Porcine LEM domain-containing 3: Molecular cloning, functional characterization, and polymorphism associated with ear size

Journal of Integrative Agriculture 2016, 15(6): 1321–1329 Available online at www.sciencedirect.com

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RESEARCH ARTICLE

Porcine LEM domain-containing 3: Molecular cloning, functional characterization, and polymorphism associated with ear size LIANG Jing1*, LI Na1, 2*, ZHANG Long-chao1, WANG Li-gang1, LIU Xin1, ZHAO Ke-bin1, YAN Hua1, PU Lei1, ZHANG Yue-bo1, SHI Hui-bi1, ZHANG Qin2, WANG Li-xian1 1

Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation, Ministry of Agriculture/Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China 2 Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture/College of Animal Science and Technology, China Agricultural University, Beijing 100193, P.R.China

Abstract Ear size exhibits remarkable diversity in pig breeds. LEM domain-containing 3 (LEMD3) on chromosome 5 is considered as an important candidate for porcine ear size. This is the first study on cloning and characterization of LEMD3 cDNA. The complete cDNA contains 4 843 bp, including a 2 736-bp open reading frame (ORF), a 37-bp 5´-untranslated region (UTR) and a 2 070-bp 3´-UTR. The complete LEMD3 gene is 126 241-bp and contains 13 exons and 12 introns. The ORF encodes a deduced LEMD3 protein of 911 amino acids, which shares 82–94% nucleic acid and 51–96% amino acid identity with other species. A phylogenetic tree constructed based on the amino acid sequences revealed that the porcine LEMD3 protein was closely related with cattle LEMD3. Resequencing of the ORF and promoter of LEMD3 from Minzhu pig and Large White revealed three single nucleotide polymorphisms (SNPs): L964C>A in the complete coding region, L4625A>G in the 3´ UTR, and L-394T>C in the promoter region. Genome-wide association study (GWAS) revealed that all of SNPs were shown significant association with ear size in Large White×Minzhu pig intercross population. With conditional GWAS, –log10(P-value) decreased by more than 80% when each of three SNPs was included as a fixed effect. These results suggested direct involvement of LEMD3 or close linkage to the causative mutation for ear size. The findings of this study might form the basis for understanding the genetic mechanism of ear size variation in pigs and provide potential molecular markers for screening ear size diversity in pig breeds. Keywords: association analysis, ear size, LEMD3, molecular cloning, pig

1. Introduction

Received 30 March, 2015 Accepted 15 September, 2015 LIANG Jing, E-mail: [email protected]; Correspondence WANG Li-xian, Fax: +86-10-62818771, E-mail: [email protected]; ZHANG Qin, E-mail: [email protected] © 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61173-X

Ear size is known to vary across pig breeds and plays an important role in characterization of pig breeds (Rothschild and Ruvinsky 1998). Traditionally, many Chinese pig breeds with large ears such as Erhualian and Minzhu were favored and selected by owners for sacrificial culture (Zhang et al. 1986). Previous studies have shown that the most significant

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quantitative trait loci (QTL) for ear size were located on Sus scrofa chromosomes (SSCs) 5 and 7 (Wei et al. 2007; Ma et al. 2009). A missense mutation (G32E) in the PPARD gene on SSC7 was considered to be responsible for the differences in porcine ear size (Ren et al. 2011). Our previous QTL fine mapping on SSC5 revealed that LEM domain-containing 3 (LEMD3) was one of the important candidates for porcine ear size (Zhang et al. 2014). Further, another study identified that LEMD3 was significantly associated with ear morphology in European pigs (Wilkinson et al. 2013). Moreover, in dogs, LEMD3 was thought to control ear size (Boyko et al. 2010; Vaysse et al. 2011). LEMD3, a member of the LEM-domain protein family, also known as MAN1, is an integral protein in the inner nuclear membrane of the nuclear envelope (PaulinLevasseur et al. 1996) and has functions to antagonize transforming growth factor-beta signaling at the inner nuclear membrane. However, since the full-length cDNA of porcine LEMD3 is not available, understanding the genetic basis of ear size diversity in pigs is difficult. This study aimed to clone the full-length cDNA of porcine LEMD3 and identify the

genetic variants associated with ear size.

2. Results 2.1. cDNA cloning and primary structure of porcine LEMD3 A 4 843-bp full-length porcine LEMD3 cDNA was obtained for the first time in this study (GenBank accession no. KP765730). The cDNA contained a 37-bp 5´-untranslated region (UTR), a 2 070-bp 3´-UTR and a 2 736-bp open reading frame (ORF) encoding a protein of 911 amino acid residues. The splice donor and acceptor consensus sequences were identified at the exon and intron boundaries by using the GT-AG rule. A total of 13 exons were identified using the Ensemble Blast search program (Table 1). Sequence comparison revealed that the nucleotide sequence of porcine LEMD3 was identical to those from Bos taurus (94%), Homo sapiens (92%), Macaca mulatta (91%), Rattus norvegicus (84%), and Mus musculus (82%), respectively.

Table 1 The exon-intron boundaries of the porcine LEM domain-containing 3 (LEMD3) gene Exon/Intron 1 2 3 4 5 6 7 8 9 10 11 12 13 1)

Exon size (bp) 1 558 38 67 68 80 146 102 103 179 82 106 79 2 204

5´ splice donor1) GAACTCACCAgtaagtatta AAAAATACAAgtaagtaaaa CAACTTGCAGgtaattgttt ATATTTGAAAgtaagcaatg TTGGAATAAGgtaaaggatt TTGTGCTTAGgtaagttgta AAGATTATAGgtatgatatt CTCATGACAGgtctgtttaa CAGGGTCAAGgtatgtattt ATCCAGTCATgtaagtatta TTCACGCGAGgtaaagtaac TGGTTTGATGgtaagaaatt TTTTAAAAAA

3´ splice acceptor1) tccttgatagTAAAAAACCC ttaaatttagGAGAGTCAAA atctttccagGAGATCATGA tttttattagGATTTAGGTC ttttaaatagGTGTGTTGGT aatattgcagGTGTAGTGA ttactttcagATGTTTTACG taaatgatagGAAAAAAATG ttttttatagCATTTCATTT taacttttagGGAAATAGGG aatatcacagGGTTGTGTAT cttacaacagGGAAATTGGT

Intron size (bp) 89 847 3 849 2 439 16 269 84 1 026 108 665 5 084 1 735 100 223

Exon sequences are revealed in uppercase letters and intron sequences in lowercase letters. Conserved gt–ag junctions are marked in boldface type.

2.2. Analysis of the deduced amino acid sequence

Table 2 The composition of amino acid encoded by porcine LEMD3 gene

The primary structure analysis of the predicted porcine LEMD3 protein revealed that the molecular weight, theoretical isoelectric point, and instability index were 100.36 kDa, 7.32, and 57.78, respectively. The amino acid composition of this protein is shown in Table 2. The content of leucine was the highest and that of tryptophan was the lowest in the predicted protein. The secondary structure of the deduced amino acid sequence is shown in Fig. 1. In the protein, alpha helix, extended strand, and random coil accounted for 33.70,

Name Ala (A) Arg (R) Asn (N) Asp (D) Cys (C) Gln (Q) Glu (E) Gly (G) His (H) Ile (I)

Count 73 68 39 53 15 35 60 76 23 28

Count percent (%) 8.0 7.5 4.3 5.8 1.6 3.8 6.6 8.3 2.5 3.1

Name Leu (L) Lys (K) Met (M) Phe (F) Pro (P) Ser (S) Thr (T) Trp (W) Tyr (Y) Val (V)

Count 88 45 20 24 55 83 44 12 23 47

Count percent (%) 9.7 4.9 2.2 2.6 6.0 9.1 4.8 1.3 2.5 5.2

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13.83, and 52.47% of the predicted secondary structure

sequence was 96, 93, 92, 84, 82, 63, and 51% identical to

composition, respectively. Therefore, the protein was clas-

those of B. taurus, H. sapiens, M. mulatta, R. norvegicus,

sified as a mixed protein. Two tertiary structures (653–780

M. musculus, Danio rerio, and Xenopus laevis, respectively.

and 778–890 aa) were detected in the deduced amino

The alignment results with LEMD3 proteins were used to

acid sequence by using SWISS-MODEL (Fig. 2). Three

construct a phylogenetic tree by using the neighbor-joining

conserved domains were predicted using the InterProS-

(NJ) method with the MEGA 5 software (Fig. 6). The por-

can sequence search program: LEM (Lap2/Emerin/Man1

cine LEMD3 showed a closer genetic relationship with that

domain) (7–50 aa), MSC (Man1-Src1p-C-terminal domain)

from B. taurus.

(509–751 aa), and RRM (RNA recognition motif) (785–864 aa; Fig. 3). Two transmembrane segments were predicted using the TMHMM Server ver. 2.0 (Fig. 4). No signal peptide

2.3. SNP identification and genome-wide association study

was found, as revealed by SignalP 4.1 server. Hydrophilichydrophobic property prediction (Fig. 5) revealed two hy-

The LEMD3 gene was screened for polymorphisms by per-

drophobic segments, which showed the same trend as the

forming comparative sequencing of Large White and Minzhu

transmembrane segments.

pigs. A total of three single nucleotide polymorphisms

Blast analysis showed that the deduced amino acid

(SNPs), which are L-394T>C, L964C>A, and L4625A>G,

Fig. 1 Predicted secondary structure of the deduced amino acid sequence.

Fig. 2 Predicted tertiary structure of the deduced amino acid sequence by SWISS-MODEL. A, 653–780 aa. B, 778–890 aa.

were identified. The L-394T>C, which was located in the promoter region, caused the disappearance of a predicted transcription factor Sp1 (–397 to –386 bp) due to the change from T to C. Prediction of microRNA (miRNA) found the SNP in 3´ UTR (L4625A>G) didn’t alternate the miRNA binding site. The polymorphism L964C>A was a synonymous mutation. The genotypes of these three SNPs were integrated into the 60K SNP genotyping data to re-perfrom GWAS. Genotypic and allelic frequencies of the SNPs in F2 population were shown in Table 3. T allele of L-394T>C, C allele of L964C>A and A allele of L4625A>G were preponderant

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Fig. 3 Predicted conserved domains of the deduced amino acid sequence by InterProScan sequence search: Lap2/Emerin/Man1 (LEM) domain (7–50), Man1-Src1p-C-terminal (MSC) domain (509–751 aa) and RNA recognition motif (RRM) (785–864 aa).

Fig. 4 The transmembrane domain prediction by TMHMM Server ver. 2.0.

Fig. 5 Hydrophilic-hydrophobic property of the porcine putative LEM domain-containing 3 (LEMD3) protein.

alleles with frequencies of 0.521, 0.522, and 0.517, respectively. From the results of GWAS in Table 4, L-394T>C, L964C>A and L4625A>G were all shown significant asso-

ciation with ear size with P-values of 1.33E-07, 8.24E-08 and 6.83E-08, respectively. The results of the conditional analysis with each detected SNP were shown in Fig. 7.

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Fig. 6 The phylogenetic tree was constructed using MEGA 5 software.

Table 3 Genotype and allele frequencies of the three single nucleotide polymorphisms (SNPs) in LEMD3 Polymorphism L-394T>C L964C>A L4625A>G

TT 0.270 CC 0.270 AA 0.264

Genotype frequency TC 0.502 CA 0.503 AG 0.505

When the genotype of each detected SNP was included as a fixed effect, the –log10(P-value) of the most significant SNP on SSC5 dropped from 8.134 to 1.535, 1.598 and 1.531 respectively, more than 80% of the drop ratio.

3. Discussion Because the full-length mRNA sequence of porcine LEMD3 cDNA is not available in GenBank, we first cloned and analyzed this sequence. Similar to human, the porcine LEMD3 contains 13 exons. The composition and structure of porcine LEMD3 mRNA and the deduced protein were similar to those of known LEMD3 mRNAs and proteins in other animals (Lin et al. 2000). The predicted LEMD3 contained three conserved domains: LEM, MSC, and RRM, same as those found in humans (PaulinLevasseur et al. 1996). The LEM domain is present in all members of the LEM protein family. RRM and MSC are DNA-binding winged helix domains and are responsible for DNA and Smad binding (Mans et al. 2004; Caputo et al. 2006; Konde et al. 2010). The two predicted tertiary structures (MSC and RRM) were located in the carboxyl-terminal region, which was involved in DNA binding (Caputo et al. 2006). The results of sequence comparison indicated that LEMD3 was highly conserved in mammals, and the NJ phylogenetic tree of LEMD3 showed the same result. Two transmembrane segments were found in LEMD3 and might span the inner nuclear membrane twice (Lin et al. 2000).

Allele frequency CC 0.229 AA 0.227 GG 0.231

T 0.521 C 0.522 A 0.517

C 0.480 A 0.478 G 0.483

Table 4 Genome-wide association of three SNPs with porcine ear size SNP L-394T>C L964C>A L4625A>G

Chr. 5 5 5

Position 32 770 008 32 771 365 32 896 451

P-value 1.33E-07 8.24E-08 6.83E-08

Binding to chromatin-associated proteins and transcriptional regulators (Smads) suggested that LEMD3 inhibits the signaling of transforming growth factor-beta (TGF-β) and bone morphogenetic protein (BMP) (Bengtsson 2007) and affect the development of bones (Hellemans et al. 2004; Ben-Asher et al. 2005; Caputo et al. 2006; Bourgeois et al. 2013). In Xenopus embryos, LEMD3 overexpression was found to antagonize the signaling of BMP (Osada et al. 2003; Raju et al. 2003). The association between TGF-β and Wnt/β-catenin pathways has an important effect on cell proliferation and differentiation of cartilage and adipocytes (Zhou et al. 2004). The external ear is composed of cartilage, connective tissue, and fat, and is covered with skin. Given its crucial role in inhibiting the activity of TGF-β, LEMD3 could indirectly regulate cartilage development and fat metabolism and could be as an important candidate for ear size in pigs. Further, mutations in the LEMD3 gene have been linked to osteopoikilosis, Buschke-Ollendorff syndrome, and melorheostosis (Mumm et al. 2007; Zhang et al. 2009; Burger et al. 2010; Yadegari et al. 2010; YusteChaves et al. 2011). In humans, LEMD3 has a possible

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5. Materials and methods 5.1. Samples and data collection

Fig. 7 –log10(P-value) of the most significant single nucleotide polymorphism (SNP) on Sus scrofa chromosome 5 (SSC5) curves of three SNPs in conditional analysis when each SNP was included as a fixed effect. The Y-axis shows –log10(P-value) of the most significant SNP on SSC5 and the X-axis indicates three detected SNPs.

role in bone development (Hellemans et al. 2004; Konde et al. 2010). In this study, three SNPs were detected in the porcine LEMD3 gene, which were genome-wide significantly associated with ear size in the F2 population. The SNP L964C>A was a synonymous mutation in the coding region and did not produce any direct change in the amino acid sequence; it might affect the gene function by causing a codon bias during translation, thereby hampering the stability of the mRNA or the controlling of gene transcription (Duan et al. 2003; Capon et al. 2004). The SNP L-394T>C in the promoter region caused the disappearance of a predicted transcription factor Sp1, which plays a critical role in various cellular processes (Sankpal et al. 2011; Safe et al. 2014; Jarvela and Hinman 2015). Another SNP L4625A>G in the 3´ UTR might play a role in gene expression. With the conditional analysis, –log10(P-value) of the most significant SNP on SSC5 dropped by more than 80% if the genotypes of three detected SNPs were added as a fixed effect. This conditional GWAS suggested direct involvement of LEMD3 or close linkage to the causative mutation for ear size.

4. Conclusion In summary, the complete mRNA sequence of porcine LEMD3 gene was obtained for the first time in this study. Sequence analysis of LEMD3 and its deduced protein was performed. In addition, SNPs of porcine LEMD3 gene were identified and were found to be associated with ear size, suggesting that the LEMD3 gene might be a good candidate for porcine ear size. This study elucidated the mRNA sequence of LEMD3 and provided the basics for further research on porcine LEMD3 gene.

All experimental animals were treated as the guidelines established by the Animal Care and Use Committee of Beijing, China. All procedures were performed according to protocols approved by the Swine Genetics and Breeding Department of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China. Ear samples were collected from five Large White pigs (age of 60 d) for rapid amplification of cDNA ends (RACE) and frozen immediately in liquid nitrogen for total RNA extraction. Ear samples for SNP identification were collected from four Large White and four Minzhu pigs, which were the parents of the Large White×Minzhu resource population generated by our group (Luo et al. 2012). A total of 302 F2 individuals were used for association analysis. All animals were slaughtered at (240±7) d. Each entire external left ear was removed after slaughtered and profiled on paper along the basis of the edge to be scanned into a digital picture (Ma et al. 2009). Subsequently, ear area was calculated for ear size using Photoshop CS6 (Adobe Systems, San Jose, CA, USA; Snider et al. 2012). The “Magnetic Lasso” tool was used to detect the edge of image to calculate the ear area by assessing the color pixel as well as by using the ruler tool (Min et al. 2005). Ear samples were also collected for genomic DNA extraction.

5.2. RNA and genomic DNA isolation Total RNA was extracted from tissue samples by using Trizol reagent (Chomczynski and Sacchi 2006). Genomic DNA was isolated from the ear samples by using the standard phenol-chloroform extraction procedure (Mullenbach et al. 1989). The concentrations and purities of total RNA and DNA were detected using the spectrophotometry and agarose gel electrophoresis. The RNA and DNA samples were stored at –80°C until analysis.

5.3. Rapid amplification of cDNA ends (RACE) For RACE, the middle fragment of LEMD3 cDNA was first cloned. First-strand cDNA synthesis was performed using 1 μg of total RNA and a PrimeScriptTM RT Reagent Kit (TaKaRa, Japan) according to the manufacturer’s instruction. Primers (Appendix) were designed based on the predicted porcine LEMD3 sequence (GenBank accession no. XM_003126352) and other conserved sequences of known vertebrate LEMD3 mRNAs. PCR was performed in a 50 μL volume, containing 2 μL LEMD3 cDNA, 200 nmol L–1 forward/reverse primers, 250 μmol L–1 dNTPs, 1 U Taq

LIANG Jing et al. Journal of Integrative Agriculture 2016, 15(6): 1321–1329

polymerase, and 25 μL 2× GC buffer I (TaKaRa, Japan). The following PCR cycling parameters were used: 94°C for 5 min, followed by 36 cycles of 94°C for 30 s, appropriate annealing temperature for 30 s, and 72°C for 1 min, with a final extension at 72°C for 10 min. The products were cloned into pMD19-T vector (TaKaRa, Japan) and sequenced by SinoGenoMax Co., Ltd., Beijing, China. For RACE PCR, first-strand LEMD3 cDNA synthesis was performed using a 3´-full RACE Core Set with PrimeScriptTM RTase (TaKaRa, Japan) and a SMARTerTM RACE cDNA Amplification Kit (Clontech, USA) according to the manufacturers’ instructions. Primers were designed based on the identified middle fragment (Appendix). For the 3′-RACE, first-round PCR was performed using a gene-specific primer 3F1, followed by a second nested PCR performed using another gene-specific primer 3F2. For 5´-RACE, the first round of PCR was performed using a gene-specific primer 5R1, followed by a second nested PCR performed using another gene-specific primer 5R2. Products were purified using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit ver. 3.0 (TaKaRa, Japan), and then cloned into pMD19-T vector (TaKaRa, Japan) and sequenced by SinoGenoMax Co., Ltd.

5.4. Sequence analysis of LEMD3 Sequence analysis of the obtained Large White LEMD3 cDNA was performed using NCBI (http://www.ncbi.nlm.nih. gov), Ensemble Blast search program (http://asia.ensembl. org/Multi/blastview), and ExPaSy software (http://www. expasy.org). The ORF and translated amino acids were detected using the NCBI ORF Finder tool (http://www.ncbi.nlm. nih.gov/projects/gorf/.html) (Zhou et al. 2010). Secondary and tertiary structures of the deduced amino acid sequences were predicted using PSIPRED ver. 3.3 (http://bioinf.cs.ucl. ac.uk/psipred/) and SWISS-MODEL (http://swissmodel.expasy.org/). InterProScan sequence search program (http:// www.ebi.ac.uk/interpro/search/sequence-search) was used for the prediction of conserved domains. The transmembrane domain was predicted using TMHMM Server ver. 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/), and signal peptides were predicted using SignalP 4.1 server (http:// www.cbs.dtu.dk/services/SignalP/). TFSEARCH (http:// www.cbrc.jp/research/db/TFSEARCH.html) and miRWalk (www.ma.uni-heidelberg.de/apps/zmf/mirwalk/) were used to predict potential transcription factor binding sites in LEMD3 gene promoter and potential miRNA sites on 3´ UTR, respectively. The following mRNA and amino acid sequences were used for multiple sequence alignment: B. taurus (NM_001192699), H. sapiens (NM_014319), M. mulatta (NM_001258094), R. norvegicus (NM_001191000), and M. musculus (NM_001081193); sequences of proteins: B. taurus

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(NP_001179628.1), H. sapiens (NP_055134.2), M. mulatta (NP_001245023.1), R. norvegicus (NP_001177929.1), M. musculus (NP_001074662.2), D. rerio (AAI63283.1), and X. laevis (NP_001082578.1). A neighbor-joining (NJ) phylogenetic tree was constructed based on the amino acid sequences of different LEMD3 by using Clustal X (2.0) and MEGA 5 software programs (Thompson et al. 1997; Tamura et al. 2007).

5.5. SNP identification and association analysis SNPs were identified by sequencing the PCR products for all exons and the 2 000-bp fragment on the upstream transcription start site. Primers for SNP amplification were designed based on GenBank no. XM_003126352 (Appendix). The PCR amplifications were performed as described above. DNAStar software was used for analyzing the polymorphic sites (Burland et al. 2000). For the 302 F2 individuals, genotype and allele frequencies were calculated for each polymorphism. Then, with these SNPs being integrated into the Illumina Porcine SNP60K Beadchip data, GWAS was re-performed using genome-wide rapid association using mixed model and regression (GRAMMAR) approach applied in our previous study (Liu et al. 2014; Zhang et al. 2014). Parity, batch, and sex were selected as fixed effects for individuals. Body weight and litter effect were considered as covariate and random effect, respectively. The genome-wide significance threshold was calculated to be 2.07E-07 (0.01/48355) (Zhang et al. 2014). Besides, adding each detected SNP as a fixed effect respectively, –log10(P-value) of the most significant SNP on SSC5 was calculated to see its drop level (Li et al. 2012). If –log10(P-value) of the most significant SNP on SSC5 dropped a lot after conditional analysis, LEMD3 is more likely to be in LD with the QTL of porcine ear size.

Acknowledgements This research was supported by the Agricultural Science and Technology Innovation Program, China (ASTIPIAS02), the National Key Technology R&D Program of China (2011BAD28B01), the Earmarked Fund for Modern Agro-Industry Technology Research System of China, and foundation from Chinese Academy of Agricultural Sciences (2014ZL006). Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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