Identification of differentially methylated regions (DMRs) of neuronatin in mice

Background Neuronatin (NNAT) is a paternal-inherited imprinted gene, first discovered in the rat neonatal brain, where it plays vital roles for neuronal growth, brain development, and metabolic regulation. The maternal imprint of NNAT has been identified in mice; however, the differentially methylated regions (DMRs) involved in the monoallelic expression of NNAT have not yet been investigated. Results In this study, we confirmed expression of two isoforms of the NNAT (α and β) in the mice brain via quantitative RT-PCR. Additionally, the methylation profile of the CpG island located in the NNAT gene locus was determined in the mice liver, brain, sperm, and the MII oocyte via bisulfite sequencing PCR. Conclusion In summary, we provide the first evidence for tissue- and gamete-specific methylation patterns of CpG3 that are located on exon 1, to be putative DMR of NNAT in mice.


Background
Genomic imprinting leads to unequal expression of paternal and maternal alleles in offspring, which is essential for normal embryogenesis, fetal growth, and functional postnatal behavior (Surani 1998;Li et al. 1999;Das et al. 2013). The differentially methylated regions (DMRs) are established during gametogenesis and regulate the parent-specific expression of imprinted genes. DMRs with allele specific methylation have also been used in cancer diagnosis (Ushijima 2005;Bonin et al. 2016). Although 125 imprinted genes have been identified in mice to date (according to the Gene imprint database; http://www. geneimprint.com/), very little has been published about the DMRs of those imprinted genes (Gu et al. 2014).
NNAT is a highly conserved imprinted gene among humans, mice, cattle, and pigs, containing two alternatively spliced transcripts (α and β) (Cheng et al. 2007;Schulz et al. 2009). Previous studies have revealed the parental-specific expression of NNAT to be associated with the methylation status of the CpG island located in the NNAT promoter sequence of pigs (Chen et al. 2014;Gu et al. 2014) and rabbits (Duan et al. 2015). A second intronic DNA sequence within the mouse NNAT with a length of 250 bp was defined as putative DMR and was revealed to act as a transcriptional activator in Drosophila (Sowpati et al. 2008). In addition, the hyper methylation of human NNAT frequently occurs in pediatric acute leukemia and Wilms Tumors (Kuerbitz et al. 2002;Hubertus et al. 2013).
All of these previously published findings suggest that there is no definite DMR of NNAT in mice. With this background, we sought to identify the DMRs of mice NNAT via quantitative real-time PCR (q-PCR) and bisulfite sequencing PCR (BSP) analyses.

Results and discussion
In this study, we proposed a model of two NNAT isoforms (α and β) using GenBank and Ensembl databases. Furthermore, putative DMRs of NNAT (CpG 1 and Open Access CpG 2, CpG 3, CpG I2) were identified via comparative sequencing analysis and methyprimer (Fig. 1a). The results of q-PCR and RT-PCR demonstrated high expression of both of the transcripts (α and β) in the brain of mice, but not in their liver, which is consistent with the UniGene database (Fig. 1b, c). Previous studies have found two transcripts of NNAT that are widely expressed in most of tissues, including in livers and kidneys of cattle (both fetal and adult) and in 2-month-old pigs (Zaitoun and Khatib 2006;Cheng et al. 2007), suggesting different NNAT expression profiles between species.
To identify the DMR of mice NNAT, methylation profiles of CpG 1, CpG 2, CpG 3, and CpG I2 were determined in both the brain and the liver using BSP. The results revealed hypermethylation of CpG1 (Fig. 2a vs. e), CpG2 (Fig. 2b vs. f ), and CpG I2 (Fig. 2d vs. h) in both brain and liver. However, we found tissue-specific methylation patterns of CpG3 in the brain (29.33%) and liver (66%) (Fig. 2c vs. g). These results point towards the methylation status of CpG3 accounting for the expression difference of NNAT in liver vs. brain. In addition, tissuespecific methylation patterns revealed CpG3 (located on exon 1) to be a putative DMR of the NNAT in mice (Fig. 2c).
Generally, DNA methylation patterns of imprinted genes were established in both sperm and oocyte, which were reprogrammed subsequent after fertilization (Seisenberger et al. 2013). To identify the gamete-specific methylation pattern of CpG3 in mice NNAT, the DNA methylation profile of CpG3 in sperm and oocyte was determined via BSP. The results reveal hypomethylation in sperm (10%) and hypermethylation in MII oocytes (90%) (Fig. 3). In summary, the gamete-specific methylation patterns suggest that the imprinting marks of NNAT are established by a sex-specific mechanism, confirming CpG3 as the DMR of NNAT.

Conclusions
Here, we studied the expression patterns of two isoforms of mice NNAT (α and β), and identified the CpG3 of mice NNAT (located on exon 1) as putative DMR.The data revealed hypermethylation of exon 1 to be associated with the silencing of mice NNAT, suggesting the NNAT gene transcriptional status to be correlated with the methylation status of exon 1, while being independent of both intron and 5′ UTR.

Tissue samples
The brain and liver were collected from two-month old ICR mice and immediately stored in liquid nitrogen until further use. Spermatozoa and MII oocytes were harvested using previously published protocols (Chen et al. 2014).

RT-PCR and quantitative real-time PCR (q-PCR)
Total RNA from liver and brain (n = 5) was isolated with TRNzol-A+ reagent (TIANGEN, Beijing, China) according to manufacturer's instructions. The cDNA was synthesized with DNAse I (Fermentas, Shanghai, China) treated total RNA via the BioRTcDNA First Stand Synthesis Kit (Bioer Technology, Hangzhou, China).

Fig. 1
Gene structure and expression analysis of NNAT in mice. a Structure of the mice NNAT locus. NNAT features two alternatively spliced transcripts (α-and β-form). Protein coding regions are shown as black, filled boxes. Circles indicate CpG islands located in NNAT and analyzed via BSP. Q: Primers for q-PCR; RT: Primers for RT-PCR; b RT-PCR was used to detect the expression of two alternatively spliced isoforms in both brain and liver. c Gene expression of NNAT in brain and liver determined via q-PCR. Data are shown as Mean ± SEM (n = 6), *p < 0.05, **p < 0.01, ***p < 0.001 The BioEasy SYBR Green I Real Time PCR Kit (Bioer Technology, Hangzhou, China) was used to perform q-PCR, using the BIO-RAD Iq5 Multicolor Real-Time PCR Detection System. The reaction conditions were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s for DNA denaturation, 60 °C for 15 s for primer annealing, and 72 °C for 30 s for extension. Relative gene expression normalized to β-actin was determined via the 2 −ΔΔCT formula. All experiments on gene expression were performed in triplicate. The gene expression data was presented as mean ± SEM. Primers used for q-PCR and RT-PCR are listed in Table 1.

Bisulfite sequencing PCR
Bisulfite sequencing PCR (BSP) was performed to determine methylation of NNAT CpG islands. The CpGenome The results are depicted for CpG 1 in brain a and liver e, for CpG 2 in brain b and liver f, for CpG 3 brain (c) and liver (g), and for CpG 3 in brain (d) and liver (h). Open and closed circles indicate unmethylated and methylated CpG sites, respectively. Numbers in parentheses represent the methylated CpG sites relative to all counted CpG sites Fig. 3 Gamete-specific methylation status of CpG 3. The methylation status of CpG 3 was analyzed via BSP for MII oocyte (a) and sperm (b). Open and closed circles indicate unmethylated and methylated CpG sites, respectively. Numbers in parentheses represent the methylated CpG sites relative to all counted CpG sites TM Turbo Bisulfite Modification Kit (Millipore, Jaffrey, NH, USA) was used for bisulfate treatment of genomic DNA of both liver and brain according to the manufacturer's instructions. Bisulfite-treatment of the DNA of sperms (n = 1 × 10 3 ) and matured oocytes (n = 100) was done according to the instructions of the EZ DNA Methylation-Direct ™ Kit (Zymo Research,CA). Nested PCR was used to amplify CpG islands, followed by T-vector cloning (positive clones, n = 10) and subsequent sequencing analysis. The BSP primers are listed in Table 2.
Authors' contributions YX, TW and ZL conceived, designed and performed the experiments and wrote the manuscript. XC and JD collected the samples. MC performed the statistical analysis. ZL provided guidance. All authors read and approved the final manuscript.