Modification of the expression profiles of Prnp and marker genes in early stage embryo by DNMT1 modulators

Abstract

The products of Prnp gene are the functional molecule in development, physiology and disease. One of the possible reasons of prion disease is the amount of cellular prion protein (PrPC) in a tissue, and controlling the expression levels of PrPC is suspected as a way of protection the prion. In addition, PrPC can affect stem cell proliferation, metastasis, drug resistant, and cancer stem cell phenotypes through several signaling pathways as a platform. Therefore, it is important to know the expression profiles during development. On the other hand, DNA methylation is a major epigenetic mechanism and plays critical roles in the silencing of retrotransposons, genomic imprinting, and cell differentiation. Even though the mechanisms of epigenetic modification are not fully unmasked during cleavage, the epigenetic modification is well studied. In this study, the expression of the Prnp was examined in the cleavage stage and the changes of developmental genes by DNMT1 modifying molecules, S-adenosyl-homocysteine (SAH), N-phthalyl-L-tryptophan (RG108), and maybridge (Myb1) in WT and bankvole Prnp KI mouse. The developmental rates were high in DNMT1 modulator treated groups with exception. In wild type mice, the development to the hatched stage was similar between group excepted SAH group. Developmental rate to hatched stage were significantly high in Myb1 group. In SAH group, the developmental rate was significantly lower after blastocyst stage compared to the other groups. On the other hand, in bank vole Knock-in mice, the developmental rates were significantly low after 4-cell stage in SAH group. The expression of the potency and lineage related genes such as MuERV-L, Cdx2, Sox2, Cx32, Dsc2, Yap, Cdh1, Pou5f1, Nanog were also modified with the inhibitor specific patterns. In WT, the SAH-treated particularly aberrant expression in these genes, as did the developmental rate decrease. On the one hand, Cdx2 expression was increased in 8-cell stage embryos treated with Myb1, and Pou5f1 gene level was also significantly increased compared to other groups after morula stage. Similarly, KI mice showed decreased expression of the above genes in SAH-treated embryos, and Myb1 treatment significantly increased Pou5f1 gene levels at the blastoderm stage of hatching progression. The expression of imprinting genes and DNMT enzyme genes were also analyzed and did not show significant changes in WT and KI mice. To investigate whether DNMT1 inhibitors affect PRNP expression, it was also identified that the expression profiles of Prnp and PrPc in mouse early embryo. In the wild type, mRNA expression did not show any significant change, but PrPc protein was significantly increased at the morula stage compared to the control. In KI mice, there was a significant increase in both mRNA and protein in RG108-treated morula stage embryos. To investigate whether the above DNMT1 inhibitors regulate expression by modulating the methylation of the CpG region of the Prnp gene, we performed bisulfite sequencing and found that Prnp expression is not regulated through this region. We then examined the effects of DNMT1 inhibitor treatment on embryo development and growth by DNMT inhibitor treated embryo transfer into surrogate mothers. Myb1 treatment significantly increased the probability of live birth after embryo transfer compared to the control group and significantly decreased in SAH-treated embryos. Organ weights of 5-week-old pups born after embryo transfer were no differences in all groups. RNA-seq results showed the high expression of carbohydrate metabolic genes in Myb1 treated embryos, immune related genes in SAH, and cell proliferation related genes in RG108. Put together, it is suggested that the gene expression is modified with the DNMT1 inhibitor specific manner and Myb1 is a useful DNMT1 inhibitor for the early stage embryo development along with the regulation of Prnp expression.