Dicer is essential for formation of the heterochromatin structure in vertebrate cells.
RNA interference is an evolutionarily conserved gene-silencing pathway in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. 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 alpha-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.
Fukagawa
Tatsuo
T
Precursory Research for Embryonic Science and Technology of Japan Science and Technology Agency, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan. tfukagaw@lab.nig.ac.jp
Nogami
Masahiro
M
Yoshikawa
Mitsuko
M
Ikeno
Masashi
M
Okazaki
Tuneko
T
Takami
Yasunari
Y
Nakayama
Tatsuo
T
Oshimura
Mitsuo
M
D000818:Animals; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D016923:Cell Death / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival; D002503:Centromere / Q000737:chemistry; D002645:Chickens; D002891:Chromosomes, Human, Pair 21; D004268:DNA-Binding Proteins; D004722:Endoribonucleases / Q000172:deficiency / Q000235:genetics* / Q000502:physiology*; D020868:Gene Silencing; D006570:Heterochromatin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008954:Models, Biological; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D034622:RNA Interference; D015183:Restriction Mapping; D019076:Transgenes
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D006570:Heterochromatin; D009687:Nuclear Proteins; D010750:Phosphoproteins; C429022:RAD21 protein, human; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases