Next Article in Journal
Phenotypic and Genetic Characteristics in a Cohort of Patients with Usher Genes
Next Article in Special Issue
Horizontal Transfer and Evolutionary Profiles of Two Tc1/DD34E Transposons (ZB and SB) in Vertebrates
Previous Article in Journal
Historical Westward Migration Phases of Ovis aries Inferred from the Population Structure and the Phylogeography of Occidental Mediterranean Native Sheep Breeds
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Genes
Volume 13
Issue 8
10.3390/genes13081422
genes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

SINE Insertion May Act as a Repressor to Affect the Expression of Pig LEPROT and Growth Traits

by
Xiaoyan Wang
1,
Chengling Chi
1,
Jia He
1,
Zhanyu Du
1,
Yao Zheng
1,
Enrico D’Alessandro
2,
Cai Chen
1,
Ali Shoaib Moawad
1,3,
Emmanuel Asare
1 and
Chengyi Song
1,*
1
College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China
2
Department of Veterinary Science, Division of Animal Production, University of Messina, 98168 Messina, Italy
3
Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, Kafrelsheikh 33516, Egypt
*
Author to whom correspondence should be addressed.
Genes 2022, 13(8), 1422; https://doi.org/10.3390/genes13081422
Submission received: 8 July 2022 / Revised: 4 August 2022 / Accepted: 5 August 2022 / Published: 10 August 2022
(This article belongs to the Special Issue Co-evolution of Mobilome and Genome)
Browse Figures
Review Reports Versions Notes

Abstract

:
Retrotransposon is an important component of the mammalian genome. Previous studies have shown that the expression of protein-coding genes was affected by the insertion of retrotransposon into the proximal genes, and the phenotype variations would be related to the retrotransposon insertion polymorphisms (RIPs). In this study, leptin (LEP), leptin receptor (LEPR), and leptin receptor overlapping transcript (LEPROT), which play important roles in the regulation of fat synthesis and body weight, were screened to search for the RIPs and their effect on phenotype and gene expression, as well as to further study the function of the insertion. The results showed that three RIPs located in intron 1 of LEPROT and intron 2 and 21 of LEPR were identified, and they were all SINEA1, which was one type of retrotransposon. The SINE insertion at the LEPROT was the dominant allele in native pig breeds. The age of 100 kg body weight of SINE+/+ Large White individuals was significantly higher than those of SINE+/− and SINE−/− individuals (p < 0.05). The LEPROT gene expression in the liver and suet of 30-day-old SINE−/− Sujiang piglets were significantly higher than those of SINE+/+ and SINE+/− piglets (p < 0.01). The dual-luciferase reporter gene assay showed that SINE insertion in PK15 and 3T3-L1 cells significantly reduced the promoter activity of the LEPROT gene (p < 0.01). Therefore, SINE insertion can be a repressor to reduce the expression of LEPROT and could be a useful molecular marker for assisted selection of growth traits in pig breeding.

1. Introduction

LEPTIN acts as a major regulator for food intake, body mass, energy homeostasis, and reproductive functioning [1,2,3]. It is secreted by fat cells and was first identified as the product of the obese (OB) gene and its effect on appetite and metabolic rate [4]. In vivo, LEPTIN must bind to its receptor (LEPTIN receptor, LEPR) to activate the downstream signaling pathway [5]. LEPR is a member of the class 1 cytokine family of receptors and is transcribed in localized areas in the brain and pituitary of the pig [6]. Leptin receptor overlapping transcript (LEPROT), which is also located upstream of LEPR in the pig genome, was first described as the product of an alternate protein using the same promoter of LEPR [7] and renamed Endospanin on account of its late endosomal/Golgi localization and membrane-spanning topology [8]. In mice, LEPROT knockdown showed high leptin sensitivity and was resistant to diet-induced obesity [9], and it was thought to be a candidate for regulating leptin sensitivity [10].
Numerous studies have been conducted on the association between polymorphism of LEPTIN, LEPR, LEPROT, and human obesity [2,11,12,13]. Consequently, the variation of these three genes related to domestic animals’ growth and carcass performance has been a major area of concern. The coding sequence of the LEPROT gene is affected by polymorphic simple sequence repeats (SSRs) with CA units ranging from 18 to 23 in miniature pig breeds compared with Duroc [14]. LEPR polymorphisms (1987C > T) were related to the fatness of Duroc finishing pigs [15]. Furthermore, using genome-wide association studies (GWAS), LEPROT and LEPR were strongly associated with bodyweight measured at 35-day-old broiler [16]. UASMS2 polymorphism in the LEPTIN gene was associated with average daily weight gain, body weight, and backfat thickness in 1378 cattle [17]. LEPROT also served as the candidate genes with biological support for ADG in pigs [18]. However, few studies have been reported about RIPs in these three genes.
Retrotransposons have been proven to be a component of the mammalian genome and have profoundly impacted the structure and function of the genome [19,20]. The insertion polymorphisms were reported to be related to the occurrence of diseases, such as cancer, autoimmunity, and neurodegeneration [21,22,23]. Retrotransposons include two major types: long terminal repeat (LTR) and non-LTR [24].
Endogenous retroviruses (ERV) are one of the superfamilies of mouse and human LTRs. Long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs) are the main order of non-LTR retrotransposons [24]. Many researchers identified retrotransposons as cis-regulatory elements to modulate the expression of proximal or distal target genes [25,26]. Moreover, this may be the primary cause of phenotype variation by RIPs in domestic animals.
Recently, research works from our laboratory have indicated RIPs distribution in the pig genome [27,28]; their role in evaluating population structure [29,30]; and their insertion function on protein-coding genes and pig phenotypic variations such as GH, TLR1, and TLR6 [31,32,33]. Therefore, considering the importance of LEPTIN, LEPR, and LEPROT on mammalian metabolism and growth, the RIPs in these three genes were identified, and the correlation between the RIPs and some pig economic performance was investigated in this study.

2. Materials and Methods

2.1. RIPs Screen and Identified

LEPTIN (ENSSSCG00000040464.2), LEPR (ENSSSCG00000025188.3), and LEPROT (ENSSSCG00000003806.4) and their flanking sequences (5 kb 5′ upstream and 3 kb 3′ downstream) were obtained from Ensembl (version Ensembl Release 101, http://asia.ensembl.org/index.html, accessed on 30 June 2020). As the bait, 3 genes in 15 assembled non-reference genomes (Landrace, Yorkshire, Pietrain, Berkshire, Hampshire, cross-breed of Yorkshire/Landrace/Duroc, Wuzhishan, Tibetan, Rongchang, Meishan, Bamei, Bama, Jinhua, Goettingen, and Ellegaard Gottingen minipigs) were downloaded from the NCBI database (version Annotation release 106, https://ncbi.nlm.nih.gov/, accessed on 1 July 2020) to screen for large structural variations (more than 50 bp) by sequence alignment using the ClustalX program. Retrotransposon insertion including SINE, LINE, and ERV among these predicted structural variations were annotated by RepeatMasker (version RepeatMasker 4.1.0 Released, http://www.repeatmasker.org/, accessed on 3 July 2020) with a customer-constructed library [28,32] and they were designated as RIPs. These RIPs were further validated by PCR amplification (Vazyme, Nanjing, China) in seven Chinese native pig breeds (Erhualian, Fengjing, Diannan small-ear, Wuzhishan, Bama, Tibetan, Meishan), three commercial pig breeds (Duroc, Landrace, Large White), one cross-breed (Sujiang), and wild boars. Two pooled DNA samples including six pigs for each breed were used. Total DNA was extracted from ear or blood tissues of each individual using the TIANamp Genomic DNA Kit (Tiangen, Beijing, China). Each DNA sample, after concentration detection by a spectrophotometer (NanoPhotometer N60 Touch, Implen Gmbh, Germany), was diluted to 50 ng/uL in concentration for pool mixture. Details of RIP evaluation for samples and primers are listed in Tables S1 and S2. All verified RIPs were sequenced at TsingKe Biological Technology Co. Ltd. (Nanjing, China).

2.2. RIP Genotyping

Nine breeds, namely, Duroc, Landrace, Large White, Erhualian, Bama, Jiangquhai, Jinghua, Wuzhishan, and Sujiang, which represented commercial pigs, Chinese native pigs, and cross pigs, were used to examine the RIP distribution. The individuals were selected according to their pedigrees, and all boars and sows without common ancestors were selected preferentially. The number of pigs used for each breed and the breed origins are listed in Table S1. The genotype, the allele frequencies, and Hardy–Weinberg equilibrium were tested using Popgene [34]. Polymorphic information content (PIC) was calculated by the following formula:
P I C = 1 − ∑ i = 1 m P i 2 − ∑ i = 1 m − 1 ∑ j = i + 1 m 2 P i 2 P j 2

2.3. Dual-Luciferase Reporter Assay

The promoter of LEPROT was predicted (version Promoter 2.0, https://services.healthtech.dtu.dk/service.php?Promoter-2.0, accessed on 25 September 2020), and one predicted region of Leprot with (852 bp) and without the SINE insertion allele (566 bp) was cloned from the Sujiang genomic DNA and verified by sequencing. The primers are listed in Table S2. Then, the 852 bp and 566 bp sequences were inserted into the pGL3-basic vectors (Promega, Madison, WI, USA) to construct LEPROTSINE+-Luc+(En) vector and LEPROTSINE—Luc+ (En) vector, which contain β-globin and Oct4 mini promoters, respectively, for enhancer/repressor activity evaluation [32,35]. In 24-well plates, PK15 and 3T3-L1 cells were cultured to approximately 70% confluence and transfected with the luciferase reporter vector using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). After 48 h, luciferase activity was evaluated using the dual-luciferase reporter system (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

2.4. Expression Analysis

Tissue samples including liver, suet, and backfat from four 30-day-old piglets for each genotype (SINE+/+, SINE+/−, and SINE−/−) were prepared. The mRNA was extracted using Trizol (Invitrogen, Shanghai, China), and 1000 ng RNA was reverse-transcribed into cDNAs using a TAKARA Kit (Takara, Tokyo, Japan). Then, quantitative real-time PCR (qPCR) was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems, New York, NY, USA) in a total volume of 20 μL containing SYBR mix (10 μL) (Takara, Tokyo, Japan), primers (4 ng), and cDNA sample (50 ng). Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and measured using the 2− ΔΔCt method. The primer sequences also are shown in Table S1.

2.5. Statistical Analysis

Statistical analyses were performed using the SPSS17.0 software package (SPSS, Chicago, IL, USA) using a one-way analysis of variance with Tukey’s post hoc test, and the data were expressed as mean ± SD. The records of body weight before slaughter, longissimus, and back fat thickness were collected in 450 Large White individuals from the Anhui Antai pig company, and the age at 100 kg body weight was adjusted using the formula recommended by the National Swine Genetic Assessment Scheme. ANOVA was used to analyze the associations between genotype and phenotype of body weight before slaughter, longissimus, and back fat thickness and the age at 100 kg body weight.

3. Results

3.1. Three RIPs Generated by Retrotransposon Insertions in the Pig LEPR and LEPROT Gene

After comparing LEPTIN, LEPR, and LEPROT genes in 1 reference genomes and 15 nonreference genomes using ClustalX, the polymorphisms of 54 structural variations that existed in more than one breed and over 50 bp were predicted and further annotated using RepeatMasker (Version 4.0.7) to find 32 predicted RIPs (Table S3). Three polymorphic RIPs were identified in the DNA pool (Figure 1A) using PCR and gel electrophoresis. The diagrams of SINE insertion in these two genes and comparison among different species are shown in Figure 1B. They were all SINEA1, located in the first intron of LEPROT, and the second and 22 introns of LEPR gene that were 286 bp, 301 bp, and 321 bp, respectively (Table 1). They were assigned as LEPROT-SINE-RIP, LEPR-SINE-RIP1, and LEPR-SINE-RIP2.

3.2. Three RIP Distributions in Different Pig Breeds

Five Chinese fat-type pig breeds (Jiangquhai, Jinhua, Rongchang, Erhualian, and Bama), three commercial lean-type breeds (Large White, Duroc, and Landrace), and one dual-type breed (Sujiang pigs originated from hybrids of Duroc, Jiangquhai, and Fengjing) were selected to investigate the insertion allele frequency. The Hardy–Weinberg test and PIC value were also performed. According to Table 2, there was a difference between fat-type pig breeds and lean-type breeds for LEPROT-SINE-RIP. Insertion frequency was clearly low in Duroc and Landrace compared with in Jiangquhai, Jinghua, and Rongchang breeds. Sujiang pigs showed a similar tendency of allele frequency to commercial breeds. Three Chinese fat-type breeds were all in disequilibrium. For LEPR-SINE-RIP1, insertion frequency and the individual homologous insertion in all breeds were relatively low. SINE insertion was only found in Large White, Duroc, Sujiang, Jiangquhai, and Jinghua, and homologous insertion individuals only were found in Large White and Jinhua pigs. The insertion frequency of LEPR-SINE-RIP1 was low in Chinese native pigs compared with commercial pigs and cross pigs. There was no insertion homologous except Wuzhishan, and no obvious difference among Chinese native pigs and commercial pigs for LEPR-SINE-RIP2. PIC value indicated that all populations were low to moderately polymorphic.

3.3. Correlation of LEPROT-SINE-RIP with Growth Performance of Large White

Because of the distribution difference between Chinese native breeds and commercial breeds, the correlation of LEPROT-SINE-RIP and LEPR-SINE-RIP1 with the growth performance of Large White pigs was investigated (Table 3). The ages at 100 kg body weight of SINE insertion homologous were higher (p < 0.05) than heterozygote and homologous without SINE insertion, which indicated that SINE insertion homologous may take more 4 days to reach 100 kg bodyweight. There was no difference (p > 0.05) among the three genotypes of LEPR-SINE-RIP1 in the Large White population for other economic traits.

3.4. Expression Pattern of LEPROT in Tissues of 30-Day-Old Piglets

The LEPROT expression pattern was further investigated in the liver, suet, and back fat of 30-day-old piglets. As shown in Figure 2, the expression of SINE−/−genotype individuals was higher (p < 0.01) than those of SINE+/+ genotype individuals in the liver and suet, which indicated that SINE insertion may decrease the expression of LEPROT in these tissues.

3.5. SINE Insertion May Serve as a Repressor in Regulating the Expression of LEPROT

There was one predicted promoter that was 289 bp away from SINE insertion. One Duroc heterozygote was taken as a DNA template. The 878 bp sequences that contained predictor promoter and SINE insertion, and the 592 bp sequences that contained only the predicted promoter were cloned by PCR. These two fragments were inserted into the pGL3 vector. The schematic diagram of vectors is shown in Figure 3A. After sequencing and dual restriction enzyme digesting, the right vectors were named LEPROTSINE+-Luc+(EN) and LEPROTSINE--Luc+(EN). PK15 and 3T3 cells were transfected with these two vectors and the pGL3 control vector. The luciferase activity assay revealed that the SINE insertion significantly suppressed LEPROT gene promoter activities in PK15 and 3T3-L1 (p < 0.05) (Figure 3B). These data supported the SINE insertion that it may act as a repressor and could be involved in regulating LEPROT.

4. Discussion

In this study, two SINE insertion polymorphisms in LEPR and one in LEPROT were identified by PCR, since 32 RIPs were predicted by comparing these three genes (LEPTIN, LEPR, and LEPROT). According to the classification by Chen et al. (2019), these three SINE insertions belonged to the young retrotransposons subfamily. There was no identified RIP by PCR in the LEPTIN gene. LEPTIN acts as a multifunctional hormone cytokine and is involved in several cellular functions throughout the body. Through binding to LEPR, LEPTIN interacts with various signal transduction pathways such as JAK-STAT3 [36], PI3K/Akt [37], and MAPK [38], etc. Perhaps, because of Leptin’s significant role in metabolism and other biological processes, retrotransposon is limited not to propagating in this gene according to natural selection. Similarly, our previous study by Chen et al. (2021) on the growth hormone gene (GH gene), which encodes a pleiotropic hormone with important roles in promoting growth, anabolic actions, and body maintenance, had no RIPs identified.
Three RIPs’ distributions in different pig breeds, including Chinese native breeds and commercial pigs, were investigated. Insertion frequency was different between two types of pig and dominant for LEPROT-SINE-RIP in Chinese native breeds and LEPR-SINE-RIP in commercial pigs. Several studies have indicated that compared to commercial pigs, native pigs showed a slower growth rate, thicker backfat, and low lean percentage [39,40,41,42,43]. Natural selection and genetic drift are also powerful forces shaping the distribution and accumulation of retrotransposons [44]. Moreover, retrotransposon insertion has contributed to a selective advantage [45]. During the breeding of commercial pigs such as Large White and Duroc, intense selective pressure on lean percentage may change the insertion frequencies of RIPs in important protein coding genes related to myogenesis and adipogenesis, which may contribute to difference in RIP distribution among native pigs and commercial pigs. In a previous study, the difference in RIP distribution among Chinese native pigs and commercial pigs were identified [30]. Considering that the variation in LEPROT and LEPR was related to growth and fat deposition, the correlation between these two RIPs and some growth and carcass traits of Large White were further identified. The age of 100 kg body weight of SINE+/+ individuals was significantly higher than that of SINE+/− and SINE−/− individuals (p < 0.05), which indicated the SINE insertion homologous grew slower than the other genotypic individuals for LEPROT-SINE-RIP. There was no significant difference in backfat thickness among the three genotype individuals. Therefore, LEPROT-SINE-RIP could serve as a useful marker in selecting faster-growing pigs.
LEPROT, also named Endospanin, is a candidate for regulating LEPTIN sensitivity [10]. The expression of LEPROT was negatively correlated with LEPTIN receptors and LEPTIN sensitivity [46]. Schwartz et al. (1996) indicated that higher serum and plasma LEPTIN levels contributed to a higher total body fat percentage. Conversely, fat type pigs express higher levels of LEPIN mRNA and protein than lean-type pigs at similar body weight [47]. The amounts of leptin at plasma were higher in Meishan than Large White sows [48]. Diet-induced adiposity in genetically lean pigs also increases the leptin concentration in plasma [49]. However, the expression of LEPROT in the liver and suet of SINE−/− individuals in the present study were higher in subjects with relatively lower LEPTIN levels. This could have been a result of the deletion frequency being dominant for LEPROT-SINE-RIP in commercial breeds, which had a lower percentage of total body fat.
At last, the SINE insertion activity on the promoter of LEPROT was analyzed. Whether in PK15 or 3T3-L1, SINE insertion repressed the activity of LEPROT promoter. As a cis-regulatory element, SINE may regulate gene expression, acting as an enhancer or promoter [50,51]. The sequences provide abundant TF binding sites such as CTCF and ADNP [51]. When they escape silencing, they could be bound by multiple transcription factors (TFs) to acquire features of the bona fide enhancer or promoter elements and in turn exert a functional impact on gene regulation [52]. However, retrotransposon could be silenced by epigenetic regulation to ensure genomic integrity, leading to the repression of nearby genes. This might be due to the spreading of epigenetic marks and certain genes maintained in a lowly expressed state in specific development [53]. It is contradictory to understand the function of enhancers or repressors acted upon by retrotransposon. Moreover, their function may relate to specific cell fate, which could be shown in that different luciferase activities in different cell lines used the pGL3 report vector with the same SINE fragment in this study and our previous study. Considering that the expression of LEPROT in liver and suet of SINE−/− individuals were higher than that of the other two genotypes, SINE insertion in the first intron of LEPROT could have acted as a repressor to decrease the expression of LERROT and affect the growth traits of Large White pigs.

5. Conclusions

In summary, three RIPs were identified in intron 1 of LEPROT and intron 2 and 21 of LEPR by gene sequence alignment, retrotransposon annotation, and PCR verification. The distribution of LEPROT-SINE-RIP was relatively high in Chinese native pigs compared with commercial pigs. SINE insertion homologous may take more 4 days to reach 100 kg bodyweight than other two genotype individuals. Additionally, the LEPROT gene expression in the liver and suet of 30-day SINE−/− Sujiang piglets were significantly higher than those of SINE+/+ and SINE+/− piglets (p < 0.01). SINE insertion in PK15 and 3T3-L1 cells significantly reduced the promoter activity of the LEPROT gene (p < 0.01) using dual-luciferase reporter gene assay. Therefore, SINE insertion can be a repressor to reduce the expression of LEPROT and could be a useful molecular marker for assisted selection of growth traits in pig breeding.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes13081422/s1, Table S1: S1 primers used for identification of RIPs, expression, and vector construction. Table S2: Number and origin of pig breeds for RIP identification and distribution. Table S3: Predicted structural variations of the porcine LEPTIN, LEPROT, and LEPR genes using Clustal X and RepeatMasker.

Author Contributions

Data curation, C.C. (Chengling Chi), Z.D., Y.Z. and A.S.M.; formal analysis, X.W.; funding acquisition, C.C. (Cai Chen) and C.S.; methodology, C.C. (Chengling Chi) and J.H.; writing—original draft, X.W.; writing—review and editing, E.D. and E.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (grant numbers 31872977 to Chengyi Song and 32002146 to Cai Chen); the Independent Innovation Fund Project of Agricultural Science and Technology in Jiangsu Province (CX(19)2016) to Chengyi Song; the Revitalization of Seed Industry (JBGS) in Jiangsu Province (JBGS(2021)028) to Chengyi Song; the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the High-End Talent Support Program of Yangzhou University to Chengyi Song.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Animal Experiment Ethics Committee of Yangzhou University (approval number: YZUDWSY2018-12).

Informed Consent Statement

Not applicable.

Data Availability Statement

All data needed to evaluate the conclusions in this paper are present either in the main text or the Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Zhang, F.; Chen, Y.; Heiman, M.; Dimarchi, R. Leptin: Structure, function and biology. Vitam. Horm. 2005, 71, 345–372. [Google Scholar] [PubMed]
  2. Obradovic, M.; Sudar-Milovanovic, E.; Soskic, S.; Essack, M.; Arya, S.; Stewart, A.J.; Gojobori, T.; Isenovic, E.R. Leptin and Obesity: Role and Clinical Implication. Front. Endocrinol. (Lausanne) 2021, 12, 585887. [Google Scholar] [CrossRef] [PubMed]
  3. Munzberg, H.; Singh, P.; Heymsfield, S.B.; Yu, S.; Morrison, C.D. Recent advances in understanding the role of leptin in energy homeostasis. F1000Research 2020, 9, 451. [Google Scholar] [CrossRef] [PubMed]
  4. Zhang, Y.; Proenca, R.; Maffei, M.; Barone, M.; Leopold, L.; Friedman, J.M. Positional cloning of the mouse obese gene and its human homologue. Nature 1994, 372, 425–432. [Google Scholar] [CrossRef] [PubMed]
  5. Zhao, S.G.; Kusminski, C.M.; Elmquist, J.K.; Scherer, P.E. Leptin: Less Is More. Diabetes 2020, 69, 823–829. [Google Scholar] [CrossRef]
  6. Barb, C.R.; Hausman, G.J.; Houseknecht, K.L. Biology of leptin in the pig. Domest. Anim. Endocrinol. 2001, 21, 297–317. [Google Scholar] [CrossRef]
  7. Bailleul, B.; Akerblom, I.; Strosberg, A.D. The leptin receptor promoter controls expression of a second distinct protein. Nucleic Acids Res. 1997, 25, 2752–2758. [Google Scholar] [CrossRef] [Green Version]
  8. Seron, K.; Couturier, C.; Belouzard, S.; Bacart, J.; Monte, D.; Corset, L.; Bocquet, O.; Dam, J.; Vauthier, V.; Lecoeur, C.; et al. Endospanins Regulate a Postinternalization Step of the Leptin Receptor Endocytic Pathway. J. Biol. Chem. 2011, 286, 17968–17981. [Google Scholar] [CrossRef] [Green Version]
  9. Couturier, C.; Sarkis, C.; Seron, K.; Belouzard, S.; Chen, P.; Lenain, A.; Corset, L.; Dam, J.; Vauthier, V.; Dubartt, A.; et al. Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity. Proc. Natl. Acad. Sci. USA 2007, 104, 19476–19481. [Google Scholar] [CrossRef] [Green Version]
  10. Londraville, R.L.; Tuttle, M.; Liu, Q.; Andronowski, J.M. Endospanin Is a Candidate for Regulating Leptin Sensitivity. Front. Physiol. 2022, 12, 2307. [Google Scholar] [CrossRef]
  11. Ghalandari, H.; Hosseini-Esfahani, F.; Mirmiran, P. The Association of Polymorphisms in Leptin/Leptin Receptor Genes and Ghrelin/Ghrelin Receptor Genes with Overweight/Obesity and the Related Metabolic Disturbances: A Review. Int. J. Endocrinol. Metab. 2015, 13, e19073. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Gasmi, A.; Mujawdiya, P.K.; Noor, S.; Piscopo, S.; Menzel, A. Lifestyle Genetics-Based Reports in the Treatment of Obesity. Arch. Razi Inst. 2021, 76, 707–719. [Google Scholar] [PubMed]
  13. Couto Alves, A.; De Silva, N.M.G.; Karhunen, V.; Sovio, U.; Das, S.; Taal, H.R.; Warrington, N.M.; Lewin, A.M.; Kaakinen, M.; Cousminer, D.L.; et al. Early Growth Genetics, Consortium. GWAS on longitudinal growth traits reveals different genetic factors influencing infant, child, and adult BMI. Sci. Adv. 2019, 5, eaaw3095. [Google Scholar] [CrossRef] [Green Version]
  14. Wang, H.Y.; Fu, Y.; Gu, P.; Zhang, Y.Y.; Tu, W.L.; Chao, Z.; Wu, H.L.; Cao, J.; Zhou, X.; Liu, B.; et al. Genome-Wide Characterization and Comparative Analyses of Simple Sequence Repeats among Four Miniature Pig Breeds. Animals 2020, 10, 1792. [Google Scholar] [CrossRef]
  15. Henriquez-Rodriguez, E.; Bosch, L.; Tor, M.; Pena, R.N.; Estany, J. The effect of SCD and LEPR genetic polymorphisms on fat content and composition is maintained throughout fattening in Duroc pigs. Meat Sci. 2016, 121, 33–39. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Dadousis, C.; Somavilla, A.; Ilska, J.J.; Johnsson, M.; Batista, L.; Mellanby, R.J.; Headon, D.; Gottardo, P.; Whalen, A.; Wilson, D.; et al. A genome-wide association analysis for body weight at 35 days measured on 137,343 broiler chickens. Genet. Sel. Evol. 2021, 53, 1–14. [Google Scholar] [CrossRef]
  17. Wang, L.Y.; Raza, S.H.A.; Gui, L.S.; Li, S.J.; Liu, X.C.; Yang, X.R.; Wang, S.H.; Zan, L.S.; Zhao, C.P. Associations between UASMS2 polymorphism in leptin gene and growth, carcass and meat quality traits of cattle: A meta-analysis. Anim. Biotechnol. 2020, 33, 279–288. [Google Scholar] [CrossRef]
  18. Cai, Z.; Christensen, O.F.; Lund, M.S.; Ostersen, T.; Sahana, G. Large-scale association study on daily weight gain in pigs reveals overlap of genetic factors for growth in humans. BMC Genom. 2022, 23, 133. [Google Scholar] [CrossRef]
  19. Richardson, S.R.; Doucet, A.J.; Kopera, H.C.; Moldovan, J.B.; Garcia-Perez, J.L.; Moran, J.V. The Influence of LINE-1 and SINE Retrotransposons on Mammalian Genomes. Microbiol. Spectr. 2015, 3, MDNA3-0061-2014. [Google Scholar] [CrossRef] [Green Version]
  20. Platt, R.N.; Vandewege, M.W.; Ray, D.A. Mammalian transposable elements and their impacts on genome evolution. Chromosome Res. 2018, 26, 25–43. [Google Scholar] [CrossRef] [Green Version]
  21. Burns, K.H. Our Conflict with Transposable Elements and Its Implications for Human Disease. Annu. Rev. Pathol.-Mech. 2020, 15, 51–70. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Proukakis, C. Somatic mutations in neurodegeneration: An update. Neurobiol. Dis. 2020, 144, 105021. [Google Scholar] [CrossRef] [PubMed]
  23. Chenais, B. Transposable Elements and Human Diseases: Mechanisms and Implication in the Response to Environmental Pollutants. Int. J. Mol. Sci. 2022, 23, 2551. [Google Scholar] [CrossRef]
  24. Wicker, T.; Sabot, F.; Hua-Van, A.; Bennetzen, J.L.; Capy, P.; Chalhoub, B.; Flavell, A.; Leroy, P.; Morgante, M.; Panaud, O.; et al. A unified classification system for eukaryotic transposable elements. Nat. Rev. Genet. 2007, 8, 973–982. [Google Scholar] [CrossRef] [PubMed]
  25. Chuong, E.B.; Elde, N.C.; Feschotte, C. Regulatory activities of transposable elements: From conflicts to benefits. Nat. Rev. Genet. 2017, 18, 71–86. [Google Scholar] [CrossRef] [Green Version]
  26. Mangiavacchi, A.; Liu, P.; Della Valle, F.; Orlando, V. New insights into the functional role of retrotransposon dynamics in mammalian somatic cells. Cell Mol. Life Sci. 2021, 78, 5245–5256. [Google Scholar] [CrossRef]
  27. Chen, C.; D’alessandro, E.; Murani, E.; Zheng, Y.; Giosa, D.; Yang, N.S.; Wang, X.Y.; Gao, B.; Li, K.; Wimmers, K.; et al. SINE jumping contributes to large-scale polymorphisms in the pig genomes. Mob. DNA 2021, 12, 17. [Google Scholar] [CrossRef]
  28. Chen, C.; Wang, W.; Wang, X.; Shen, D.; Wang, S.; Wang, Y.; Gao, B.; Wimmers, K.; Mao, J.; Li, K.; et al. Retrotransposons evolution and impact on lncRNA and protein coding genes in pigs. Mob. DNA 2019, 10, 19. [Google Scholar] [CrossRef]
  29. Chen, C.; Wang, X.Y.; Zong, W.C.; D’alessandro, E.; Giosa, D.; Guo, Y.F.; Mao, J.D.; Song, C.Y. Genetic Diversity and Population Structures in Chinese Miniature Pigs Revealed by SINE Retrotransposon Insertion Polymorphisms, a New Type of Genetic Markers. Animals 2021, 11, 1136. [Google Scholar] [CrossRef]
  30. Wang, X.; D’alessandro, E.; Chi, C.; Moawad, A.S.; Zong, W.; Chen, C.; Song, C. Genetic Evaluation and Population Structure of Jiangsu Native Pigs in China Revealed by SINE Insertion Polymorphisms. Animals 2022, 12, 1345. [Google Scholar] [CrossRef]
  31. Wang, W.; Chen, C.; Wang, X.; Zhang, L.; Shen, D.; Wang, S.; Gao, B.; Mao, J.; Song, C. Development of Molecular Markers Based on the L1 Retrotransposon Insertion Polymorphisms in Pigs (Sus scrofa) and Their Association with Economic Traits. Russ. J. Genet. 2020, 56, 183–191. [Google Scholar] [CrossRef]
  32. Wang, X.Y.; Chen, Z.X.; Murani, E.; D’alessandro, E.; An, Y.L.; Chen, C.; Li, K.; Galeano, G.; Wimmers, K.; Song, C.Y. A 192 bp ERV fragment insertion in the first intron of porcine TLR6 may act as an enhancer associated with the increased expressions of TLR6 and TLR1. Mob. DNA 2021, 12, 20. [Google Scholar] [CrossRef] [PubMed]
  33. Chen, C.; Zheng, Y.; Wang, M.L.; Murani, E.; D’alessandro, E.; Moawad, A.S.; Wang, X.Y.; Wimmers, K.; Song, C.Y. SINE Insertion in the Intron of Pig GHR May Decrease Its Expression by Acting as a Repressor. Animals 2021, 11, 1871. [Google Scholar] [CrossRef]
  34. Yeh, F.C.; Yang, R.C.; Boyle, T. POPGENE version 1.32: Microsoft Windows– Based Freeware for Population Genetic Analysis, Quick User Guide. Master’s Thesis, Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton, AB, Canada, 1999. [Google Scholar]
  35. Chan, S.; Shen, D.; Sang, Y.; Wang, S.; Wang, Y.; Chen, C.; Gao, B.; Song, C. Development of enhancer-trapping and -detection vectors mediated by the Tol2 transposon in zebrafish. PeerJ 2019, 7, e6862. [Google Scholar] [CrossRef]
  36. Triantafyllou, G.A.; Paschou, S.A.; Mantzoros, C.S. Leptin and Hormones: Energy Homeostasis. Endocrinol. Metab. Clin. N. Am. 2016, 45, 633–645. [Google Scholar] [CrossRef]
  37. Macedo, T.J.S.; Menezes, V.G.; Barberino, R.S.; Silva, R.L.S.; Gouveia, B.B.; Monte, A.P.O.; Lins, T.L.B.G.; Santos, J.M.S.; Bezerra, M.E.S.; Wischral, A.; et al. Leptin decreases apoptosis and promotes the activation of primordial follicles through the phosphatidylinositol-3-kinase/protein kinase B pathway in cultured ovine ovarian tissue. Zygote 2021, 29, 445–451. [Google Scholar] [CrossRef]
  38. Han, Y.C.; Ma, B.; Guo, S.; Yang, M.; Li, L.J.; Wang, S.J.; Tan, J. Leptin regulates disc cartilage endplate degeneration and ossification through activation of the MAPK-ERK signalling pathway in vivo and in vitro. J. Cell Mol. Med. 2018, 22, 2098–2109. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Lan, Y.H.; Mckeith, F.K.; Novakofski, J.; Carr, T.R. Carcass and muscle characteristics of Yorkshire, Meishan, Yorkshire × Meishan, Meishan × Yorkshire, Fengjing × Yorkshire, and Minzhu × Yorkshire pigs. J. Anim. Sci. 1993, 71, 3344–3349. [Google Scholar] [CrossRef]
  40. Song, B.; Zheng, C.; Zheng, J.; Zhang, S.; Zhong, Y.; Guo, Q.; Li, F.; Long, C.; Xu, K.; Duan, Y.; et al. Comparisons of carcass traits, meat quality, and serum metabolome between Shaziling and Yorkshire pigs. Anim. Nutr. 2022, 8, 125–134. [Google Scholar] [CrossRef]
  41. Liu, J.; Nong, Q.; Wang, J.; Chen, W.; Xu, Z.; You, W.; Xie, J.; Wang, Y.; Shan, T. Breed difference and regulatory role of CRTC3 in porcine intramuscular adipocyte. Anim. Genet. 2020, 51, 521–530. [Google Scholar] [CrossRef]
  42. Lei, L.; Wang, Z.; Li, J.; Yang, H.; Yin, Y.; Tan, B.; Chen, J. Comparative Microbial Profiles of Colonic Digesta between Ningxiang Pig and Large White Pig. Animals 2021, 11, 1862. [Google Scholar] [CrossRef]
  43. Poklukar, K.; Candek-Potokar, M.; Batorek Lukac, N.; Tomazin, U.; Skrlep, M. Lipid Deposition and Metabolism in Local and Modern Pig Breeds: A Review. Animals 2020, 10, 424. [Google Scholar] [CrossRef] [Green Version]
  44. Bourque, G.; Burns, K.H.; Gehring, M.; Gorbunova, V.; Seluanov, A.; Hammell, M.; Imbeault, M.; Izsvak, Z.; Levin, H.L.; Macfarlan, T.S.; et al. Ten things you should know about transposable elements. Genome Biol. 2018, 19, 199. [Google Scholar] [CrossRef] [PubMed]
  45. Lanciano, S.; Mirouze, M. Transposable elements: All mobile, all different, some stress responsive, some adaptive? Curr. Opin. Genet. Dev. 2018, 49, 106–114. [Google Scholar] [CrossRef]
  46. Roujeau, C.; Jockers, R.; Dam, J. Endospanin 1 Determines the Balance of Leptin-Regulated Hypothalamic Functions. Neuroendocrinology 2019, 108, 132–141. [Google Scholar] [CrossRef]
  47. Ramsay, T.G.; Yan, X.; Morrison, C. The obesity gene in swine: Sequence and expression of porcine leptin. J. Anim. Sci. 1998, 76, 484–490. [Google Scholar] [CrossRef]
  48. Barbe, A.; Kurowska, P.; Mlyczynska, E.; Rame, C.; Staub, C.; Venturi, E.; Billon, Y.; Rak, A.; Dupont, J. Adipokines expression profiles in both plasma and peri renal adipose tissue in Large White and Meishan sows: A possible involvement in the fattening and the onset of puberty. Gen. Comp. Endocrinol. 2020, 299, 113584. [Google Scholar] [CrossRef]
  49. Yang, X.F.; Qiu, Y.Q.; Wang, L.; Gao, K.G.; Jiang, Z.Y. A high-fat diet increases body fat mass and up-regulates expression of genes related to adipogenesis and inflammation in a genetically lean pig. J. Zhejiang Univ. Sci. B 2018, 19, 884–894. [Google Scholar] [CrossRef]
  50. Nakanishi, A.; Kobayashi, N.; Suzuki-Hirano, A.; Nishihara, H.; Sasaki, T.; Hirakawa, M.; Sumiyama, K.; Shimogori, T.; Okada, N. A SINE-derived element constitutes a unique modular enhancer for mammalian diencephalic Fgf8. PLoS ONE 2012, 7, e43785. [Google Scholar] [CrossRef] [Green Version]
  51. Ichiyanagi, T.; Katoh, H.; Mori, Y.; Hirafuku, K.; Boyboy, B.A.; Kawase, M.; Ichiyanagi, K. B2 SINE Copies Serve as a Transposable Boundary of DNA Methylation and histone Modifications in the Mouse. Mol. Biol. Evol. 2021, 38, 2380–2395. [Google Scholar] [CrossRef]
  52. Sundaram, V.; Wysocka, J. Transposable elements as a potent source of diverse cis-regulatory sequences in mammalian genomes. Philos Trans. R Soc. Lond. B Biol. Sci. 2020, 375, 20190347. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Robbez-Masson, L.; Rowe, H.M. Retrotransposons shape species-specific embryonic stem cell gene expression. Retrovirology 2015, 12, 45. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Genes 13 01422 g001 550
Figure 1. RIP identification and insertion loci diagram in LEPROT and LEPR. (A) The electrophoretogram of PCR production of three loci in LEPROT and LEPR genes in 12 breeds or population DNA pool. (B) Insertion loci diagram and sequence alignment for different species in LEPROT and LEPR.
Figure 1. RIP identification and insertion loci diagram in LEPROT and LEPR. (A) The electrophoretogram of PCR production of three loci in LEPROT and LEPR genes in 12 breeds or population DNA pool. (B) Insertion loci diagram and sequence alignment for different species in LEPROT and LEPR.
Genes 13 01422 g001
Genes 13 01422 g002 550
Figure 2. Relative expression of LEPROT gene in tissues of 30-day-old Sujiang piglets. * showed p < 0.05; ** showed p < 0.01.
Figure 2. Relative expression of LEPROT gene in tissues of 30-day-old Sujiang piglets. * showed p < 0.05; ** showed p < 0.01.
Genes 13 01422 g002
Genes 13 01422 g003 550
Figure 3. SINE insertion served as a repressor to affect the promoter of LEPROT. (A) Schematic diagram of the recombinant vector structure. (B) The results of promoter activity verification. * showed p < 0.05; ** showed p < 0.01.
Figure 3. SINE insertion served as a repressor to affect the promoter of LEPROT. (A) Schematic diagram of the recombinant vector structure. (B) The results of promoter activity verification. * showed p < 0.05; ** showed p < 0.01.
Genes 13 01422 g003
Table
Table 1. The details of three RIPs in LEPROT and LEPR genes.
Table 1. The details of three RIPs in LEPROT and LEPR genes.
LociInsertion in Exon/Intron/UTRLength (bp)DirectionTypeChromosome (Sus Scrofa11.1)
LEPROT-SINE-RIPIntron1286-SINEA16:146978072-146978073
LEPR-SINE-RIP1Intron2301+SINEA16:146873751-146874052
LEPR-SINE-RIP2Intron21321+SINEA16:146808226-146808547
Table
Table 2. Genotype and allele frequency of three RIPs in the RIP polymorphic breeds.
Table 2. Genotype and allele frequency of three RIPs in the RIP polymorphic breeds.
Polymorphic SiteBreedsNumberGenotype FrequencyAllele FrequencyHardy–Weinberg EquilibriumPolymorphic Information Content
+/++/−−/−+−
LEPROT-SINE-RIPLarge White45014.0066.0020.0047.0053.00<0.010.374
Duroc2412.5066.6720.8345.8354.170.090.373
Landrace24037.5062.5018.7581.250.260.258
Sujiang2429.1737.5033.3347.9252.080.220.375
Jiangquhai2462.5020.8316.6772.9227.080.020.317
Jinhua2475.00025.0075.0025.00<0.010.305
Rongchang2429.1770.83064.5835.420.010.353
LEPR-SINE-RIP1Large White4294.9033.5761.5421.6878.320.810.282
Duroc24045.8354.1722.9277.080.150.291
Sujiang24062.5037.5031.2558.750.030.490
Jiangquhai18016.6783.338.3391.670.660.141
Jinhua244.1729.1766.6718.7581.250.830.258
LEPR-SINE-RIP2Large White429062.7037.3031.3568.65<0.010.338
Duroc24075.0025.0037.5062.500.0030.359
Landrace24095.834.1747.9252.08<0.010.375
Sujiang24066.6733.3333.3366.670.010.346
Bama24087.5012.5043.7556.250.00010.371
Jiangquhai24033.3366.6716.6783.330.330.239
Erhualian2408.3391.674.1795.830.830.077
Jinhua24037.5062.5018.7581.250.260.258
Wuzhishan2425.0058.3316.6754.1745.830.390.373
Table
Table 3. Correlation between LEPROT-SINE-RIP polymorphism and growth traits of Large White pigs.
Table 3. Correlation between LEPROT-SINE-RIP polymorphism and growth traits of Large White pigs.
GenotypeNumberBody Weight before SlaughterThickness of BackfatAge at 100 kg Body Weight100 kg Day-Old Backfat Thickness
SINE+/+6399.23 ± 1.26 A11.17 ± 0.37164.81 ± 1.12 a11.20 ± 0.32
SINE+/−297105.10 ± 0.66 B11.53 ± 0.18160.49 ± 0.59 B11.02 ± 0.15
SINE−/−90104.04 ± 1.20 B11.64 ± 0.34160.59 ± 1.08 b11.22 ± 0.30
Note: Different superscript lowercase letters indicated difference between groups (p < 0.05). Different superscript capital letters indicated significant difference between groups (p < 0.01).
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Wang, X.; Chi, C.; He, J.; Du, Z.; Zheng, Y.; D’Alessandro, E.; Chen, C.; Moawad, A.S.; Asare, E.; Song, C. SINE Insertion May Act as a Repressor to Affect the Expression of Pig LEPROT and Growth Traits. Genes 2022, 13, 1422. https://doi.org/10.3390/genes13081422

AMA Style

Wang X, Chi C, He J, Du Z, Zheng Y, D’Alessandro E, Chen C, Moawad AS, Asare E, Song C. SINE Insertion May Act as a Repressor to Affect the Expression of Pig LEPROT and Growth Traits. Genes. 2022; 13(8):1422. https://doi.org/10.3390/genes13081422

Chicago/Turabian Style

Wang, Xiaoyan, Chengling Chi, Jia He, Zhanyu Du, Yao Zheng, Enrico D’Alessandro, Cai Chen, Ali Shoaib Moawad, Emmanuel Asare, and Chengyi Song. 2022. "SINE Insertion May Act as a Repressor to Affect the Expression of Pig LEPROT and Growth Traits" Genes 13, no. 8: 1422. https://doi.org/10.3390/genes13081422

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop