Expression patterns and DNA methylation profile of GTL2 gene in goats

Abstract Gene trap locus 2 (GTL2), a long non-coding paternal imprinting gene, participates in various biological processes, including cell proliferation, differentiation, and apoptosis, by regulating the transcription of target mRNA, which is tightly related to the growth of the organic and maintenance of function. In this study, DNA methylation patterns of CpG islands (CGI) of GTL2 were explored, and its expression level was quantified in six tissues, rumen epithelium cells, and skeletal muscle cells in goats. GTL2 expression levels were measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and the methylation model was confirmed by bisulfite-sequencing PCR (BSP). CGI methylation of GTL2 indicated a moderate methylation (ranging from 81.42 to 86.83%) in the brain, heart, liver, kidney, lung, and longissimus dorsi. GTL2 is most highly expressed in brain tissues, but there is no significant difference in the other five tissues. In addition, in the rumen epithelium cell proliferation, GTL2 expression was highest at 60 h, followed by 72 h, and almost unchanged at 12–48 h. In the skeletal muscle cell differentiation, GTL2 expression was highest at 0 and 24 h, significantly decreasing at 72 and 128 h. Pearson correlation analysis did not indicate a clear relationship between methylation and GTL2 expression levels, suggesting that other regulatory factors may modulate GTL2 expression. This study will provide a better understanding of the expression regulation mechanism of genes in the delta-like homolog 1 gene (DLK1)-GTL2 domain.


Introduction
The specific spatiotemporal expression of imprinted genes plays an essential role in the growth of the embryo development of the individual nervous system after birth, the maintenance of normal metabolism, 1 and the transport of nutrients between the fetuses and their mothers. 2Gene trap locus 2 (GTL2) is a maternally imprinted gene located in the delta-like homolog 1 gene (DLK1)-GTL2 imprinted domain, which is closely related to fetal growth as well as postnatal development and metabolism. 3GTL2, a long non-coding RNA (lncRNA), regulates the transcription of target mRNA by encoding an untranslated RNA.Its transcription product is a small non-coding RNA, interacting with important pathway proteins, such as cAMP and P53.Interactions occur, thereby affecting the proliferation of cells, the development of the body, and the maintenance of functions. 4LncRNAs can regulate gene expression through transcriptional and post-transcriptional regulation at different levels, thereby participating in various biological processes like cell proliferation, differentiation, and apoptosis. 5,6NA methylation usually occurs on the cytosine of mammalian CpG dinucleotides.DNA methylation is involved in various cellular physiological processes, including cell differentiation, genome stability, and X chromosome inactivation, [7][8][9] and dynamically regulates the expression of specific genes during individual development. 10Studies have shown that DNA methylation inhibits the expression of specific genes. 11The expression of imprinted genes is controlled by sexspecific DNA methylation markers in reproductive cells. 12The imprinted gene GTL2 is typically expressed by the maternal allele and regulated by intergene germline-derived DMR (IG-DMR) designated by paternal methylated DMR. 13 Studies have shown that the expression level of GTL2 is regulated by DNA methylation level during skeletal muscle development in sheep. 14Current studies have explored the potential relationship between gene expression and methylation levels in promoter regions with CpG islands, but studies have yet to be conducted on CpG islands within genes.
Currently, the research on imprinted genes mainly focuses on humans, 15 mice, 15 pigs, 16 cattle, 17 goats, 18 and other species.Nevertheless, there is a rare report on the regulatory mechanism of GTL2 expression in goats.This study focused on the methylation level of CpG islands and the expression level of GTL2 in Jianzhou Da'er goats and analyzed their relationships.The purpose is to explore whether the expression of GTL2 is affected by other imprinting regulatory elements and enrich and refine the expression and imprint regulation mechanism of GTL2 in the imprinted domain of DLK1-GTL2.

Animal management and sample information
The Jianzhou Da'er goats were provided by the Jianyang Dageda Farm (Chengdu, Sichuan, China).All goats were kept in indoor pens with slots and raised on a regular diet (concentrate ratio to forage, 35:65) twice per day at 07:00-08:00 and 17:00-18:00.Three 2-month-old ewes were randomly selected to collect the six tissue samples, including brain, heart, lung, liver, kidney, and longissimus dorsi.The tissues were immediately frozen in liquid nitrogen and stored at À80 C before RNA isolation.In addition, small pieces of rumen and longissimus dorsi were sampled for cell culture.Three biological replicates were used in this study.

Cell culture, proliferation, and differentiation
The rumen epithelium cells and skeletal muscle cells were separately cultured in the DMEM growth medium (supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin).They were developed under 5% of Carbon dioxide, 37 C. When the cell density reached 80 to 90%, cells were inoculated into cell culture plates.The rumen epithelium cells were cultured in a proliferation medium and collected at 12, 24, 36, 48, 60, and 72 h after subculture, respectively.When the confluence of skeletal muscle cells reached 70% after subculture, the medium was replaced with DMEM differentiation medium (supplemented with 2% horse serum and 1% penicillinstreptomycin) to induce the differentiation of the skeletal muscle cells, which were collected at the time point 0, 24, 72, and 168 h after differentiation.At corresponding periods, RNA was extracted from rumen epithelium cells and skeletal muscle cells for subsequent qRT-PCR detection.

RNA extraction and cDNA synthesis
Following the manufacturer's instructions, the RNA was extracted from tissue and cell samples using RNA Easy Fast Tissue/Cell Kit (TIANGEN, Beijing, China).Then, use NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Beijing, China) to measure RNA concentration and purity.In addition, determine the integrity of the extracted RNA by 1.5% (w/v) agarose gel electrophoresis (Fig. S1).The cDNA was then synthesized from 1 mg RNA using the PrimeScript TM RT reagent Kit with gDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's instructions.

Primer design and GTL2 cloning
Firstly, the conserved region of GTL2 was determined by alignment using the relative sequences from humans (Homo Sapiens, AB032607.1),cattle (Bos Taurus, NR_037684.1),pig (Sus scrofa, EF468461.1),and sheep (Ovis aries, AY017220.1).Secondly, one primer pair (named GTL2_1F/1R, Table 1) was designed to amplify the partial mRNA sequence of GTL2 using the Primer Premier 5.0 software (PREMIER Biosoft Intl., CA, USA).PCR amplification was performed using the synthesized cDNA as a template with the above cloning primers.The PCR condition was as follows: an initial denaturing step at 95 C for 5 min, followed by 35 cycles of denaturing at 95 C for 30 s, annealing at 57 C for 30 s and extending at 72 C for 60 s, with a final extension step at 72 C for 5 min.The 40 mL reaction system contained 20 mL 2Â Taq PCR Master Mix (Takara), 1.6 mL Forward primer (10 mM), 1.6 mL Reverse primer (10 mM), 2 mL cDNA, and 14.8 mL ddH 2 O.The amplification product (Fig. S1) was recovered by glue purification, and the purified product was ligated with the pMD 19-T vector and converted into DH5a competent cells.Lastly, positive clones were selected for GTL2 sequencing using an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA).

Quantitative real-time PCR to assess GTL2 expression
According to the above-obtained mRNA sequence, the primer pair (GTL2_QP_F/R, Table 1) was designed to quantify the expression of GTL2 in different tissues, the rumen epithelium cells, and skeletal muscle cells.The beta-actin (ACTB, NM_001314342.1)was used as a housekeeping gene for normalizing the values of GTL2.The qRT-PCR assay was performed in the CFX96 TM real-time PCR detection system (Bio-Rad, CA, USA) in a volume of 10 mL consisting of 5 mL 2X SYBR Premix Ex Taq II (Vazyme, Nanjing, China), 0.3 mL each of forward and reverse primer (10 mM), 1 mL cDNA, and 3.7 mL nuclease-free water.The cycling conditions were as follows: 95 C for 30 s, 40 cycles of 10 s at 95 C and 30 s at 60.0 or 59.7 C, 60 s at 72 C for annealing, and a final extension for 7 min.
A melting curve was performed to assess the specificity of primers.Each reaction was performed with three biological replicates and triplicate technical replicates.Relative gene expression was calculated with the 2 ÀDDCt method. 19

DNA extraction and methylation detection
Genomic DNA was extracted from the six tissues using the TIANamp Genomic DNA Kit (TIANGEN).Its concentration was determined by A NanoDrop ND-2000 spectrophotometer (Thermo NanoDrop Technologies, DE, USA).In addition, its purity and integrity were assessed by electrophoresis (Fig. S1).Bisulfite-treated DNA was obtained using the EZ DNA Methylation Gold TM Kit (Zymo, Orange, CA, USA), followed by PCR amplification with methylated primers.The online methylation primer design tool Methprimer (http://www.urogene.org/methprimer/)was used to predict the CpG islands of GTL2 and design the methylation primers (GTL2_BSP_1F/1R, GTL2_BSP_2F/2R, Table 1).All primers used in this study were synthesized by Sangon Bioengineering Co., Ltd (Shanghai, China).The cycle conditions used were as follows: an initial denaturing at 94 C for 4 min, followed by 40 cycles of denaturing at 94 C for 20 s, annealing at 56 C for 30 s, extension 72 C for 1 min, with a final extension at 72 C for 3 min.The PCR product was confirmed by 1.5% agarose gel electrophoresis, followed by Shrimp alkaline phosphatase (SAP).After the unincorporated dNTPs were dephosphorylated with shrimp alkaline phosphatase.T polymerase was carried out simultaneously by the Digestion and RNase A digestion.The enzymedigested product was resin purified for 60 min.DNA methylation levels of these products were quantified using the MassARRAY platform (Sequenom, San Diego, CA, USA), and methylation data were analyzed using Epitperer v1.0 software (Sequenom).

Statistical analysis
The methylation degree of the GTL2 coding region in different tissues was calculated using the QUantification tool for Methylation Analysis (QUMA, http://quma.cdb.riken.jp/).The methylation level and gene expression data were tested using the Bonferroni test.Then the significance analysis of the differences in methylation levels and gene expression levels between tissues was performed using SAS (version 4.0, USA) and one-way ANOVA.In addition, the significance analysis of the differences in multiple specific CpG site methylation was performed using SAS (version 4.0, USA).By comparison, 15 specific methylation sites were selected for correlation analysis with mRNA expression.Pearson correlation analysis was used to analyze the correlation between GTL2 expression and methylation level.p < 0.05 indicates a significant statistical difference, and the results of methylation level and gene expression level were shown as mean ± standard.

Specific expression of caprine GTL2 in tissues and cells
GTL2 expression was examined in six tissues by qRT-PCR.GTL2 is extensively expressed in the brain, heart, liver, kidney, lung, and LD of Jianzhou Da'er goats.As shown in Fig. 1A, the differential expression between different tissues was significantly different; the expression of GTL2 was significantly increased in the brain (p < 0.05), while the expression in LD, heart, lung, and kidney was significantly decreased.At the same time, deficient expression levels were detected in the heart, $30-fold lower than in the liver.Furthermore, we performed quantitative validation during the periods of proliferation in the rumen epithelium cells and differentiation of skeletal muscle cells.In the rumen epithelium cell proliferation, GTL2 expression was highest at 60 h, followed by 72 h, and almost unchanged at 12-48 h (Fig. 1B).In the skeletal muscle cells differentiation, GTL2 expression was highest at 0 and 24 h, and significantly decreased at 72 and 168 h (Fig. 1C).

GTL2 methylation level and its tissue specificity
Using online predicted tools, 37 CpG sites were found to be located between 62 and 453 bp (CpG island No. 1) of GTL2, and 38 CpG sites were located between 458 and 901 bp (CpG island No. 2) of GTL2 (Fig. 2).By examining the amplified products of the sulfite- treated DNA (Fig. 3A), we found that goat GTL2 was all located in the exons of goat GTL2 and confirmed the prediction of two CpG islands.At the same time, multiple specific CpG sites were detected, in which the blue site represented the detected specific methylation site and the red site described the undetected specific CpG site.Besides, as shown in Fig. 3B, the methylation level of GTL2 in six tissues was 81.42-86.83%.The methylation patterns of GTL2 CpG islands showed no significant difference between tissues.The average methylation level of the 5 0 distal CpGs was lower than that of the 3 0 distal CpGs, which indicated that the methylation degree of the 5 0 distal CpGs of the coding sequence of the GTL2 was higher than that of the 3 0 distal CpGs.Interestingly, there were significant differences between most of the sites in the two CpG islands (Fig. 4).

Correlation analysis of GTL2 tissues expression and methylation
The tissue expression and methylation level of GTL2 were correlated by Pearson correlation analysis.As shown in Table 2, the correlation coefficient of the tissue expression level and methylation level of GTL2 was À0.732-0.853.However, there was no significant correlation between methylation level and GTL2 expression level in the six tissues.At the same time, there were few significant correlations between specific CpG sites and mRNA expression (Fig. 5).

Discussion
To preliminarily explore the physiological role of goat GTL2, we evaluated the tissue specificity expression of GTL2 in the above tissues of the Jianzhou Da'er goat.The results showed that goat GTL2 is widely expressed in all tissues tested, indicating that GTL2 is a common imprinted gene that regulates the development and function of nearly all tissues and organs.According to our experimental results, GTL2 was widely expressed in all tissues studied and was significantly higher in the brain than in other tissues.Previous studies have suggested that GTL2 is specifically expressed in the liver, 20 Blastocysts, 21 and placenta. 22This experiment found that caprine GTL2 was highly expressed in the brain.The result was consistent with the experimental results in cattle 22,23 and pigs.GTL2 has been reported to be involved in cell proliferation and differentiation.Our study also showed that GTL2 was highly expressed during the rumen epithelium cell proliferation and skeletal muscle cell differentiation by the quantitative analysis of GTL2.
Previous studies have noted that different parentalderived alleles of GTL2 are affected by varying  methylation levels to present tissue-specific expression.Generally, the allele of GTL2 maternal origin is preferentially expressed, but the expression of its paternal allele is not completely suppressed in some tissues. 24mprinted genes showed maternally derived allelic or biallelic expression in different cattle tissues.Methylation analysis of two regulatory regions of an imprinted gene (DIO3) in various tissues of cattle and their association with gene expression showed that the differences of imprinted genes in different tissues were distinct.Still, the observed tissue-specific genes showed no significant relationship between expression levels. 25Our study clarified the DNA methylation of GTL2 and compared the methylation status of differently methylated areas in related goat tissues.There was no significant difference in the expression of GTL2 methylation in different tissues.
Changes in methylation profiles often predict changes in the expression of related genes.Abnormal methylation profiles can trigger strange gene expression and lead to immunodeficiency disease in humans, 26 abnormal thyroid hormone levels in lambs, 27 developmental failure in swine and rabbits, 28,29 and the occurrence of mastitis in dairy cows. 30Walker et al. showed that the expression of many genes in bovine endometrium in early pregnancy was significantly correlated with DNA methylation levels, and most of the genes found were negatively correlated. 31However, studies by Schroeder et al. showed that mammalian upper methylated domains (PMDs) and hypermethylated domains (HMDs) and gene body DNA methylation were positively correlated with gene expression levels. 32owever, in this study, there was no significant correlation between GTL2 methylation levels and gene expression levels in the six tissues.There was a significant negative correlation between a few specific CpG sites and gene expression.

Conclusion
In conclusion, this study showed that the expression of GTL2 in the brain was significantly higher than that in the heart, kidney, lung, and longissimus dorsi.Still, the gene methylation level was not notably different among the six tissues.The gene expression level was not significantly related to the methylation level, which implies that the essential elements regulating gene imprinting were not within the GTL2.

Author contributions
TZ conceived and designed the experiments.YL collected the samples and executed the experiments.ZG, YL, and TZ

Figure 1 .
Figure 1.Quantitative expression of GTL2 in different tissues and cells of goat.(A) Different tissues.(B) Rumen epithelium cells.(C) Muscle skeletal cells.The ' Ã ' symbol indicates a p-value less than 0.05.

Figure 2 .
Figure 2. Prediction of GTL2 methylation: the CpG island and the CpG site distribution map of the cloned GTL2 sequence.

Figure 3 .
Figure 3. Analysis of GTL2 methylation level.(A) DLK1-DIO3 imprinting domain pattern and GTL2 CpG site distribution map.(B) GTL2 two CpG island methylation levels in six tissues.

Figure 4 .
Figure 4. Significant methylation difference among the CpG sites in goat GTL2.(A) The first CpG island.(B) The second CpG island.The ' Ã ' symbol indicates a p-value < 0.05, and the ' ÃÃ ' symbol indicates a p-value < 0.01.

Figure 5 .
Figure 5.The relationship between CpG methylation and mRNA expression of GTL2 in goats.The ' Ã ' symbol indicates a p-value < 0.05, and the ' ÃÃ ' symbol indicates a p-value < 0.01.

Table 1 .
Primer information was used in this study.

Table 2 .
GTL2 methylation level, tissue expression level, and correlation analysis.
The ' Ã ' symbol indicates a p-value less than 0.05.