regulated by posttranslational modifications (Wijchers et al., 2006). The expression pattern of members of the forkhead gene family is temporally and spatially specific.

Foxn1 protein contains a regulatory domain that acts as an activator of transcription; it is located between the C-terminal amino acids 402 and 455 (Schüddekopf et al., 1996; Lelièvre et al., 2012), the mouse activation domain comprises aa 509-563 (Schorpp et al.,1997). It is selectively expressed in thymic and skin epithelial cells. Over- and ectopic-expression of Foxn1 in the early life adversely influences immature TECs,T and B cells, and skin epithelial development (Ruan et al., 2014). Mutations in Foxn1 gene result in failure in thymus development, hairless nude skin and short life (Abitbol et al., 2015; Bryson et al., 2013). On the other hand, Foxn1 upregulation in the thymus of aged mice results in the regeneration in thymus function,which is demonstrated by an increase in the rate of thymopoiesis and production of native T cells. Foxn1 maintains TECs to support T-cell development via mcm2 (Ma et al., 2012). miR-18b and miR-518b have been shown to upregulate Foxn1 (Kushwaha et al., 2014). When Foxn1 is absent, its metazoan ancestor, Foxn4, exhibits substantial thymopoietic activity (Swann et al., 2014). Umbilical cord-derived mesenchymal stem cells (UC-MSCs) provide a proper microenvironment for the reconstitution and functional maturation of the thymus in Foxn1(-/-) mice (Liu et al., 2014).

The pig (Sus scrofa) is one of the most important economic animals that serves as a meat source for humans. It is also one of the most important animal models for human disease. The present study cloned Min pig (a native pig breed of China) Foxn1 gene and conducted sequence analysis and mRNA expression profiling. The findings of the present study could provide additional information that might be used in the future researches on Foxn1 gene.

Materials and Methods

Animal and sample collection

A 30-day-old Min pig was sacrificed and used in cloning Foxn1 gene. Ear tissue was stored at –20℃until DNA analysis. Heart, liver, spleen, lung, kidney,adrenal gland, thymus, skin, subcutaneous fat,longissimus dorsi, large intestine, small intestine,stomach, mesenteric lymph node, throat, ovary and tongue tissues were collected and stored at –70℃until RNA extraction. DNA was extracted from the ear tissue sample following the standard phenolchloroform method. The total RNA was extracted from different tissues using TRIzol reagent, following the manufacturer's instructions.

Cloning of pig Foxn1 gene

Based on the reference sequence of the pig Foxn1 gene (GenBank Acc. No.: NC_010460) in NCBI,which was derived from automated computational analysis, 10 primer pairs were designed to clone its DNA sequence. One primer pair (CDSF and CDSR)was designed to clone the entire CDS. The sequence and location of the primers are shown in Table 1. PCR reaction system (the total reaction volume: 25 μL)included 1 μL of genomic DNA (75 to 150 ng), 2.5 μL 1×Taq reaction buffer with MgCl2, 2 μL of dNTPs(0.2 mmol · L-1), 1 μL each primer (0.2 μmol · L-1),0.2 μL of rTaq DNA polymerase (1 U) and 17.3 μL RNase-free H2O. PCR was conducted by predenaturing at 95℃ for 5 min; followed by 30 cycles of 95℃ for 1 min, 55-60℃ for 1.5 min, and 72℃ for 1 min; and a final extension at 72℃ for 10 min.All the PCR products were detected by 1% agarose electrophoresis and purified using a gel extraction kit. The ligation reaction system contained 0.5 μL of pMD18-T vector, 5 μL of the purified PCR products and 4.5 μL of the ligation buffer, which was then incubated at 4℃ for at least 12 h. All the reagents were purchased from TaKaRa. Cloning products were sequenced by Huada Biotechnology Co., Ltd.

Sequence analysis of Foxn1 gene

Overlapping fragments that were amplified by PCR were assembled using the software, DNAMAN version7. Exons and introns were divided based on NCBI (http://www.ncbi.nlm.nih.gov/) reference sequence. The complete CDS of Foxn1 gene was translated by using EditSeq program of DNAStar software. Foxn1 protein phosphorylation sites were predicted by NetPhos 2.0 (http://www.cbs.dtu.dk/services/NetPhos/). The transmembrane domain of the deduced amino acid sequence was predicted by TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/). The conserved domain was predicted by Prosite (http://prosite.expasy.org/scanprosite/).The homologous nucleotide sequence was clustered by BLAST (BLASTP, TBLASTX, http://www.ncbi.nlm.nih.gov /BLAST). MEGA5 version was used to generate an unweighted maximum likelihood phylogenetic network. Jones-Taylor-Thornton(JTT+G) model was utilized, and 100 replicates were used to generate the bootstrap values.

Expression of Foxn1 in different tissues

RNA samples extracted from different tissues were subjected to reverse transcription (RT)-PCR using BcaBEST RNA PCR kit. The reverse transcription system included 3 μg of the total RNA, 3 μL of the oligo-dT primer (50 μmol · L-1), 3 μL of random 6-mers (100 μmol · L-1), and RNase-free dH2O to a final volume of 30 μL. The reaction mixture was kept at 37℃ for 15 min, then at 85℃ for 5 s. One primer pair (Foxn1F and Foxn1R) was used to detect the expression of Foxn1 in various tissues (Table 1).β-actin (β-actinF and β-actinR) was used as the control. PCR product was detected by 1% agarose gel electrophoresis.

Results

DNA and cDNA cloning and sequence analysis of Foxn1 gene

After PCR products were assembled by using DNAMAN version7, a 14 730 bp fragment of pig Foxn1 DNA was obtained (GenBank Acc. No.:KJ778072), which included eight exons and seven introns. The location of exons from 1 to 8 was 1-123,518-976, 3 732-3 842, 5 512-5 642, 7 800-7 896,11 390-11 597, 11 746-12 237 and 14 411-14 730.

The complete CDS of pig Foxn1 gene was 1 941 bp in length, the start codon was ATG, and the stop codon was TGA. Its A+T and C+G contents were 36.94% and 63.06%, respectively. The gene was predicted to encode a 646-amino acid protein, with a deduced molecular weight of 68.7 ku and an isoelectric point of 5.81. Foxn1 polypeptide sequence consisted of 60 negatively charged residues (Asp+Glu) and 42 positively charged residues (Arg+Lys), indicating that the protein should have an overall negative charge.Hydropathicity correlation analysis revealed that the protein was highly hydrophilic. Thirty-five phosphorylation sites were predicted by using NetPhos 2.0.TMHMM analysis indicated that the protein apparently had no obvious transmembrane domain, suggesting that Foxn1 was neither a membranous acceptor nor could be located within the membrane. A conserved domain of forkhead 3 was detected between amino acids 269-365 (Fig. 1).

Fig. 1 Phylogenetic tree and alignment of Foxn1 amino acid sequences from Min pig and other species

Molecular phylogenetic analysis of Foxn1 gene

The complete CDS sequences of Foxn1 gene of 44 species were downloaded from NCBI. These species included 41 mammals, one avian, one amphibian and one osteichthyes species. The deduced amino acid sequence of Min pig Foxn1 gene was compared to 43 Foxn1 gene sequence from other animals using DNAMAN 7.0 software. CDS of Min pig Foxn1 gene was 92.43%, 92.38%, 92.35%, 92.17% and 92.12% identical with that of the water buffalo,cattle, Balaenoptera acutorostrata scammoni, alpaca and Arabian camel, respectively. The amino acid sequences of Min pig Foxn1 protein were 94.58%,94.43%, 93.96%, 93.81% and 93.34% identical with those of the water buffalo, cattle, alpaca, Arabian camel and pacific walrus, respectively.

A molecular phylogenetic tree was constructed using the deduced protein sequences of Min pig and other species (Fig. 1). Pig Foxn1 protein sequence showed the highest homology with that of the sheep,chiru, water buffalo and cattle.

Tissue expression profile of Foxn1

The present study selected 17 tissues such as the thymus, heart and liver to determine the expression levels of Foxn1 in each tissue. Fig. 2 showed that Foxn1 was only expressed in the thymus, skin and tongue. To prove whether Foxn1 was expressed in the tongue, the fragment of Foxn1 in the tongue was cloned and sequenced, and the results showed that it was really Foxn1 gene.

Fig. 2 Expression levels of Min pig Foxn1 mRNA in different tissues

Discussion

Foxn1 (formerly known as Whn) was first reported in nude mice in 1994 (Nehls et al., 1994). The nude mice showed developmental problems involving the thymus and skin. Further studies indicated that mutations occurring within DNA-binding domain and carboxyterminal domain resulted in nonfunctional Foxn1 proteins (Brissette et al., 1996). In the epidermis and the hair bulb, Foxn1 played an important role in the process of keratinocyte differentiation (Potter et al.,2010; Darnell et al., 2014). In the thymus, Foxn1 promoted the differentiation of immature epithelial cells into functional cortical and medullary TECs,which was essential for the development of T-cells(Romano et al., 2013; Romano et al., 2012). It had been demonstrated that the upregulation of Foxn1 could substantially reverse age-related thymic involution (Bredenkamp et al., 2014). On the other hand, over- or ectopic-expression of Foxn1 in the early life adversely influenced immature TECs, T and B cells and skin epithelial development (Zhang et al.,2012).

The present study analyzed the characteristics of pig Foxn1 gene. DNA analysis indicated that pig Foxn1 gene included eight exons and seven introns,which were in agreement with those of other animals.However, comparative analysis of the amino acid sequence of Foxn1 in pig, chicken, western clawed frog and zebrafish indicated that except for the conserved domain, the rest of the sequences were significantly different. Phylogenetic reconstruction revealed that pig Foxn1 protein sequence was closely related to that of artiodactyla animals, such as the water buffalo, cattle, sheep and chiru. In addition,the animals belonging to the same order were mainly clustered within the same branch, except for members of Rodentia (thirteen-lined ground squirrel and house mouse).

During fetal life, Foxn1 was expressed in several mesenchymal and epithelial cells, including those of the liver, lung, intestine, kidney and urinary tract.While after birth, its expression was restricted to stromal thymus and skin cells. It had been known that Foxn1 was necessarily required for the normal development, function and maintenance of hair follicles and thymic epithelial cells (TECs). However,the molecular mechanisms by which Foxn1 expression and activity were regulated were only incompletely understood (Gallo et al., 2017). This study found that the transcription of pig Foxn1 mRNA was y organspecific as it was only expressed in the thymus,skin and tongue. Previous reports had described the expression of Foxn1 in the thymus, skin and nail(Mecklenburg et al., 2004), but not in the tongue,which suggested that the function of Foxn1 in this specific organ remained unknown.

Conclusions

In conclusion, Min pig Foxn1 gene complete CDS was isolated, and the sequence homology and phylogeny were analyzed. RT-qPCR was used to detect the spatial expression differences. The results suggested that Foxn1 was conservative among vertebrates. Its expression pattern had obvious tissue specificity.