DNA Methylation And Histone Modifications Regulate The Expression Of Bovine ZAR1 Gene

Zygote arrest 1(ZAR1) gene plays a crucial role in maternal zygotic transition(MZT). Bovine ZAR1 is a single copy gene, located on chromosome 6, the full-length is 4126bp containing 1487bp, with 4 extrons and 3 introns, including 1155bp open reading frame, encoding precursor protein of 384 amino acid. The protein has an typical plant homeobox zing finger PHD motif involved in transcription regulation. It has been postulated that DNA methyltransferases interacting with HDACs repress transcription through PHD motif. ZAR1 coding region is proved including numerous palindrome repeats forming stem-loops and cruciform structure, which may interact directly with proteins. Short palindrome repeats are characteristic of DNA-binding domain of nuclear receptors. UGUA sequence and polyadenylation element were detected within 3'-UTR of ZAR1 mRNA, associated with ZAR1 translation regulation.Mouse ZAR1 was expressed specifically in ovary but not in testis. Although ZAR1^-/- mice are viable and grossly normal, ZAR1^-/- females are infertile. Ovarian development and oogenesis and the early stages after fertilization seems unimpaired, but most embryos from ZAR1^-/- females arrest at the one-cell stage. Fewer than 20% of the embryos derived from ZAR1^-/- females develop to the two-cell stage and no embryos succeed to develop to the four-cell stage. ZAR1 was found in variant tissues including ovary, testis, skeletal muscle, and myocardium in cattle as well as in embryo development, which is not restricted in ovary. There is no report about the epigenetic regulation of the gene. In this study, RT-PCR and bisulfite-sequencing PCR and chromatin immuno-precipitation assay(ChIP) were used to detect the dynamic change of DNA methylation and histone modification in the regulatory region of bovine ZAR1 gene. Real time PCR was applied to detect ZAR1 dynamic expression pattern in bovine oocytes and IVF embryos.1. expression of ZAR1 gene in bovineRT-PCR was used to detect the expression of ZAR1 in bovine heart, kidney, liver and testis. The result showed that the expression of ZAR1 had a tissue-specific pattern that is abundant in testis, fewer in kidney and heart, but not detectable in liver.2. DNA methylation status of ZAR1 geneDNA methylation is one of the modes regulating gene expression, mainly methylated only at 5'cytosines in the CpG dinucleotide changed into guanosine. Without changing DNA sequence, DNA methylation influences the function of gene through regulating the gene expression. In this study 11 CpG sites in the 5' regulation region of ZAR1 gene were chosen to examine their DNA methylation status by bisulfite-sequencing PCR (BSP). The result showed that the methylation level in heart was significantly higher than in the other three tissues, demonstrating ZAR1 expression is associated with DNA methylation.3. Histone modification pattern of ZAR1 geneHistone modification is another important epigenetic regulation mechanism, which modulate gene expression through transforming the conformation of chromosome and thereby impact the interaction between the transcription factors and DNA. Generally H3 acetylation is related to gene activation, while H3K9 methylation is considered to be associated with gene silence.ChIP was used to investigate the histone modification of 5' regulatory region in bovine heart, kidney, liver and testis. The result showed that acetylation level of H3 was higher in heart and lower in liver. The high level of H3 acetylation may lead to the expression in heart: the low level of H3 acetylation may be involved in the non-expression of ZAR1 in liver . H3K9 methylation was not detectable in tested tissues confirming H3K9me is associated with gene silencing and this result was consistent with the general expression of ZAR1 gene in bovine. It was reported that the major targets of H3K9 methylation were satellite domain and coding region, rather than the promoter region. Consequently, it is possible that H3K9 methylation was not the major modification form in the region detected in this study.4. ZAR1 expression pattern in bovine oocytesThe methods of studying mRNA level include Northern blot, RT-PCR, real time PCR, gene chip and so on. Real time PCR is remarkable in qualitative analysis, exhibiting more specificity and sensitivity. Real time PCR was used to detect ZAR1 mRNA level during oocyte maturation(GV,PMI,MI,AI-TI,MII). The level of ZAR1 mRNA is high in GV and PMI phases with significant difference and lower in MI, AI-TI and MII phases without significant difference.5. ZAR1 expression pattern in bovine IVF embryosZAR1 is a critical gene during embryo development and thereby it'll be helpful for the further research to detect ZAR1 expression pattern in bovine IVF embryos by real time PCR. The results showed that ZAR1 exhibited higher expression level in 2-cell, 4-cell and 8-cell with significant difference and lower level in morula and blastula without significant difference.In conclusion, ZAR1 plays a critical role in maternal zygotic transition(MZT). This study showed the expression of ZAR1 had a tissue-specific pattern in bovine, which is detectable in heart, kidney and testis, but not in liver. The investigation of DNA methylation and histone modification status in the 5' regulation region of ZAR1 demonstrated both of them are correlated with the tissue-specific expression patten. The levels of ZAR1 mRNA present dynamic changes in oocyes maturation and early embryo development: ZAR1 mRNA is higher expressed in GV phase, decreases during oocyte maturation. It is notable that ZAR1 mRNA evidently increased in 2-cell stage, compared with MII, which is inferred that ZAR1 has a compact correlation with MZT.