The methylation of cytosine at CG sites in the mammalian genome is dynamically reprogrammed during gametogenesis and preimplantation development. It was previously shown that oocyte-derived DNMT1 (a maintenance methyltransferase) is essential for maintaining and propagating CG methylation at imprinting control regions in preimplantation embryos. In mammalian somatic cells, hemimethylated-CG-binding protein UHRF1 plays a critical role in maintaining CG methylation by recruiting DNMT1 to hemimethylated CG sites. However, the role of UHRF1 in oogenesis and preimplantation development is unknown. In the present study, we show that UHRF1 is mainly, but not exclusively, localized in the cytoplasm of oocytes and preimplantation embryos. However, smaller amounts of UHRF1 existed in the nucleus, consistent with the expected role in DNA methylation. We then generated oocyte-specific Uhrf1 knockout (KO) mice and found that, although oogenesis was itself unaffected, a large proportion of the embryos derived from the KO oocytes died before reaching the blastocyst stage (a maternal effect). Whole genome bisulfite sequencing revealed that blastocysts derived from KO oocytes have a greatly reduced level of CG methylation, suggesting that maternal UHRF1 is essential for maintaining CG methylation, particularly at the imprinting control regions, in preimplantation embryos. Surprisingly, UHRF1 was also found to contribute to de novo CG and non-CG methylation during oocyte growth: in Uhrf1 KO oocytes, transcriptionally-inactive regions gained less methylation, while actively transcribed regions, including the imprinting control regions, were unaffected or only slightly affected. We also found that de novo methylation was defective during the late stage of oocyte growth. To the best of our knowledge, this is the first study to demonstrate the role of UHRF1 in de novo DNA methylation in vivo. Our study reveals multiple functions of UHRF1 during the global epigenetic reprogramming of oocytes and early embryos.

Highlights d A forward genetic screen identifies Zfp281 as a factor for ESto-TS-like conversion d Zfp281 shapes the transcriptome of TS cells and regulates early placental development d Zfp281 interacts with MLL or COMPASS subunits and binds to the promoters of target genes d ZNF281 stabilizes the transcriptome in undifferentiated human TS cells

DNA methylation at CG sites is important for gene regulation and embryonic development. In mouse oocytes, de novo CG methylation requires preceding transcription-coupled histone mark H3K36me3 and is mediated by a DNA methyltransferase DNMT3A. DNMT3A has a PWWP domain, which recognizes H3K36me2/3, and heterozygous mutations in this domain, including D329A substitution, cause aberrant CG hypermethylation of regions marked by H3K27me3 in somatic cells, leading to a dwarfism phenotype. We herein demonstrate that D329A homozygous mice show greater CG hypermethylation and severer dwarfism. In oocytes, D329A substitution did not affect CG methylation of H3K36me2/3-marked regions, including maternally methylated imprinting control regions; rather, it caused aberrant hypermethylation in regions lacking H3K36me2/3, including H3K27me3-marked regions. Thus, the role of the PWWP domain in CG methylation seems similar in somatic cells and oocytes; however, there were cell-type-specific differences in affected regions. The major satellite repeat was also hypermethylated in mutant oocytes. Contrary to the CA hypomethylation in somatic cells, the mutation caused hypermethylation at CH sites, including CA sites. Surprisingly, oocytes expressing only the mutated protein could support embryonic and postnatal development. Our study reveals that the DNMT3A PWWP domain is important for suppressing aberrant CG hypermethylation in both somatic cells and oocytes but that D329A mutation has little impact on the developmental potential of oocytes.

Transcription is a dynamic process. To detect the dynamic relationship among protein clusters of RNA polymerase II and coactivators, gene loci, and transcriptional activity, we insert an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site. By optimizing the individual elements, we develop the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNA polymerase II and BRD4 are observed proximal to the transcription start site of Nanog when the gene is transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 tend to be located near the transcription start site, even without transcription activity. Thus, the STREAMING-tag system reveals the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding transcriptional regulation in living cells.