Abstract
Regulation of DNMT1 is critical for epigenetic control of many genes and for genome stability. Using phylogenetic analysis we characterized a block of 27 nucleotides in the 3′UTR of Dnmt1 mRNA identical between humans and Xenopus and investigated the role of the individual elements contained within it. This region contains a cytoplasmic polyadenylation element (CPE) and a Musashi binding element (MBE), with CPE binding protein 1 (CPEB1) known to bind to the former in mouse oocytes. The presence of these elements usually indicates translational control by elongation and shortening of the poly(A) tail in the cytoplasm of the oocyte and in some somatic cell types. We demonstrate for the first time cytoplasmic polyadenylation of Dnmt1 during periods of oocyte growth in mouse and during oocyte activation in Xenopus. Furthermore we show by RNA immunoprecipitation that Musashi1 (MSI1) binds to the MBE and that this element is required for polyadenylation in oocytes. As well as a role in oocytes, site-directed mutagenesis and reporter assays confirm that mutation of either the MBE or CPE reduce DNMT1 translation in somatic cells, but likely act in the same pathway: deletion of the whole conserved region has more severe effects on translation in both ES and differentiated cells. In adult cells lacking MSI1 there is a greater dependency on the CPE, with depletion of CPEB1 or CPEB4 by RNAi resulting in substantially reduced levels of endogenous DNMT1 protein and concurrent upregulation of the well characterised CPEB target mRNA cyclin B1. Our findings demonstrate that CPE- and MBE-mediated translation regulate DNMT1 expression, representing a novel mechanism of post-transcriptional control for this gene.
Highlights
Maternal stores of DNMT1 mRNA and protein, accumulated in the egg during oogenesis in vertebrates, are responsible for maintenance methylation in the early embryo, which is reliant on these stores prior to the handover of developmental control to the zygotic genome in the maternal-to-zygotic transition (MZT)
We demonstrate that MSI1 expression peaks in growing mouse oocytes, correlating with Dnmt1 polyadenylation, and that MSI interacts with endogenous Dnmt1 mRNA by RNA-immunoprecipitation
We previously identified a cytoplasmic polyadenylation element (CPE) in the 39UTR of the Dnmt1 gene which is conserved between human and mouse (11)
Summary
Maternal stores of DNMT1 mRNA and protein, accumulated in the egg during oogenesis in vertebrates, are responsible for maintenance methylation in the early embryo, which is reliant on these stores prior to the handover of developmental control to the zygotic genome in the maternal-to-zygotic transition (MZT). A special isoform of DNMT1 is expressed only in oocytes (DNMT1o), transcribed from a unique 59 exon, and is more stable than the isoform expressed in somatic cells (DNMT1s) [1]. The maternal stores of DNMT1o appear to be sufficient to allow progression to the blastocyst stage in mouse. DNA methylation in mammalian oocytes is important for the regulation of imprinted genes, disruption of which causes several human disease syndromes [2]. Imprinted genes are active from only one parental chromosome, either the paternal or maternal allele, and the alleles show differential DNA methylation. Deletion of DNMT1o in mouse oocytes causes loss of genomic imprints in offspring and the postimplantation death of resulting embryos [3,4]
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