Elsevier

Gene

Volume 676, 15 November 2018, Pages 243-248
Gene

Research paper
DNA methylation status of CRABP2 promoter down-regulates its expression

https://doi.org/10.1016/j.gene.2018.07.049Get rights and content

Highlights

  • The CRABP2 plays an important role in beef quality, yield and fat deposition.

  • The appropriate DNA methylation is essential for normal gene function.

  • The Jianxian Red cattle exhibited a higher mRNA level and DNA methylation level than the hybrid cattle during in muscle and fat tissues.

  • A useful molecular marker for muscle developmental and as a model for studies in other species.

Abstract

As an important epigenetic modification DNA methylation is catalyzed by DNA methylation transferases (DNMTs) and occurs mainly in CpG islands. DNA methylation plays an important role in regulates gene expression, cell differentiation, genetic imprinting and tumor therapy. Retinoic acid-binding proteins (RAC) is vital for the absorption, transport, metabolism and maintenance of homeostasis of retinoic acid, which in turn regulates the differentiation and proliferation of cells by regulating the transcription of many target genes, therefore, these proteins influence differentiation and proliferation of adipocytes and muscle fibroblasts. Thus, cellular retinoic acid binding protein 2 (CRABP2) may be a candidate gene which affects beef quality, yield and fat deposition. The aim of this study was to evaluate the expression and the methylation pattern on the differentially methylated region (DMR) of the promoter of CRABP2. The DNA methylation pattern was tested by bisulfite sequencing polymerase chain reaction (BSP), the quantitative real-time PCR (qPCR) was used to analysis the expression of CRABP2 gene. The results showed that the DNA methylation level was higher in purebred cattle breed than that in hybrid cattle breeds which was negative correlation with the expression of the gen. These results indicate that the methylation status of the CRABP2 DMR can regulate mRNA expression. What's more, there are different methylation and expression patterns in different breeds and tissues which may influence the phenotype, and the results may be a useful parameter to investigate the function of CRABP2 in muscle and fat developmental in Chinese cattle.

Introduction

As a main epigenetic modification, DNA methylation is the process of covalent attachment of methyl and cytosine provided by S-adenosylmethionine (SAM) under the action of DNA methyltransferases (DNMTs) (Cheng and Blumenthal, 2008). In animal models, this modification typically occurs in the CpG-rich and symmetrical region of the DNA strand, often referred to as CpG islands, most of which are found in the promoter region of housekeeping genes and developmental genes (Illingworth and Bird, 2009), and DNA methylation in this area will pass through a CpG binding domain proteins block the binding of transcription factors and RNA polymerase to the template strand and thereby inhibit the transcriptional process(Bogdanovic and Veenstra, 2009), further, affect the corresponding region of the gene expression level, and eventually causing the change of corresponding biological function. It's not only affecting the genes expression of cellular, but also can be inherited as cells divide (Feng et al., 2006). Up to now, the main regulatory mechanisms of methylation on gene expression have been found including: (1) Affecting the conformation of DNA molecules. (2) Sterically hindered effect, Methyl of 5-methylcytosine can prevent the transcription factor binding with DNA, thus interfering the normal function of transcription factor play. (3) Protein specificity, after CpG site methylation, its recruitment and binding of specific proteins to achieve the regulation of gene expression. (4) Affecting the transcription level. RNA-mediated DNA methylation leads to gene silencing at the transcriptional level (Chen et al., 2001). Many DNA methylation occurred in promoter regions, and methylation level is usually negatively correlated with the gene expression, indicating its restrain gene transcription (Li et al., 2011). Different tissues showed different methylation patterns to accommodate their unique structure and function (Tajbakhsh et al., 1997). DNA methylation plays an important role in the development of muscle and fat, there was a slight change in the methylation level of the genome from myoblasts to myotubes but a significant increase in the genomic Further analysis revealed that these highly hypermethylated promoter region genes (transcription factors ID4 and ZNF238) Associated with muscle contraction and other muscular processes (Miyata et al., 2014).

Retinoic acid binding protein 2 (CRABP2) plays an important role in the uptake, transport, metabolism and maintenance of homeostasis of Retinoic acid, in turn, regulates cell differentiation and proliferation by regulating the transcription of many target genes. CRABP2 plays a vital role in differentiation of skeletal muscle, and affects the differentiation and proliferation of muscle cells and muscle fibroblasts, affecting animal growth and development (Yuan et al., 2013). Retinoic acid (RA) is a key regulator of gene expression during embryonic development and plays an important role in cell growth, differentiation and visual systems (Manolescu et al., 2010; Chaudhuri et al., 1999), CRABP2 is a nuclear shuttle protein that translocates RA into the nucleus and interacts with its receptor RAR.RXR, as a co-activator of RAR.RXR in combination with a RA-responsive element of a specific gene to regulate gene expression (Wolf, 2000), CRABP2 is also involved in post-transcriptional regulation of RNA, the binding protein HuR binds to the 3 ‘UTR region to stabilize the RNA and against RNA degradation, whereas CRABP2 interacts with HuR to enhance its binding to the RNA of interest and improve the stability of these transcripts (Vreeland et al., 2014). The expression of CRABP2 was found change significantly in various tumors, or increase or decrease. Since the CRABP2 gene plays an important role in the development of muscle and fat, and DNA methylation affects gene expression in post-transcriptional regulation. Once, we designed experiments to study the CRABP2 methylation effects on the expression of the gene, in order to provide basis for the improvement of Chinese cattle.

Firstly, CRABPs were identified on chickens (Sani and Hill, 1974), later, one after another in mice, rats, cattle and people were cloned out. In vertebrate cells, CRABPs contain two highly homologous proteins, the cellular retinoic acid binding protein 1 (CRABP1) and the cellular retinoic acid binding protein 2 (CRAPB2) (Aström et al., 1991; Banaszak et al., 1994). The cattle CRABP2 gene is located on chromosome 3 and consists of 4 exons with a full cDNA length of 430 bp, and encoding 138 amino acids. Yuan J et al. found CRABP2 expression upward in the process of C2C12 differentiation, through lentiviral transfection technology found that CRABP2 will accelerate the process of C2C12 cells differentiate into myotubes in vitro (Yuan et al., 2013), so, the CRABP2 gene plays an important role in muscle development. Campos, B et al. detected extensive CpG methylation upstream of the CRABP2 gene locus in a study sample comprising 100 astrocytic gliomas of WHO Grade II to IV. Compared to nontumorous control samples, tumors revealed increased CpG methylation, and methylation levels were inversely correlated to CRABP2 mRNA expression (Campos et al., 2012).

For the first time, to the study compare the DNA methylation profiles in the DMR of the promoter of the CRABP2 gene in muscle and fat tissues from three cattle breeds (Jiaxian Red cattle, Jiaxian Red cattle × Angus, Jiaxian Red cattle × Simmental) and their relationships to mRNA expression patterns of adult. The objective was to identify the relation between epigenetic modifications and gene expression changes in cattle, which possibly contributed to animal breeding and genetics.

Section snippets

Tissue collection and preparation of DNA and RNA

The experimental animals used in this study were a famous elite native breed of Chinese Jiaxian Red cattle and its Crossbred cattle. 9 two-year-old adult cattles (there are three individuals for each breed) were used in this study. There is no direct and collateral blood relationship within the last 3 generations among the three cattle from each group. The tissues were collected into sterile physiological saline immediately after removed from the slaughtered cattles at a local abattoir, and

Expression profile

We used the cDNA from two tissues (muscle and fat) of nine cattle (Jiaxian Red cattle, Jiaxian Red cattle × A ngus, Jiaxian Red cattle × Simmental) to detect the mRNA distribution of bovine CRABP2. The relative expression levels of the CRABP2 gene in two tissues from three cattle breeds are shown in Fig. 1. The results of qPCR analysis showed that CRABP2 has a broad tissue distribution in all examined breeds. On the other hand, the results indicate that the expression levels of CRABP2 were

Discussion

As an important genetic modification, DNA methylation modulates gene expression through methyl modification without changing the gene sequence, and can be passed on to offspring. It has an important impact on the regulation of gene expression and critical in establishing patterns of gene repression during development (Cedar and Bergman, 2009). In recent years, the research on DNA methylation has become a hot spot, new achievements in the research of methylation on tumor treatment and growth and

Conclusions

This study is the first to report changes of methylation and expression patterns in the CRABP2 in different tissues and breeds of cattle. The hybridization adult bovine group exhibited a lower mRNA level and significantly higher DNA methylation level than the purebred bovine group in muscle, and this characteristic may be useful as a molecular marker for muscle developmental in cattle and as a model for studies in other species, potentially contributing to an improvement of marker-assisted

Acknowledgements

This study was supported by the Program of National Natural Science Foundation of China (31601926, 31772574), Natural Science Basic Research Program of Shanxi (2017JM3024, 2017JM3012), Postdoctoral Science Foundation of China (2015M570856), National Beef Cattle and Yak Industrial Technology System (CARS-37), Key Scientific Research Projects for Higher Education of Henan (17A230005), Openness and cooperation projects science and technology of Henan (172106000041), Science and Technology Project

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    These authors contributed equally to this work.

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