Mol Biol Rep DOI 10.1007/s11033-014-3182-x

Molecular cloning and characterization of the promoter region of the porcine apolipoprotein E gene Jihan Xia • Bingjun Hu • Yulian Mu Leilei Xin • Shulin Yang • Kui Li



Received: 17 December 2013 / Accepted: 17 January 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Apolipoprotein E (APOE), a component of lipoproteins plays an important role in the transport and metabolism of cholesterol, and is associated with hyperlipoproteinemia and Alzheimer’s disease. In order to further understand the characterization of APOE gene, the promoter of APOE gene of Landrace pigs was analyzed in the present study. The genomic structure and amino acid sequence in pigs were analyzed and found to share high similarity in those of human but low similarity in promoter region. Real-time PCR revealed the APOE gene expression pattern of pigs in diverse tissues. The highest expression level was observed in liver, relatively low expression in other tissues, especially in stomach and muscle. Furthermore, the promoter expressing in Hepa 1–6 was significantly better at driving luciferase expression compared with C2C12 cell. After analysis of porcine APOE gene

promoter regions, potential transcription factor binding sites were predicted and two GC signals, a TATA box were indicated. Results of promoter activity analysis indicated that one of potential regulatory elements was located in the region -669 to -259, which was essential for a high expression of the APOE gene. Promoter mutation and deletion analysis further suggested that the C/EBPA binding site within the APOE promoter was responsible for the regulation of APOE transcription. Electrophoretic mobility shift assays also showed the binding site of the transcription factor C/EBPA. This study advances our knowledge of the promoter of the porcine APOE gene. Keywords Apolipoprotein E  Transcription  Promoter  Pig  C/EBPA

Introduction

J. Xia  B. Hu  Y. Mu  L. Xin  S. Yang (&)  K. Li Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science (IAS), Chinese Academy of Agricultural Science (CAAS), Beijing 100193, People’s Republic of China e-mail: [email protected] J. Xia e-mail: [email protected] B. Hu e-mail: [email protected] Y. Mu e-mail: [email protected] L. Xin e-mail: [email protected] K. Li e-mail: [email protected]

Apolipoprotein E (APOE), a component of the plasma lipoproteins, plays an important role in the transport and metabolism of cholesterol and other lipids [1]. The majority of circulating APOE is produced by hepatocytes [2], and astrocytes are responsible for it synthesis in the brain, where it is the major apolipoprotein. APOE is a component of many lipoprotein receptors and a ligand for many lipoprotein receptors. APOE is a major protein of the very low-density lipoproteins, also is present in almost all kinds of lipoproteins, including chylomicron, chylomicron remnants, intermediate-density lipoproteins and the cholesterol-rich subclass of high-density lipoproteins [3]. Therefore APOE plays an important role as a ligand of lipoprotein receptors [4]. Apolipoprotein E is also known to play an important role in lipid metabolism [5, 6]. In human, the APOE genetic polymorphism was controlled by

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three alleles E2, E3 and E4, for six phenotypes [7]. There are three homozygous phenotypes (E4/4, E3/3 and E2/2) and three heterozygous phenotypes (E4/3, E3/2 and E4/2). ApoE alleles (e2, e3, e4) are associated with Alzheimer’s diseases and cardiovascular disease [8]. In addition, APOE is involved in processes of degeneration and regeneration after nerve lesions as well as in the pathogenesis of Alzheimer’s disease (AD) [9]. In pigs, the APOE gene was situated on the long arm of chromosome 6 in the q2.1 region by fluorescent in situ hybridization [10, 11]. The porcine APOE gene are composed of four exons and three introns that contain a (CG)13 microsatellite in intron 3 [12]. There are three codominant alleles controlling the APOE genetic polymorphism at a single locus in pigs [12]. It encodes a pre-APOE protein comprised of a signal peptide of 18-amino-acids and a mature protein of 299 amino acids ending with a 30 untranslated 125 bp sequence [10]. APOE is synthesized and secreted primarily in liver, brain and tissue macrophages throughout the body, where APOE plays critically important roles [3]. Pigs an important model organism had been widely used for human health research because of the metabolic and immune similarity between pigs and human [13]. The study of disease related genes and their related regulating elements of pigs are important for the swine disease model. To further study APOE, here we cloned the promoter from -2,335 to ?277 and predicted potential transcription factor binding sites, investigating its potential biological role by analyzing tissue expression profile and C/EBPA regulated transcription. C/EBPA protein dimers directly activate transcription from lineage specific many gene promoters and viral enhancers [14]. The knowledge of the porcine APOE in this study will contribute useful information for further research. Table 1 Primers information in experiments

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Primer name

Materials and methods Isolation of pig APOE promoter Homologous ESTs of pig were available from GenBank using a BLASTN program based on human APOE gene sequence. Total DNA was extracted from liver tissues of landrace pigs using reagent (Roche, Beijing, China) and treated with Dnase-free RNase (Roche, Beijing, China). The specific primers (APOE-2335/? 277-R/F on Table 1) were designed to amplify pig APOE promoter from -2,335 to ?277. Sequence analysis of pig APOE gene Multiple sequence alignments were performed using Clustal X2. Prediction of putative transcriptional binding sites was performed using TFSEARCH program and Transcriptional Element Search Software (TESS). In order to analyze Motif of C/EBPA binding sites for the APOE promoter, C/EBPA binding site consensus sequences of rat (TESS accession no.R08109) [15] and human (TESS accession no. R08103) [16] were analyzed. Motif analysis of C/EBPA binding sites in select APOE promoter sequence was performed. Sequence logos depict distributions for the APOE promoter C/EBPA binding site (The JASPAR Database). Tissue distribution of pig APOE Total RNA was extracted from three landrace pigs using Trizol reagent (Invitrogen, Beijing, China) and treated with RNase-free DNase. First strand cDNA synthesis was performed using MMLV reverse transcriptase (Promega, Madison, USA) according to the manufacturer’s instructions.

Primer sequence (50 -30 )

PCR (Tm) (oC)

Size (bp)

59

2,612

60

192

60

166

APOE-2335/? 277-F

GTGACCGTGGCCCGTT

APOE-2335/? 277-R

GTCGGCTCTGGCTCGT

qAPOE-F

ATGAGGGTTCTGTGGGTTG

qAPOE-R

CACTTGGTCAGACAGGGACT

GAPDH-R

AGGGCATCCTGGGCTACACT

GAPDH-F

TCCACCACCCTGTTGCTGTAG

pAPOE-1627/? 277-F

ACTCAATGAAGTGTCGGTTTT

57

1,904

pAPOE-1627/? 277-R pAPOE-1171/? 277-F

GTCGGCTCTGGCTCGT CCCCGGATGATGGGGTTA

59

1,448

pAPOE-1171/? 277-R

GTCGGCTCTGGCTCGT 58

946

58

536

pAPOE-669/? 277-F

TGTGGCTGTGGCTGTGGC

pAPOE-669/? 277-R

GTCGGCTCTGGCTCGT

pAPOE-259/? 277-F

CTCGGGAGTTACTGGGTG

pAPOE-259/? 277-R

GTCGGCTCTGGCTCGT

Mol Biol Rep

Tissue distribution of pigs APOE relative to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed by real-time PCR. Brain, lung, liver, stomach, spleen, heart, ileum, longissimus dorsi and hypothyroid tissues were collected from mature landrace. The primer pairs (qAPOE-F/R and GAPDH-F/R, on Table 1) were designed. The PCR reaction was performed on a 7500 realtime PCR System (Applied Biosystems) using SYBR Premix Ex Taq TM with ROX (TaKaRa, Dalian, China). Reaction conditions were denaturation for 2 min at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. Samples were analyzed in triplicate to ensure statistical significance. Dates were analyzed based on the stable expression level of the GAPDH gene according to method of Livak and Schmittgen [17]. Plasmid construction Genomic DNA template was prepared from a landrace pig liver tissue. An 2612 bp amplicon (-2,335 to ?277, with the first nucleotide ‘‘G’’ in DNA corresponding to the first base incorporated into RNA designated as ?1) was cloned using LA Taq DNA Polymerase and ligated into the pMD18-T vector (TaKaRa, Dalian, China) for subsequent manipulation as described previously [18]. Briefly, constructs containing variable length of truncated pig APOE promoter were individually amplified using different forward primers and a common reverse primer. The forward primers and reverse primer contained kpnI and BglII recognition sequences, respectively, in order to facilitate plasmid ligation (APOE-2335/? 277-F/R, pAPOE-1627/ ? 277-F/R, pAPOE-1171/? 277-F/R, pAPOE-669/? 277F/R, pAPOE-259/? 277-F/R, Table 1). The amplified fragments were then inserted into the multiple cloning site of the pGL3-basic vector in order to generate luciferase reporter constructs. For site mutation, four primers and three PCR reactions were conducted to create a site-specific mutation by overlap extension. The mutation plasmids were constructed as described above. All plasmids were sequenced to confirm proper insertion prior to transfection experiments. Cell culture, cell transfection and luciferase assays Mice hepatic cells (Hepa 1–6) were utilized to perform luciferase reporter assays. Cells were seeded on coverslips 24-well plates for luciferase assays and cultured in dulbecco’s modified eagle’s medium (DMEM, Hyclone, USA) supplemented with 10 % (v/v) fetal bovine serum under humidified air containing 5 % CO2 at 37 °C. When cells had reached 80 % confluence, transfections were performed using Lipofectamine 2000 (Invitrogen, Beijing, China) according to the manufacture’s instructions. At 32 h after transfection, luciferase assays were performed using

Dual Luciferase activity. Luciferase assays were performed using Dual Luciferase Assay kits (Promega, Madison, USA) and a Luminometer TD-20/20. Firefly luciferase levels were normalized against Renilla luciferase levels. Electrophoretic mobility shift assays Nuclear proteins were extracted from Hepa 1–6 cells (Nuclear Extract Kit, Thermo). The sequences used for EMSAs were 50 -CCCCTTAACCCAGAAATCCCAGAC30 , and synthesized (Invitrogen, Beijing, China). The oligonucleotides were 50 -labeled with Biotin (Invitrogen, Beijing, China). The 50 -labelled probes were double stranded in subsequent experiments. The binding mixture included Binding buffer, Glycerol, Mgcl2, Poly (dI-dC), NP-40 (Thermo Scientific, Rockford, IL USA), and nuclear extract according to the Thermo EMSA Kit’s instructions. The mixture was maintained at room temperature for 10 min, and 20 fmol of Biotin end-labeled oligonucleotide probes was added, and incubated for a further 20 min at room temperature in the presence or absence of a 200-fold molar excess of unlabelled probes. DNA–protein complexes were fractionated by electrophoresis on 6 % nondenaturating polyacrylamide gels.

Results Cloning of pig APOE promoter and sequence analysis The APOE promoter of 2612 (-2,335 to ?277) bp was cloned with special pair primers (Table 1). The 600 bp (from -600 to ?1) region of the porcine APOE promoter was BLASTN against human, mice, rat and chimpanzee promoter at same regions (from -600 to ?1). There are similarity (63.01 %), and two GC signal, a TATA box were found in the five sequence (Fig. 1), which suggested functional importance of these fragments across the five species. Sequence analysis of porcine APOE promoter segments revealed that the flanking region harboured potential binding sites for multiple transcription factors including AP-1 and Sp1, in addition to the hepatic specific transcription factors HNF4 and HNF1 (Fig. 1). APOE expressed in different tissues The results of real-time PCR showed that pig APOE was expressed highly in liver. The high pig APOE expression was observed in the liver and very low expression was detected in other tissues, especially in muscle tissues such as heart and longissimus dorsi. Pig APOE mRNA levels were very similar in the other tissues examined (Fig. 2).

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Fig. 1 Homology of human, mouse, rat and chimpanzee APOE and porcine APOE promoter. The porcine promoter sequence (600 bp) was aligned with human, mouse, rat and chimpanzee APOE sequence. The similarity is 63.01 %. Two GC signals, a TATA box were indicated

50 region of porcine APOE gene promoter

Fig. 2 Relative expression of APOE mRNA in different the landrace tissues as detected using real-time PCR and the comparative Ct (DDCt) value method. Results were normalized against GAPDH relative to heart. Bars represent the mean ± SD of three independent experiments. The statistical significance of the differences values were analyzed by the Student’s t test, **p \ 0.001

Fig. 3 The activity of the porcine APOE promoter is significantly higher in Hepa 1–6 than C2C12. Data are expressed as mean ± SD of three independent experiments. The statistical differences in luciferase activity were analyzed by the Student’s t test, *p \ 0.05, **p \ 0.001

APOE promoter expression in Hepa 1–6 and C2C12 cell In order to analyze the APOE promoter expression in different cells, we used the porcine APOE promoter region of 2,612 (-2,335 to ?277) to assemble fusion constructs and a firefly luciferase reporter. The promoter expressing in Hepa 1–6 was significantly better at driving luciferase expression, and reporter levels were much higher than that in C2C12 cell (Fig. 3).

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Sequence with porcine APOE 50 -flanking was analyzed from -723 to ?277. Sequence analysis indicated several potential transcription factor binding sites. The TATA-like box is located at the 50 -flanking region of the porcine APOE gene. There is a region with high probability of being a C/EBPA target. This DNA was used for the luciferase reporter gene construct designated pC/EBP-Luc. There are five putative Sp1 sites and two GC signals and GATA-1 sites (Fig. 4). C/EBPA regulated pig APOE promoter activity in Hepa 1–6 cells In order to verify the transcriptional regulation of porcine APOE by C/EBPA as predicted, we undertook a mutation in the position (-411 to -402) of APOE and tested the ability of the mutation sequence to drive the expression of a luciferase reporter. Two luciferase reporter plasmids with or mutation the conserved C/EBPA binding site from the APOE promoter sequence were constructed and transfected Hepa 1-6 cells. After co-transfection and luciferase assays, we observed that the C/EBPA mutation and deleption of pAPOE-669/? 277 had decreased significantly than the pAPOE-669/? 277 promoter activity (Fig. 6). The C/EBPA binding site consensus sequence was predicted by multiple sequence alignment among pig, rat and human (Fig. 5). Luciferase assays of pigs APOE promoter activity We generated a series of deletion mutants for the APOE promoter via PCR-based approaches using the APOE2335/277 constructs as templates. The amplified mutant fragments were then sub-cloned into the pGL3-basic vector and our results are shown in Fig. 6. For the APOE promoter fragments, the deletion constructs pAPOE-1171/277 displayed highest promoter activity. Further deletions pAPOE-669/277 and pAPOE-259/277 resulted in a gradual decrease in promoter activity. The APOE promoter activity of the C/EBPA binding sites deletion and mutations with pAPOE-669/277 deceased significantly (Fig. 6).

Mol Biol Rep Fig. 4 Sequence of the 50 untranslated and promoter region from nucleotide -723 to ?277. Numbering of the sequence begins with the transcription initiation start site. There were a TATA box and two GC signals. Potential transcription factor binding sites were predicted and indicated with underline

Fig. 5 Motif analysis of C/EBPA binding sites in select APOE promoter sequence. Multiple sequence alignment among pig, rat and human (left panel) revealed a C/EBPA binding site consensus

sequence. The sequence logos depicted distributions for the APOE promoter C/EBP binding site (right panel)

We synthesized specific oligonucleotides containing the C/EBPA elements presented in the APOE promoter, and then tested them with a C/EBPA consensus sequence, the control in EMAS experiments with nuclear extracts from Hepa 1–6 cells. As shown in Fig. 7, incubation of Hepa 1–6 nuclear extracts with the C/EBPA consensus sequence produced a DNA–protein band shift. In contrast, the C/EBPA oligonucleotide unlabeled probe failed to form such a complex in this experiment. These DNA–protein complexes were determined to be specific to the C/EBPA sites by successful competition assays using excess unlabeled consensus (Fig. 7).

polymorphism located in a single locus in pigs [24]. Pigs and human APOE gene CDS (Pig GenBank accession no.NM_214308.1 and Human NM_000041.2 respectively), showed high homology (81.69 %) and similar genomic structures, which all harbored four exons and three introns. The study of swine APOE promoter regulator elements helps to analysis these diseases. To our knowledge, the current report is the first study cloning and sequence analysis of APOE gene promoter region in pigs. We isolated the promoter (-2,335 to ?277) regions of porcine APOE by PCR. Pigs APOE promoter regions (from -600 to ?1) has similarity (63.01 %) against human, mouse, rat and chimpanzee in promoter same regions and we found two GC signal, a TATA box (Fig. 1). We speculated that the similarity and the GC signal, TATA box would be important for the APOE gene expression in different species. The porcine APOE promoter segments harboured potential binding sites for multiple transcription factors including AP-1, Sp1 and the hepatic specific transcription factors such as HNF4 and HNF1 (Fig. 4) which contributed to specific expression in liver. In this study, we found pig APOE mainly distributed in liver of nine tissues, but extremely low expression were

Discussion Apolipoprotein (APOE) is a major determinant in lipoprotein metabolism, cardiovascular disease, Alzheimer’s disease [19, 20], cognitive function, immunoregulation and even infectious diseases [21, 22]. In human, there are three homozygous phenotypes (E4/4, E3/3, and E2/2) and three heterozygous phenotypes (E4/3, E3/2, and E4/2) [23]. In pigs, three codominant alleles controling the APOE genetic

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Fig. 6 Luciferase assays of pigs APOE promoter activity. Five luciferase reporter plasmids expressing successive truncations of the APOE promoter sequence were constructed and transfected into Hepa 1–6 cells. The resulting firefly luciferase activity was normalized to Renilla luciferase activity and the relative values were presented as fold induction over the activity of the pGL3-basic vector. The location of each 50 -deletion sequence was indicated at the left of each bar. Boxes represent the location of the putative APOE binding site within the promoter regions. The mutated putative APOE binding site is labeled with an ‘‘X’’. The relative luciferase activity values represent the mean ± SD of three independent experiments. Statistical differences in luciferase activity were assessed using the Student’s t test, *p \ 0.05, **p \ 0.001

significantly higher in hepatocytes than C2C12 cells showed this specificity. Moreover, transgenic mice expressing 11b-HSD1 activity selectively in the liver under transcriptional control of hepatic regulatory sequences derived from the human APOE gene [25]. We thus speculated that the porcine APOE promoter would highly derive gene expression specifically in hepatocytes. We generated a series of deletion APOE promoter constructs, and co-transfected with various promoter regions fused to firefly luciferase with a Renilla luciferase expression control vector in Hepa 1–6 cells (Fig. 6). Promoter mutation and deletion pAPOE -669/? 277 analysis suggested that a C/EBPA binding site was essential for a high level of expression of the APOE gene for the first time. The CCAAT/enhancer binding protein C/EBPA was transcription factors that played integral roles in the development and function of many organ systems including liver, hematopoietic [26, 27]. There is a putative C/EBPA binding site in the promoter of pig APOE gene. The C/EBPA binding sites have been identified in APOE promoter (Fig. 5). Although, Shixin et al. reported that the SNP at position -155 among these three (-440, -155, ?501) was found to exert a marked influence on the transcription of the porcine APOE gene [9], the C/EBPA regulation position (-411 to -402) was not related these polymorphism sites. We surmised that the C/EBPA regulation position was essential for the APOE expression, and thus the position did not contain polymorphism sites. Furthermore, gel retardation assays were performed to validate the binding of C/EBPA in the core promoter region of porcine APOE. Competition experiments with Biotin end-labeled oligonucleotide probes also confirmed the C/EBPA element binding sites in the core region of the APOE (Fig. 7). Thus the present results suggested that porcine APOE was a regulatory target gene of C/EBPA, and shed insight on further investigation into the function of swine APOE.

Conclusion Fig. 7 EMSA analysis of the C/EBP binding sites in the pig APOE promoters. Biotin end-labeled oligonucleotide probes for the C/EBPA consensus binding sites were incubated with Hepa 1–6 nuclear extracts. Competition experiments were performed using a 200-fold excess of unlabeled C/EBPA consensus probes. Arrow indicate the resulting the band shift

detected in other tissues such as heart, ileum and spleen tissues (Fig. 2). So we believe that the promoter of swine APOE may be used as a pretty good pigs liver specific promoter. Further, the result of the porcine APOE promoter region of 2,612 (-2,335 to ?277) to assemble fusion constructs and firefly luciferase reporters expressed

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In conclusion, this study provided information that helped to understand the promoter of the porcine apolipoprotein E molecular characterization and potential transcriptional regulation. Two GC signals, a TATA box were indicated at the promoter of APOE sequence both in human, mouse, rat and chimpanzee. Real-time PCR revealed a high and specific expression in the liver, relatively low in other tissues. Results of promoter activity analysis indicated the C/EBPA was essential for a high level of expression of the APOE gene. Promoter mutation and deletion analysis further suggested that a C/EBPA binding site within the APOE promoter was responsible for the regulation of APOE transcription.

Mol Biol Rep Acknowledgments This research was supported by the National Key Technology Support Program (2012BAI39B04, 2011BAI15B02), The National Basic Research Program (2011CBA01005), National Science and Technology Major Project (2013ZX08010-003), National Natural Science Foundation of China (31372276), Innovation Project of Chinese Academy of Agricultural Sciences and The National High Technology Research and Development Program of China (SQ2011AAJY2795). Disclosure Statement

The authors declare no competing interests.

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Molecular cloning and characterization of the promoter region of the porcine apolipoprotein E gene.

Apolipoprotein E (APOE), a component of lipoproteins plays an important role in the transport and metabolism of cholesterol, and is associated with hy...
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