In mammals, transcription in the zygote begins after fertilization. This transcriptional wave is called zygotic gene activation (ZGA). During ZGA, epigenetic modifications, such as DNA methylation and histone modifications, are dynamically and drastically reconstructed in a sequence-specific manner. However, how such orchestrated gene upregulation is regulated remains unknown. Recently, using microinjection techniques, we have revealed that a class of long noncoding RNAs, named promoter-associated noncoding RNAs (pancRNAs), mediates specific gene upregulation through promoter DNA demethylation during ZGA. Here, we describe the experimental methods available to control the expression levels of pancRNAs and to evaluate epigenetic status after pancRNA manipulation.
Microinjection Long noncoding RNA Mouse DNA methylation
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
We thank Dr. Elizabeth Nakajima for proofreading the manuscript. We thank Katsuhiko Hayashi for discussion. This work was supported by Grants-in-Aid [No. 15H04603] to T.I. from Japan Society for the Promotion of Science (JSPS). N.H. is supported by a JSPS Research Fellowship.
Aoki F, Worrad DM, Schultz RM (1997) Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo. Dev Biol 181:296–307CrossRefPubMedGoogle Scholar
Latham KE, Garrels JI, Chang C et al (1991) Quantitative analysis of protein synthesis in mouse embryos. I. Extensive reprogramming at the one-and two-cell stages. Development 112:921–932PubMedGoogle Scholar
Li E (2002) Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet 3:662–673CrossRefPubMedGoogle Scholar
Farthing CR, Ficz G, Ng RK et al (2008) Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes. PLoS Genet 4:e1000116CrossRefPubMedPubMedCentralGoogle Scholar
Imamura T, Yamamoto S, Ohgane J et al (2004) Non-coding RNA directed DNA demethylation of Sphk1 CpG island. Biochem Biophys Res Commun 322:593–600CrossRefPubMedGoogle Scholar
Tomikawa J, Shimokawa H, Uesaka M et al (2011) Single-stranded noncoding RNAs mediate local epigenetic alterations at gene promoters in rat cell lines. J Biol Chem 286:34788–34799CrossRefPubMedPubMedCentralGoogle Scholar
Hamazaki N, Uesaka M, Nakashima K et al (2015) Gene activation-associated long noncoding RNAs function in mouse preimplantation development. Development 142:910–920CrossRefPubMedPubMedCentralGoogle Scholar
Zhang Y, Rohde C, Tierling S et al (2009) DNA methylation analysis by bisulfite conversion, cloning, and sequencing of individual clones. Methods Mol Biol 507:177–187Google Scholar
Li L-C, Dahiya R (2002) MethPrimer: Designing primers for methylation PCRs. Bioinformatics (Oxford, England) 18:1427–1431CrossRefGoogle Scholar