Expression characterization and transcription regulation analysis of porcine Yip1 domain family member 3 gene

Objective The Yip1 domain family (YIPF) proteins were proposed to function in endoplasmic reticulum (ER) to Golgi transport and maintenance of the morphology of the Golgi, which were homologues of yeast Yip1p and Yif1p. YIPF3, the member 3 of YIPF family was a homolog of Yif1p. The aim of present study was to investigate the expression and regulation mechanism of porcine YIPF3. Methods Quantitative realtime polymerase chain reaction (qPCR) was used to analyze porcine YIPF3 mRNA expression pattern in different tissues and pig kidney epithelial (PK15) cells stimulated by polyinosine-polycytidylic acid (poly [I:C]). Site-directed mutations combined with dual luciferase reporter assays and electrophoretic mobility shift assay (EMSA) were employed to reveal transcription regulation mechanism of porcine YIPF3. Results Results showed that the mRNA of porcine YIPF3 (pYIPF3) was widely expressed with the highest levels in lymph and lung followed by spleen and liver, while weak in heart and skeletal muscle. Subcellular localization results indicated that it expressed in Golgi apparatus and plasma membranes. Upon stimulation with poly (I:C), the level of this gene was dramatically up-regulated in a time- and concentration-dependent manner. pYIPF3 core promoter region harbored three cis-acting elements which were bound by ETS proto-oncogene 2 (ETS2), zinc finger and BTB domain containing 4 (ZBTB4), and zinc finger and BTB domain containing 14 (ZBTB14), respectively. In which, ETS2 and ZBTB4 both promoted pYIPF3 transcription activity while ZBTB14 inhibited it, and these three transcription factors all played important regulation roles in tumorigenesis and apoptosis. Conclusion The pYIPF3 mRNA expression was regulated by ETS2, ZBTB4, and ZBTB14, and its higher expression in immune organs might contribute to enhancing ER to Golgi transport of proteins, thus adapting to the immune response.


INTRODUCTION
The Yip domain family (YIPF) proteins are mammalian homologues of yeast Yip1p and Yif1p; both have five similar transmembrane segments (Yip domain). They are the two mem bers of an integral Golgi membrane protein complex that bind to the Ypt/Rab GTPases [1]. Rab GTPases are key regulators of membrane traffic [2]. The name comes from that the Rab family are annotated as Ypt proteins in yeast cells. Yip1p, Yptinteracting protein, was first identified as a Golgi membrane protein and was found to bind the Rab GTPases includ ing Ypt1p and Ypt31p [3]. Another integral membrane protein, Yif1p (Yip1pinteracting factor), was later identified by its ability to interact with Yip1p in a twohybrid mass screen [1].
There are nine YIPF members in mammalian cells. Phylogenetic analyses showed that YIPF4, YIPF5/YIP1A, YIPF6, and YIPF7/YIP1B are Yip1p homologs, while YIPF1, YIPF2, YIPF3, YIF1A, and YIF1B are Yif1p homologs [4]. Knockdown of Yip1p homologs (YIPF4, YIPF5, YIPF6) caused significant reduction in expression levels of their partner Yif1p homologs (YIPF3, YIF1A, YIPF1/YIPF2, respectively), suggesting that Yif1p homologs are unstable in the absence of Yip1p homologs [5]. The knockdown of the partner (YIPF4/YIPF3, YIPF5/YIF1A) caused fragmentation of the Golgi apparatus, suggesting their involvement in main tenance of the Golgi structure [6]. Moreover, the Yip1p•Yif1p complex was also required for establishing the fusion com petence of endoplasmic reticulumderived vesicles [7], and loss function of the complex caused a block of endoplasmic reticulum (ER) to Golgi transport. Besides above nine YIPE members, researchers have identified a subunit of the Yip1p-Yif1p complex in a thermosensitive yeast strain and named it as Yos1p. Depletion or inactivation of Yos1p could block transport between the ER and the Golgi complex [8].
Human YIPF3 (hYIPF3) protein, once named killer lineage protein (KLIP1), was expressed in nucleated hematopoietic cells, from early embryonic hematopoietic stem cells to ma ture adult blood lymphoid lineages. Moreover, this gene was expressed in fetal and adult GPA + erythroblasts, the fetal liver CD34 + subset, fetal spleen, and adult bone marrow CD56 + natural killer (NK) and CD19 + B cells, which may contribute to improving hematopoiesis and immune functions [9]. It suggested that YIPF3 might be associated with immune re sponse; however, it is still a piece of blank paper about YIPF3 function in pigs.
In recent years, the incidence of swine diseases has been increasing. Porcine reproductive and respiratory syndrome [10] and African swine fever [11] have caused great losses to the pig industry. Numerous studies have identified differences in immune response across a range of parameters between pigs reared under the same environmental conditions [12]. Studies targeting the genetics of disease resistance are essen tial for pig health improvement.
As to the commercial swine industry, application of disease resistance breeding is so important that many animal scientists focus on improving genetic resistance. With this in mind, we investigated the pYIPF3 gene to gain insight into its potential biological roles and transcript regulation traits.

Ethics statement
All the animal treatment processes and the protocols we used were approved by the Animal Care and Use Committee of Guangdong Province, China. The approval ID or permit numbers are SCXK (Guangdong) 20110029 and SYXK (Guangdong) 20110112.

5'-flanking region cloning
To obtain the 5' flanking region of this gene, pYIPF3 mRNA sequence (GenBank No. NM_001245980) was blasted in the high throughput genomic sequences database (http://blast. ncbi.nlm.nih.gov/Blast.cgi). Then a porcine genomic DNA clone (GenBank No. CU928765.2) containing the gene was isolated and approximately 2,000 bp of 5'flanking sequences was amplified with the primers YIPFproF/R (Table 1).

Tissue expression patterns
Tissues were obtained from three Landrace barrows. Total RNA was extracted from the tissues using Trizol reagent (In vitrogen, Carlsbad, CA, USA). RNA quality was assessed by agarose gel electrophoresis and the ND2000 Spectropho tometer (Thermo Scientific, Waltham, MA, USA) and was discarded if the 260/280 ratio was not between 1.8 and 2.1. The RNA was then transcribed into cDNA using MMLV reverse transcriptase (Promega, Madison, WI, USA). Real time polymerase chain reaction (RTPCR) was performed using Hieff qPCR SYBR Green Master Mix (YEASEN Bio, Shanghai, China) on a LightCycler 480 System (Roche, Basel, Switzerland). Betaactin gene (βactin) was used for endoge nous control. The PCR was then performed in triplicate and the gene expression levels were quantified relative to the ex pression of endogenous control. The primers used in this study were list in Table 1.

Treatment of PK15 cells with polyinosine-polycytidylic acid
To further illustrate the YIPF3 expression changes at tran scription level after virus infection, a stimulation experiment of PK15 cells with polyinosinepolycytidylic acid (poly I:C) was performed. Poly I:C, an interferon inducer, is a synthetic mimetic of viral dsRNA and induces immune responses simi lar to a viral infection [13]. PK15 cells were plated in 6well plates at an amount of 2.5×10 5 cell/well. When cells reached about 80% confluence, the medium was replaced with fresh challenge media, with or without poly I:C (SigmaAldrich, St Louis, MO, USA) at a final concentration of 2 μg/mL and 10 μg/mL, respectively. Cells were harvested at 6, and 12 h. Triplicate cell samples were collected at each time point. In dividual RNA from the three samples at each time point was measured in duplicate.

Subcellular localization of pYIPF3 protein in PK15 cells
In order to gain knowledge about the subcellular localization of porcine YIPF3 protein, coding sequence of pYIPF3 gene was amplified using YIPFCF/R primers (Table 1) and subcloned into the EcoRI/XhoI site of the pEGFPC1 vector (Clontech, Takara Bio Inc., Mountain View, CA, USA) to yield a mam malian expression plasmid pYIPF3GFP. After sequencing verification, the plasmids were extracted using an Endofree Plasmid Mini Kit (Omega BioTek, Inc., Norcross, GA, USA). PK15 cells were seeded onto cover slips in 6well plates. Tran sient transfections of pYIPF3GFP and pEGFPC1 (control) were performed with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Twentyfour hours after transfection, the cells were fixed at 37°C for 15 min with 3.7% formaldehyde, and then incubated with 10 μM Hoechst 33342 for 10 min at room tem perature. Finally, the images were obtained and analyzed using the Olympus FluoView FV1000 Confocal Microscope (Olym pus, Tokyo, Japan).

Dual-luciferase reporter assay and site-directed mutagenesis
To determine transcription start sites (TSS) of pYIPF3, a primerextension RTPCR method was utilized [14,15], which is a procedure that employs a same antisense primer located in exon 1, and various overlapping sense primers ranging from nt -27 to nt +51 ( Table 1). The PCR products were subcloned into the XhoI/HindIII site of pGL3Basic (Promega, USA).  The plasmids were cotransferred into 293T cells in quadru plet with an internal control pRLTK (Promega, USA). Empty pGL3Basic vector with pRLTK was also transfected in parallel as an external control. Twentyfour hours after transfection, the activities of firefly luciferase in pGL3 and Renilla luciferase in pRLTK were measured on BioTek Synergy HT MultiMode Microplate Read (BioTek, Winooski, VT, USA). According to the result, fragments with highest activity were selected to be analyzed using the PROMO web tool and TFSEARCH. Transcription factor binding sites with high scores and interesting characteristics were mutated using QuickChange Lightning SiteDirected Mutagenesis Kit (Strat agene, LaJolla, CA, USA) with three pairs of primers (Table  1), multisitedirected mutagenesis was also performed using multisitedirected mutagenesis kit (Stratagene, USA) with above primers. After sequencing verification, firefly luciferase and Renilla luciferase activities of these mutational vectors were measured as above.

Electrophoretic mobility shift assay
Nuclear protein extracts were prepared from 293T cells using Nuclear and Cytoplasmic Extraction Reagents (Thermo Sci entific, USA). The protein concentration of each nuclear extract was detected by Pierce BCA Protein Assay Kit (Thermo Sci entific, USA). All probes with 22 nucleotides were labeled at 5′ end with Biotin (Table 1, Probes). In the competition experi ments, unlabeled oligonucleotides or mutated oligonucleotides without Biotin were added in 50fold excess. When testing the effect of antibody on the mobility complex, 5 μL of the antibody was added to the nuclear extracts prior to the labeled probes. The primary antibodies of ETS2 (sc351 X, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ZBTB4 (sc514883, Santa Cruz Biotechnology, USA) were added to the binding reactions to perform super gel shift experiments. LightShift Chemiluminescent EMSA Kit (Thermo Scientific, USA) was used in the electrophoretic mobility shift assay (EMSA), and the protocol was performed according to the paper [16].

Porcine YIPF3 highly expressed in lymph
Analysis of the pYIPF3 cDNA sequence revealed that it en coded a protein of 347 residues with a highly conserved Yip1 domain in mammals. Further, there are five transmembrane domain distributed between residues Ile149 and His294 (data not shown) as predicted by TMHMM (http://www.cbs.dtu. dk/services/TMHMM/).
The expression pattern of pYIPF3 mRNA showed that it has highest expression level in lymph, followed by lung, spleen and liver. Lower expression levels were detected in backfat, kidney, stomach, small intestine, brain and heart, whereas almost no expression in longissimus dorsi and biceps femoris ( Figure 1).

Porcine YIPF3 mainly localized in Golgi apparatus
The YIPF3 fusion protein was found to distributed in Golgi apparatus and cytomembrane (Figure 2), which was consis tent with its transmembrane domain predicted by TMHMM. Moreover, many documents about the location and function of yeast Yip1p and Yif1p also supported this result. Green flu orescence was detected throughout the control cells transfected with GFP vector alone (data now shown).

Porcine YIPF3 was upregulated after stimulation with polyinosine-polycytidylic acid
As described previously, hYIPF3 gene usually expresses in fetal spleen, adult bone marrow CD56 + NK and CD19 + B cells, which drove us to further investigate its response under mimic viral infections. Our results demonstrated that pYIPF3 mRNA in control cells was only occasionally detected. Transfection of poly (I:C) led to a significantly higher expression compared with the control. Moreover, the levels of mRNAs were mark edly induced under administration of high concentration (10 μg/mL) poly I:C rather than low concentration (2 μg/mL) (Fig  ure 3). In contrast to 6hour poly (I:C) stimulation, the 12 hour stimulation provided higher mRNA expression levels. As a result, it seems that poly (I:C) triggered pYIPF3 expres sion in a time and concentrationdependent manner.

Determination of transcription start sites
Like human homologous genes, pYIPF3 has 9 exons, and the total length of the gene body is about 4,833 bp ( Figure 4A). In which, the potential TSS was denoted as +1. Electrophoresis showed that all sense primers up to primer 6 yielded PCR products, indicating that these sequences were part of pYIPF3 mRNA, whereas the 7th sense primers failed to yield PCR products indicating that it was not part of pYIPF3 mRNA ( Figure 4B). Based on the RTPCR results, it was concluded  that the TSS might distribute on regions nt -27 to nt -14, lo cated between the 3′ ends of primers YIPFTSS(6)F and YIPF TSS(7)F. Agreeing with this, the "C" nucleotide of TSS ( Figure  4C) predicted by the promoter predictor (http://www.fruit fly.org/seq_tools/promoter.html) was actually found in this region with highest score 0.98.

Transcription activity analysis of pYIPF3 promoter
To find the important region required for transcription activity within the 5'flanking sequence of pYIPF3 gene, the DNA clone extending from nt -1,789 to nt +74, was truncated into serial fragments and inserted into the pGL3Basic vector (Fig  ure 4C). As shown in Figure 5A, all constructs pGL31877 to pGL3286, but not pGL3130, presented high transcription activity. This result suggested that pGL3130 lacks important activation elements mediating pYIPF3 transcription. The longer construct pGL3286 displayed a highest transcription activity than the others, indicating that the core promoter is located from -198 to -42 ( Figure 5A). Using the prediction tool of transcription factor binding site, we found that this fragment harbors three potential binding sites for multiple transcription factors including ETS2 (at -150 to -143 of an tisense strand), ZBTB4 (at -130 to -123) and ZBTB14 (at -88 to -79 of antisense strand), which are all highly conserved among three species ( Figure 5B). Deletion of ETS2 or ZBTB4 binding sites of pGL3286 caused 39% or 52.5% drop of tran scription activity, respectively, and almost abolishment of promoter activity was observed in the double deletion plasmid  Table 1). The Lane 2 is from YIPF-TSS(2)F, and so on. (C) Structure of the pYIPF3 promoter region. Underlined sequences indicate primers for pGLB-1877~130 constructs. The left arrow represents the transcription factor binding to the antisense strand, while the right arrow represents binding to the sense strand. Exons are in uppercase and 5' flank region of TSS in lowercase. YIPF3, Yip1 domain family member 3; PCR, polymerase chain reaction; TSS, the transcription starts site; ATG, translation initiation codon; TGA, translation stop codon; TIS, translation initiation site. The number of oligonucleotides is relative to the TSS (C is assigned number +1) of pYIPF3.
pGL3286mut3 ( Figure 5C). These experiments highlight the transcription enhance roles for both ETS2 and ZBTB4. On the contrary, mutation of ZBTB14 binding site resulted in prominently increase of transcription activity even when either of ETS2 and ZBTB4 binding sites was mutated simul taneously ( Figure 5C).

Transcription binding sites confirmed by electrophoretic mobility shift assay
Our study found that the three binding sites were crucial for promoter activity of pYIPF3. Thus, these binding sites were used as probes in EMSA. Obviously, the probes correspond ing to the three ciselements were combined by unknown proteins, because they all had a lag in the lane 2 compared to lane 1 ( Figure 6). Compared with lanes 2, the bands of all lanes 3 in Figure 4 became less intense in terms of competition with unlabeled wildtype probe. It indicated that the binding of probes for proteins was specific. Consistent with expectations, the mutation probes did not weaken the bands ( Figure 6A, 6B).
As there was no suitable antibody for ZBTB14, super shift assays were only performed for ETS2 and ZBTB4 potential binding sites. The results clearly showed that the bands were significantly lagged when the antibodies were added (lane 7 in Figure 6A and 6B), which indicated that the probes can be combined by ETS2 and ZBTB4 proteins, respectively. When immunoglobulin G antibody was added, there was no similar lag band, which further indicated that the binding was spe cific (lane 9 in Figure 6A, B).

DISCUSSION
Our results provided evidence that porcine YIPF3 is located in Golgi apparatus, and its mRNA showed a higher expres sion levels in lymph, lung, spleen and liver which are important organs related to immune system. As differential expression of genes in different tissues suggests a direct or indirect rela tionship between these genes' function and these tissues [17], high expression of pYIPF3 in these immune organs suggested that these tissues need more YIPF3 to maintain their func tion in normal physiological activities. Consistently, there are always a lot of immune related genes highly expressed in immune organs [18]. In addition, when PK15 cells were stimulated by poly (I:C) for different times with different final concentrations, the mRNA expression profiles of pYIPF3 displayed in a time and concentration dependent manner. The expression pattern of pYIPF3 in response to poly (I:C) was consistent with many immunerelated genes such as interferon (IFN) [19], guanylate binding protein 1 (GBP1), and GBP2 genes [20].
Transcription element analysis and sitedirected muta genesis assay showed that deletion of either ETS2 or ZBTB4 binding site would lead to decrease in transcription activity of pYIPF3 promoter. ETS2, belonging to the ETS family which could regulate telomerase activity [21], was highly expressed in prostate cancers [22]. As an important transcription factor with mitogenic and oncogenic activity, high expression of ETS2 predicted poor prognosis in acute myeloid leukemia patients undergoing allogeneic hematopoietic stem cell trans plantation [23], but also its downregulation would inhibit the invasion and metastasis of renal cell carcinoma cells [24]. As a very important transcription activator, ETS2, which was closely related to immunity, might play an important role in the immune response that YIPF3 participated in.
In, general, ZBTB4 usually acted as a transcription repressor, and was frequently downregulated in cancer [25]. Depletion of ZBTB4 promoted cell cycle arrest in response to activation of p53 and suppressed apoptosis through regulation of P21CIP1 [26]. Moreover, ZBTB4 restoration would suppress the tumor growth in mice [27]. But in this study, ZBTB4 showed more transcription activation for YIPF3 expression. The specific role remains to be verified by further experiments.
ZBTB14, also known as ZF5, usually acted as a transcrip Binding sites confirmation for ZBTB4. NE, nuclear extracts from 293T cells. Labeled pETS2 represents the binding sequence of ETS2 was labeled by Biotin. mETS2 means the binding sequence of ETS2 has been mutated. Similar explanation for pZBTB4 in B and for pZBTB14 in C. Competitive assays were performed using 50-fold molar excesses of unlabeled oligonucleotides. Negative IgG is used for testing nonspecific binding. ETS2, ETS proto-oncogene 2; ZBTB4, zinc finger and BTB domain containing 4; IgG, immunoglobulin G.
tional repressor, which inhibited DNA binding and mediated dimerization [28]. In addition, ZBTB14 was regarded as novel regulators of immunometabolic signals from skeletal muscle [29]. Here, our experiments further confirmed that ZBTB14 acted as a suppressor. Only with its repression function, can ETS2 and ZBTB4 guarantee the stable expression of pYIPF3.
In summary, our study showed that pYIPF3 was highly ex pressed in immune organs and gave a response to immune stimulation. Further, ETS2, ZBTB4, and ZBTB14 all played important regulation roles for pYIPF3 transcription. Com bined with its localization of Golgi apparatus, it led us to a hypothesis about porcine YIPF3 in the immune system: The high expression of YIPF3 contributed to enhancing ER to Golgi transport of proteins, thus adapting to the immune stimulation. The function and regulation profiles about pYIPF3 provides useful information in pig breeding, especially for disease resistance.