Genomic Imprinting at the Porcine DIRAS3 Locus

Simple Summary DNA methylation associated with one of the two alleles from parents is an important mechanism that causes a silencing of that allele, leading to expression of another allele only. There has been a lack of detailed studies on DNA methylation and expression patterns that are related to the DIRAS3 gene in pigs. The objective of this study was to provide a comprehensive overview of DNA methylation and expression associated with the DIRAS3 gene in pigs by generating an embryonic pig model and analyzing next-generation sequencing using pig embryos and adult pigs. Our results clearly showed the presence of DNA methylation near the DIRAS3 gene in pigs and high expression of DIRAS3 in the hypothalamus from adult pigs and expression of only one allele in all the tested tissues including the hypothalamus. In summary, our findings suggested DNA methylation might be related to those unique gene expression patterns during the development of pigs. Abstract The epigenetic mechanisms underlying genomic imprinting include DNA methylation and monoallelic expression of genes in close proximity. Although genes imprinted in humans and mice have been widely characterized, there is a lack of detailed and comprehensive studies in livestock species including pigs. The purpose of this study was to investigate a detailed methylation status and parent-of-origin-specific gene expression within the genomic region containing an underexamined porcine DIRAS3 locus. Through whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) of porcine parthenogenetic embryos and analyses of public RNA-seq data from adult pigs, DNA methylation and monoallelic expression pattern were investigated. As a result, maternal hypermethylation at the DIRAS3 locus and hypothalamus-specific and monoallelic expression of the DIRAS3 gene were found in pigs. In conclusion, the findings from this study suggest that the presence of maternal hypermethylation, or imprints, might be maintained and related to monoallelic expression of DIRAS3 during pig development.


Introduction
Genomic imprinting plays a crucial role in mammalian development and growth [1]. The epigenetic mechanisms underlying genomic imprinting include DNA methylation during mammalian embryonic development [2,3]. Differentially established DNA methylation in two parental germlines consists of epigenetic imprints and causes monoallelic expression of genes in close proximity [4]. For example, a differentially methylated region (DMR) at the SGCE/PEG10 locus serves as the imprinting control region (ICR) which forces paternal allele-specific expression in humans [5,6], mice [7,8], pigs [9], and sheep [10]. Although genes imprinted in humans and mice have been widely characterized, there is a lack of detailed and comprehensive studies in livestock species including pigs. Our recent approach of the use of whole-genome bisulfite sequencing (WGBS) and RNA sequencing Animals 2021, 11, 1315 2 of 10 (RNA-seq) of porcine embryos that underwent parthenogenesis has facilitated verification of the imprinting status of known imprinted clusters and identification of novel imprinted genes [9,11]. This facilitation was attributed to a direct sequencing comparison between parthenogenetic and biparental porcine embryos. As a result, a detailed methylation status and parent-of-origin-specific gene expression within genomic regions containing underexamined target loci, such as the DIRAS3 locus, could be further investigated.
The distinct subgroup of the Ras family member 3 (DIRAS3) gene, also known as ARHI and NOEY2, encodes a small GTPase, which belongs to the Ras superfamily and serves as a tumor suppressor against various cancers including breast and ovarian cancers [11,12]. A maternal imprint and paternal monoallelic expression of the human DIRAS3 gene have been reported to establish a functional allele that acts as a negative regulator of growth and development [13,14]. In pigs, although paternal expression of the DIRAS3 gene at the fetal stage [15] and in adult skeletal muscles in which expression of DIRAS3 is very low [16] was reported, DNA methylation imprints at the locus has not been investigated. In addition, according to the genotype-tissue expression (GTEx) project (https://gtexportal. org/home/gene/DIRAS3, accessed 15 January 2021), in an adult human, the DIRAS3 gene was expressed the most in the hypothalamus among 54 sampled normal tissues and expressed in high levels in the nucleus accumbens (basal ganglia), ovary, and pituitary, whereas expressions in the other 50 normal human tissues were very low. However, detailed information about tissue-specific and/or monoallelic expression of the DIRAS3 gene in adult pigs is still missing. Therefore, we explored DNA methylation using our parthenogenetic model and the expression of DIRAS3 in the adult pig hypothalamus, as well as other major tissues (adipose, liver, lung, skeletal muscle, and spleen) by processing raw RNA-seq data from this study and from gene expression omnibus (GEO) series.
Here, we aimed to determine concurrence of paternal allele expression and a maternal DMR within the promoter, exon, and intron regions of the porcine DIRAS3 gene using RNA-seq and WGBS of parthenogenetic and biparental control embryos. In addition, tissuespecific and monoallelic expression of the DIRAS3 gene in adult pigs was investigated by analyzing RNA-seq data. Our comprehensive analysis of DNA methylation and imprinted gene expression status of the porcine DIRAS3 gene provides advanced groundwork for future studies of the DIRAS3 locus.

Sample Collection and Data Used
Animal procedures were approved by the IACUC of the National Institute of Animal Science of Korea (approval number NIAS2015-670). Porcine oocytes were collected from 50 normal Landrace × Yorkshire × Duroc (LYD) pigs and matured in vitro and then parthenogenetic embryos were generated by electrical stimulation described in our previous reports [9,17]. After placing into oviducts of surrogate gilts, parthenogenetic embryos were developed until day 21. Furthermore, fertilized control porcine embryos were collected on day 21 after natural mating as described previously [9,17]. A publicly available dataset under accession number GSE158430 in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) was accessed on 29 November 2020. Then, raw RNA-seq data for adipose tissue, hypothalamus, liver, lungs, skeletal muscles, and spleen of two 6-month-old adult male pigs (P348 and P350) (SubSeries GSE158412), raw H3K4me1 ChIP-seq data of the P348 hypothalamus (SubSeries GSE158427), and raw CTCF ChIP-seq data of the P350 hypothalamus (SubSeries GSE158416) were downloaded. We named P348-Pig 1, p350-Pig 2.
To produce the transcriptome, RNA and cDNA library construction and RNA-seq were performed by TNT Research (Jeonju, Korea) as described [9,17], with total RNA isolated from the whole collected CN (n = 3) and PA (n = 3) embryos using the TRIzol reagent (Sigma-Aldrich, St Louis, MO, USA) following the manufacturer's instructions. In brief, RNA samples treated with DNase I to avoid genomic DNA contamination were electrophoresed in 1.2% agarose gels to evaluate the integrity of RNA, which was then confirmed by the 28S/18S rRNA ratio (>2.0) and the RNA integrity number (RIN) (>7.0) using an Agilent 2100 BioAnalyzer. Using the ratios of A260/A280 and A260/A230 (1.8-2.0), the concentrations of RNA were assessed. One µg of total RNA was used to construct cDNA libraries with TruSeq RNA Sample Prep Kit v.2 (Illumina, San Diego, CA, USA). Quantification and qualification of the cDNA libraries were assessed by quantitative real-time PCR (qPCR) and using an Agilent 2100 Bioanalyzer, respectively. An Illumina HiSeq2500 RNA-seq platform was used to sequence the library products (101 nt pairedend). After quality checking, adapter trimming, and filtering by FastQC and Trim Galore, about 76.8 (CN1), 73.0 (CN2), 77.2 (CN3), 80.0 (PA1), 79.3 (PA2), and 80.3 (PA3) million cleaned reads were retrieved. STAR aligner (v.2.7.5) [19] was used to align the reads to the reference genome (Sscrofa11.1) with default parameter settings and produce BAM files of aligned reads. To normalize read coverages, deepTools (v3.5.0) [20] was used.

Bioinformatics and Statistical Analysis
All WGBS and RNA-seq alignments were visualized using the R/Bioconductor package Gviz (v1.32.0) [21] and/or Integrative Genomics Viewer (IGV) (v2.8.13) [22]. The downloaded raw RNA-seq and ChIP-seq data of adult pigs were analyzed to check tissuespecific and monoallelic expression. The downloaded reads were quality-checked by FastQC and trimmed using Trim Galore. STAR aligner [19] was used to map cleaned reads to the reference pig genome (Sscrofa11.1). Read coverages and alignments in those adult pigs were examined using BAM files and normalized using deepTools [20]. Differential expression was analyzed by quantifying the raw reads against the pig transcriptome using Salmon (v1.3.0) [23] and analyzing those quantified reads using the R/Bioconductor package DESeq2 (v.1.28.1) [24], and then the adjusted p-value < 0.05 was regarded as statistically significant. To call a DMR with the threshold of >10 CpGs, the maximum distance between CpGs < 300 bp, the mean methylation ratio difference (∆ ave) > 0.2 and the false discovery rate (FDR) < 0.05, metilene (v0.2-8) [25] was used. The shapiro.test function in the R package stats (v4.0.5) [26] was used to conduct the Shapiro-Wilk test to evaluate normality of the data. The Kruskal-Wallis test was performed using the kruskal.test function in the R package stats (v4.0.5) [26] to determine whether there is any significant difference between tissue groups regarding gene expression levels in adult pigs.

Differential Expression and Methylation at the DIRAS3 Locus of Porcine Embryos
To verify paternal expression of the DIRAS3 gene in day 21 porcine embryos, we compared DIRAS3 expression between our model of parthenogenetic embryos without the paternal allele and biparental control embryos. By analyzing RNA-seq data, an exclusive expression of DIRAS3 in the biparental control embryos with the paternal allele was found, but not in the parthenogenetic embryos ( Figure 1a, Table 1). Surrounding noncoding genes (the porcine LOC110261211 and LOC110261446 genes encoding noncoding RNAs) were not expressed in both embryos. Furthermore, unlikely for humans, expression of an overlapping transcript (e.g., GNG12-AS1 in humans) was not detected. Further differential gene expression analysis revealed that the expression of DIRAS3 was more than 85-fold higher in the control embryos than in the parthenogenetic embryos (adjusted p < 0.05) (Figure 1a). In addition, our analysis of WGBS showed that a significantly higher DNA methylation at the DIRAS3 locus occurred in the parthenogenetic embryos with two maternal alleles compared with the biparental control embryos (Figure 1b, Table 1, Table S1). This maternal hypermethylation established a DMR throughout the DIRAS3 locus including a CpG island. Taken together, our combined results of RNA-seq and WGBS provided strong evidence of maternal imprints that give rise to a paternal expression of the porcine DIRAS3 gene. than 85-fold higher in the control embryos than in the parthenogenetic embryos (adjusted p < 0.05) (Figure 1a). In addition, our analysis of WGBS showed that a significantly higher DNA methylation at the DIRAS3 locus occurred in the parthenogenetic embryos with two maternal alleles compared with the biparental control embryos ( Figure 1b, Table 1, Table  S1). This maternal hypermethylation established a DMR throughout the DIRAS3 locus including a CpG island. Taken together, our combined results of RNA-seq and WGBS provided strong evidence of maternal imprints that give rise to a paternal expression of the porcine DIRAS3 gene.

Sequence Elements and Differentially Methylated CpGs within the Porcine DIRAS3 Locus
Since DNA hypermethylation has been recognized as an epigenetic regulator of gene expression that potentially silences or downregulates tumor suppressor genes such as DIRAS3 [27], detailed maternal hypermethylation status was profiled in the porcine DIRAS3 locus. Unlike humans in whom CpG islands are separated into three pieces at the promoters and the gene body, the DIRAS3 locus in pigs contains one consecutive CpG island spanning between a part of the promoter region and a large portion of the gene body (exon 1, intron, and exon 2) (Figure 2). Because differentially methylated CpGs (or Cs) were distributed throughout the CpG island and even farther in the promoter and exon 2, a DMR was also consecutive throughout the porcine DIRAS3 locus. The consecutiveness was detected even with conservative criteria of at least ten differentially methylated CpGs with the maximum distance between CpGs of less than 300 bp, which was changed from the default maximum distance of 500 bp in the DMR caller, metilene (Figure 2). On the other hand, in humans, three separate DMRs that corresponded to three CpG islands were reported [28]. In sum, in pig embryos, maternal hypermethylation occurred throughout the promoter and the gene body of the DIRAS3 gene beyond the CpG island, establishing one DMR. exon 2, a DMR was also consecutive throughout the porcine DIRAS3 locus. The consecu-tiveness was detected even with conservative criteria of at least ten differentially methylated CpGs with the maximum distance between CpGs of less than 300 bp, which was changed from the default maximum distance of 500 bp in the DMR caller, metilene ( Figure  2). On the other hand, in humans, three separate DMRs that corresponded to three CpG islands were reported [28]. In sum, in pig embryos, maternal hypermethylation occurred throughout the promoter and the gene body of the DIRAS3 gene beyond the CpG island, establishing one DMR. Figure 2. Distribution of differential methylation (i.e., maternal hypermethylation) in the DIRAS3 locus of the porcine parthenogenetic (PA) embryos compared to the control (CN) embryos. The nucleotide sequence of the genomic DNA covers a promoter (black), exon 1 (brown, underlined), intron (black), and exon 2 (brown, underlined) of the porcine DIRAS3 gene from the beginning to the end. Brown bold faces in exon 2 indicate a translated region. This partial genomic DNA sequence in chromosome 6 (chr6 or NC_010448.4:144575648-144578820) of the reference pig genome (Sscrofa11.1 or susScr11) was retrieved from the NCBI Nucleotide database (https://www.ncbi.nlm.nih.gov/nuccore, accessed 15 January 2021). The start and end of the CpG island (chr6:144576300-144578127) and DMR (displayed in Figure 1) are denoted with green and red brackets, respectively. Differentially methylated CpGs (or Cs) between PA and CN (i.e., hypermethylated in PA) are highlighted with red. Highlighted Gs indicate methylated Cs on the minus strand (coding strand). To call a DMR with a threshold, metilene was used, as mentioned in the Materials and Methods.  Figure 2. Distribution of differential methylation (i.e., maternal hypermethylation) in the DIRAS3 locus of the porcine parthenogenetic (PA) embryos compared to the control (CN) embryos. The nucleotide sequence of the genomic DNA covers a promoter (black), exon 1 (brown, underlined), intron (black), and exon 2 (brown, underlined) of the porcine DIRAS3 gene from the beginning to the end. Brown bold faces in exon 2 indicate a translated region. This partial genomic DNA sequence in chromosome 6 (chr6 or NC_010448.4:144575648-144578820) of the reference pig genome (Sscrofa11.1 or susScr11) was retrieved from the NCBI Nucleotide database (https://www.ncbi.nlm.nih.gov/nuccore, accessed 15 January 2021). The start and end of the CpG island (chr6:144576300-144578127) and DMR (displayed in Figure 1) are denoted with green and red brackets, respectively. Differentially methylated CpGs (or Cs) between PA and CN (i.e., hypermethylated in PA) are highlighted with red. Highlighted Gs indicate methylated Cs on the minus strand (coding strand). To call a DMR with a threshold, metilene was used, as mentioned in the Materials and Methods.

Tissue-Specific and Monoallelic Expression of the DIRAS3 Gene in Adult Pigs
We further examined the expression of DIRAS3 in adult pigs to show whether imprinted gene expression maintains at the adult stage. First, we explored whether tissuespecific expression patterns exist. Considering that the DIRAS3 gene in humans was expressed predominantly in the brain (hypothalamus, nucleus accumbens), as well as in the ovary and the pituitary (as mentioned above), we predicted that this expression pattern may be conserved in pigs. Our analysis of RNA-seq data from two six-month-old male pigs revealed the DIRAS3 gene expression appeared to be hypothalamus-specific among the analyzed major pig tissues (Table 2, Figure 3a,b). However, tissue distribution was non-normal according to the Shapiro-Wilk test, and therefore a non-parametric test (the Kruskal-Wallis test) was performed. The result showed that mRNA expressions of both LOC110261211 and DIRAS3 were not statistically different between the analyzed tissues ( Table 2). On the other hand, these TPM values from the hypothalamus were approximately five to eight times higher than those values from the control embryos in Figure 1a. Among other tissues including adipose tissue, liver, lung, skeletal muscle, and spleen, DIRAS3 expression was observed as slightly higher in adipose tissue, but the absolute expression level was low (Figure 3a,b). Secondly, further analysis of read coverages and alignments showed that only reference alleles (A alleles in pig 1 and C allele in pig 2) were expressed at heterozygous sites in all the tested tissues instead of the alternative alleles shown in published ChIP-seq data from genomic DNA (gDNA) of the same pigs, indicating monoallelic expression of the DIRAS3 gene (Figure 3c,d). Overall, these data suggest that the imprinted expression of DIRAS3 is maintained at the adult stage in which DIRAS3 expression shows a tendency of hypothalamus-enriched expression.   0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 For both LOC110261211 and DIRAS3, the distribution significantly differed from normal distribution according to the Shapiro-Wilk test (p-values were 0.0423 and 1.267 × 10 −5 , respectively). The p-values from the Kruskal-Wallis test were 0.148 (LOC110261211) and 0.08204 (DIRAS3), which are lower than the significance level (0.05) indicating that there were no significant differences between the tissue groups.

Discussion
Our findings in this study suggest that the DIRAS3 gene in porcine embryos is subjected to silencing of its promoter region in the maternal allele through DNA hypermethylation, which is one of the genomic imprinting mechanisms including direct silencing of promoters, inhibition of long-range communication between promoters and enhancers, repression of long-noncoding RNA (lncRNA), isoform-dependent silencing, and histone modifications [29][30][31]. This direct silencing via maternal DNA hypermethylation might lead to paternal allele-specific expression of DIRAS3 in biparental control embryos, but not in parthenogenetic embryos, where it is deficient in the paternal allele. As such, our generation of parthenogenetic embryos enabled detection of paternal expression by all-or-none fashion (i.e., all biparental controls having a paternal allele, and no parthenotes without any paternal allele), along with a construction of the methylome and the transcriptome.
Further analysis of DNA sequence elements and methylated CpGs showed that both the regions of CpG island and maternal methylation were more widely spread in pigs than in humans. Since aberrant DNA methylation on the paternal allele has been identified as a major inhibitor of the imprinted expression of DIRAS3 [14], a detailed overview of methylated regions is needed. It was reported that in human patients with breast cancer and ovarian cancer, hypermethylation in both CpG islands, I and II, was associated with reduced expression of DIRAS3 [32,33]. This CpG island II is located at the proximal promoter and exon 1 of the human DIRAS3 isoform [28,32] and corresponds, in part, to the pig CpG island, suggesting paternal hypermethylation in this region could be related to reduced DIRAS3 expression. Given that the porcine DMR was extended 652 bp upstream and 86 bp downstream beyond the CpG island (Figure 2), there might be additional gene regulation via DNA methylation.
Furthermore, our data analysis revealed that in adult male pigs, the expression of the DIRAS3 gene tended to be hypothalamus-specific and monoallelic, suggesting maintenance of the imprinted gene expression during development. Despite the tendency of hypothalamus specificity, non-normal distribution and non-statistical significance (p = 0.08204) of the distribution of DIRAS3 might be due to the low sample size. Considering tissuespecific DIRAS3 expression patterns shown in the GTEx project (predominant in the human hypothalamus, nucleus accumbens, ovary, and pituitary) and given the hypothalamusspecific tendency in the current study, the reason why a previous study reported ubiquitous expression of porcine DIRAS3 in tested tissues [16] might be due to saturation of RT-PCR. There are also conflicting reports on the imprinted status of DIRAS3 in pig placenta, as one report mentioned a lack of imprinted isoform in the placenta [15], while another report displayed monoallelic expression of DIRAS3 in the placenta [34]. Based on our plot of RNA-seq read coverages, there was only one expressed transcript in the DIRAS3 locus, unlike humans in whom a longer isoform exists according to the Ensembl database (http://www.ensembl.org/, accessed 15 January 2021). To clarify the imprinted status of one DIRAS3 transcript in the pig placenta, future studies will need to investigate both monoallelic expression and methylation status and link them to provide comprehensive profiling.
Overall, the porcine DIRAS3 locus has similarities and differences compared to the human ortholog. The presence of maternal hypermethylation (imprints), tissue-specific expression tendency in the hypothalamus, and its monoallelic expression pattern were similar. Differences covered the locations and numbers of the CpG island and DMR, as well as non-existence of an overlapping GNG12-AS transcript in pigs. Further investigation across species in a comparative manner will improve our understanding on the underlying genetic and epigenetic mechanisms related to the DIRAS3 locus.

Conclusions
Although genes imprinted in humans and mice have been widely characterized, including tumor suppressor genes such as DIRAS3 or ARHI, there is a lack of detailed and comprehensive studies in livestock species including pigs. The findings from this study suggest that the presence of maternal hypermethylation, or imprints, might be maintained and related to monoallelic expression of DIRAS3 during pig development. Our approach based on parthenogenesis and various next-generation sequencing data promotes comprehensive and detailed identification of imprinted genes and loci in a genomic context.