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Gene activation-associated long noncoding RNAs function in mouse preimplantation development.

Hamazaki N, Uesaka M, Nakashima K, Agata K, Imamura T - Development (2015)

Bottom Line: Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes.Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation.Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Global COE Program, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan Division of Basic Stem Cell Biology, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

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Effect of knockdown of pancRNAs on partnered gene expression and on DNA methylation. (A) Expression levels of the indicated pancRNAs and their genes measured by qPCR in siRNA-injected 2-cell and 4-cell embryos. Il17d expression was not detectable (N.D.) in 2-cell embryos. (B) DNA methylation levels of the corresponding promoters in siRNA-injected embryos. The regions analyzed are displayed in Fig. 1A. (C) Effect of pancRNA knockdown on blastocyst formation. Asterisks indicate significant differences compared with si Control samples. The numbers of embryos used for injection of si Control, si pancIl17d, si pancMospd3 and si pancTbc1d22a siRNAs were 261, 251, 78 and 70, respectively. *P<0.05; **P<0.01; ***P<0.001. Error bars indicate s.e.m.
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DEV116996F3: Effect of knockdown of pancRNAs on partnered gene expression and on DNA methylation. (A) Expression levels of the indicated pancRNAs and their genes measured by qPCR in siRNA-injected 2-cell and 4-cell embryos. Il17d expression was not detectable (N.D.) in 2-cell embryos. (B) DNA methylation levels of the corresponding promoters in siRNA-injected embryos. The regions analyzed are displayed in Fig. 1A. (C) Effect of pancRNA knockdown on blastocyst formation. Asterisks indicate significant differences compared with si Control samples. The numbers of embryos used for injection of si Control, si pancIl17d, si pancMospd3 and si pancTbc1d22a siRNAs were 261, 251, 78 and 70, respectively. *P<0.05; **P<0.01; ***P<0.001. Error bars indicate s.e.m.

Mentions: Since we previously found that pancRNAs could activate gene expression in rat differentiated cell lines (Tomikawa et al., 2011), we tested whether these developmentally expressed pancRNAs could be involved in the gene upregulation in early mouse embryos by knocking them down using siRNAs. We found that microinjection of siRNA for pancIl17d, pancMospd3 or pancTbc1d22a into the pronucleus suppressed expression of the partner mRNA at the 2-cell and 4-cell stage, when partner expression normally begins (Fig. 3A), and this suppression was accompanied by a lack of decline in the methylation level in the respective promoter region (Fig. 3B; supplementary material Fig. S8). At the Il17d locus, this knockdown effect could be rescued by co-injection of the pancIl17d overexpression vector (Fig. 3B). Overexpressed pancIl17d might work as a sponge for the siRNA, and these pancRNAs might mediate acquisition of the hypomethylated status of the corresponding promoters and potentiate subsequent gene expression after fertilization.Fig. 3.


Gene activation-associated long noncoding RNAs function in mouse preimplantation development.

Hamazaki N, Uesaka M, Nakashima K, Agata K, Imamura T - Development (2015)

Effect of knockdown of pancRNAs on partnered gene expression and on DNA methylation. (A) Expression levels of the indicated pancRNAs and their genes measured by qPCR in siRNA-injected 2-cell and 4-cell embryos. Il17d expression was not detectable (N.D.) in 2-cell embryos. (B) DNA methylation levels of the corresponding promoters in siRNA-injected embryos. The regions analyzed are displayed in Fig. 1A. (C) Effect of pancRNA knockdown on blastocyst formation. Asterisks indicate significant differences compared with si Control samples. The numbers of embryos used for injection of si Control, si pancIl17d, si pancMospd3 and si pancTbc1d22a siRNAs were 261, 251, 78 and 70, respectively. *P<0.05; **P<0.01; ***P<0.001. Error bars indicate s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4352986&req=5

DEV116996F3: Effect of knockdown of pancRNAs on partnered gene expression and on DNA methylation. (A) Expression levels of the indicated pancRNAs and their genes measured by qPCR in siRNA-injected 2-cell and 4-cell embryos. Il17d expression was not detectable (N.D.) in 2-cell embryos. (B) DNA methylation levels of the corresponding promoters in siRNA-injected embryos. The regions analyzed are displayed in Fig. 1A. (C) Effect of pancRNA knockdown on blastocyst formation. Asterisks indicate significant differences compared with si Control samples. The numbers of embryos used for injection of si Control, si pancIl17d, si pancMospd3 and si pancTbc1d22a siRNAs were 261, 251, 78 and 70, respectively. *P<0.05; **P<0.01; ***P<0.001. Error bars indicate s.e.m.
Mentions: Since we previously found that pancRNAs could activate gene expression in rat differentiated cell lines (Tomikawa et al., 2011), we tested whether these developmentally expressed pancRNAs could be involved in the gene upregulation in early mouse embryos by knocking them down using siRNAs. We found that microinjection of siRNA for pancIl17d, pancMospd3 or pancTbc1d22a into the pronucleus suppressed expression of the partner mRNA at the 2-cell and 4-cell stage, when partner expression normally begins (Fig. 3A), and this suppression was accompanied by a lack of decline in the methylation level in the respective promoter region (Fig. 3B; supplementary material Fig. S8). At the Il17d locus, this knockdown effect could be rescued by co-injection of the pancIl17d overexpression vector (Fig. 3B). Overexpressed pancIl17d might work as a sponge for the siRNA, and these pancRNAs might mediate acquisition of the hypomethylated status of the corresponding promoters and potentiate subsequent gene expression after fertilization.Fig. 3.

Bottom Line: Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes.Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation.Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Global COE Program, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan Division of Basic Stem Cell Biology, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

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