Genetic evidence for Dnmt3a‐dependent imprinting during oocyte growth obtained by conditional knockout with Zp3‐Cre and complete exclusion of Dnmt3b by chimera formation

Abstract

In the male and female germ-lines of mice, both of the two de novo DNA methyltransferases Dnmt3a and Dnmt3b are expressed. By the conditional knockout experiments using the TnapCre gene, we previously showed that deletion of Dnmt3a in primordial germ cells disrupts paternal and maternal imprinting, however, Dnmt3b mutants did not show any defect. Here, we have knocked out Dnmt3a after birth in growing oocytes by using the Zp3-Cre gene and obtained genetic evidence that de novo methylation by Dnmt3a during the oocyte growth stage is indispensable for maternal imprinting. We also carried out DNA methylation analysis in the mutant oocytes and embryos and found that hypomethylation of imprinted genes in Dnmt3a-deficient oocytes was directly inherited to the embryos, but repetitive elements were re-methylated during development. Furthermore, we show that Dnmt3b-deficient cells can contribute to the male and female germ-lines in chimeric mice and can produce normal progeny, establishing that Dnmt3b is dispensable for mouse gametogenesis and imprinting. Finally, Dnmt3-related protein Dnmt3L is not only essential for methylation of imprinted genes but also enhances de novo methylation of repetitive elements in growing oocytes.

HDAC1 and HDAC2 in mouse oocytes and preimplantation embryos: Specificity versus compensation

“…57,58 DNA methyltransferases A (DNMT3A) and its cofactor DNA methyltransferases L (DNMT3L) are required to establish DNA methylation in growing oocytes. 59 Maternalspecific de novo DNA methylation likely contributes to gene regulation in the oocyte and marks specific genes for activity in the embryo, as in the case of imprinted genes. 60 Indeed, Hdac1/2 mutant oocytes fail to establish normal imprinting DNA methylation marks.…”

Genomic imprinting and its relevance to congenital disease, infertility, molar pregnancy and induced pluripotent stem cell

“…[20][21][22] This enzyme forms a complex with the structurally related, germ-cell-specific cofactor Dnmt3L, which enhances the catalytic activity of Dnmt3a and is important for imprint establishment (Figure 1). 21,[23][24][25] The other de novo methyltransferase Dnmt3b is not involved, except at one particular ICR (see later).…”

Genomic imprinting in mammals: its life cycle, molecular mechanisms and reprogramming

“…It was later confirmed that the mutant oocytes indeed lack DNA methylation at these germline DMRs [39]. It was also established that DNMT3B is dispensable for the establishment of the maternal imprints [39].…”

“…In these studies, embryos derived from the mutant oocytes displayed loss of DNA methylation at the maternal alleles of the DMRs that are normally maternally methylated, and biallelic expression or silencing of the imprinted genes associated with these DMRs [36][37][38][39]. It was later confirmed that the mutant oocytes indeed lack DNA methylation at these germline DMRs [39]. It was also established that DNMT3B is dispensable for the establishment of the maternal imprints [39].…”

“…When the genes coding for these proteins were respectively knocked out in the germline of mice, it was found that DNMT3A and DNMT3L are required for the establishment of the maternal imprints in growing oocytes [36][37][38][39]. In these studies, embryos derived from the mutant oocytes displayed loss of DNA methylation at the maternal alleles of the DMRs that are normally maternally methylated, and biallelic expression or silencing of the imprinted genes associated with these DMRs [36][37][38][39]. It was later confirmed that the mutant oocytes indeed lack DNA methylation at these germline DMRs [39].…”