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The pluripotency of naïve mouse embryonic stem cells (mES) is regulated by multiple signaling pathways, with inhibition of protein kinase C (PKCi) playing a particularly important role in maintaining naïve mES. However, the regulatory function of nucleosome remodeling and deacetylase (NuRD) complex in mES cultured in a PKCi system is unknown. We found that, compared with 2iL-derived mES, PKCi-derived mES showed low mRNA expression of NuRD complex subunits, including MBD3, HDAC1/HDAC2, MTA1, and RbAP46/RbAP48. Western blot showed that PKCi-derived mES expressed lower protein levels of MBD3 and HDAC2 at passage 3, as well as MBD3, HDAC2, and MTA1 at passage 10, indicating that PKCi suppressed NuRD complex expression. Knockdown of MBD3 increased PKCi-derived mES pluripotency by increasing NANOG and OCT4 expression and colony formation. By contrast, overexpression of MBD3 or removal of PKC inhibitor-induced differentiation of mES, results in reduced NANOG, OCT4, and REX1 expression and colony formation, increased differentiation-related gene expression, and differentiation into flat cells. Knockdown of MBD3 in mES upon PKC inhibitor removal partially reversed cell differentiation. Our results show that the regulatory NuRD complex and its MBD3 subunit influence the naïve pluripotency of mES cultured in a PKCi system.

In mammals, epigenetic marks on the X chromosomes are involved in dosage compensation. Specifically, they are required for X chromosome inactivation (XCI), the random transcriptional silencing of one of the two X chromosomes in female cells during late blastocyst development. During natural reproduction, both X chromosomes are active in the female zygote. In somatic-cell cloning, however, the cloned embryos receive one active (Xa) and one inactive (Xi) X chromosome from the donor cells. Patterns of XCIhave been reported normal in cloned mice, but have yet to be investigated in other species. We examined allele-specific expression of the X-linked monoamine oxidase type A (MAOA) gene and the expression of nine additional X-linked genes in nine cloned XX calves. We found aberrant expression patterns in nine of ten X-linked genes and hypomethylation of Xist in organs of deceased clones. Analysis of MAOA expression in bovine placentae from natural reproduction revealed imprinted XCI with preferential inactivation of the paternal X chromosome. In contrast, we found random XCI in placentae of the deceased clones but completely skewed XCI in that of live clones. Thus, incomplete nuclear reprogramming may generate abnormal epigenetic marks on the X chromosomes of cloned cattle, affecting both random and imprinted XCI.