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Dicer is essential for formation of the heterochromatin structure in vertebrate cells

Nature Cell Biology volume 6, pages 784–791 (2004)Cite this article

Abstract

RNA interference is an evolutionarily conserved gene-silencing pathway in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs1. The biological function of the RNAi-related pathway in vertebrate cells is not fully understood. Here, we report the generation of a conditional loss-of-function Dicer mutant in a chicken–human hybrid DT40 cell line that contains human chromosome 21. We show that loss of Dicer results in cell death with the accumulation of abnormal mitotic cells that show premature sister chromatid separation. Aberrant accumulation of transcripts from α-satellite sequences, which consist of human centromeric repeat DNAs, was detected in Dicer-deficient cells. Immunocytochemical analysis revealed abnormalities in the localization of two heterochromatin proteins, Rad21 cohesin protein and BubR1 checkpoint protein, but the localization of core kinetochore proteins such as centromere protein (CENP)-A and -C was normal. We conclude that Dicer-related RNA interference machinery is involved in the formation of the heterochromatin structure in higher vertebrate cells.

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Figure 1: Generation of a Dicer−/− clone containing a chicken Dicer transgene under the control of a tet-repressible promoter.
Figure 2: Dicer-deficient cells show premature sister chromatid separation and chromosome mis-segregation.
Figure 3: Localization of a cohesin protein (Rad21), kinetochore proteins (CENP-A and -C) and a checkpoint protein (BubR1) in Dicer-deficient cells.
Figure 4: Centromeric repeat sequences of human chromosome 21 produced long RNA transcripts in Dicer-deficient cells.
Figure 5: Localization of heterochromatin proteins (HP1s) in Dicer-deficient cells.

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Acknowledgements

The authors are very grateful to K. Kita and Y. Fukagawa for technical assistance. This work was supported by the PRESTO of JST and by a Grant-in-Aid for Scientific Research on Priority Areas — Genome Biology, Cancer Cell Biology and Cell Cycle — from the Ministry of Education, Science, Sports and Culture of Japan. The work in the laboratory of M.O. was supported partly by a Human Genome and Tissue Engineering programme grant from the Minister and Labour Science Research Grants.

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Authors and Affiliations

  1. Precursory Research for Embryonic Science and Technology (PRESTO) of Japan Science and Technology Agency (JST), National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, 411-8540, Shizuoka, Japan

    Tatsuo Fukagawa, Masahiro Nogami & Mitsuko Yoshikawa

  2. Institute of Comprehensive Medical Science, Fujita Health University, Toyoake, 470-1101, Aichi, Japan

    Masashi Ikeno & Tuneko Okazaki

  3. Department of Biochemistry, Miyazaki Medical College, Kiyotake, 889-1692, Miyazaki, Japan

    Yasunari Takami & Tatsuo Nakayama

  4. Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Nishimachi 86, Yonago, 683-8503, Tottori, Japan

    Mitsuo Oshimura

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  1. Tatsuo Fukagawa
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Correspondence to Tatsuo Fukagawa.

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Fukagawa, T., Nogami, M., Yoshikawa, M. et al. Dicer is essential for formation of the heterochromatin structure in vertebrate cells. Nat Cell Biol 6, 784–791 (2004). https://doi.org/10.1038/ncb1155

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