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

Abstract

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.

On the origin and functions of RNA-mediated silencing: from protists to man

“…Moreover, in organisms that contain a single Dicer gene such as S. pombe and vertebrates, Dicer-null mutants are RNAi-defective but viable at the cellular level (Giraldez et al 2005;Kanellopoulou et al 2005;Martienssen et al 2005;Murchison et al 2005;Volpe et al 2002). Though these mutants commonly show reactivation of transposons and/or repetitive sequences, deficient (hetero)chromatin formation, and/or abnormal chromosome segregation; and vertebrate germ cells fail to differentiate (Fukagawa et al 2004;Kanellopoulou et al 2005;Martienssen et al 2005;Murchison et al 2005). Second, if a particular function is now widely represented in all examined eukaryotic supergroups, the most parsimonious scenario would suggests that it was already operative in the last common ancestor of eukaryotes.…”

“…Interestingly, the mechanism(s) of RNAi-mediated (hetero)chromatin formation also appears to have diverged in present day eukaryotes since, for instance, an RdRP has been implicated in this process in S. pombe Verdel et al 2004;Volpe et al 2002) but it occurs in the absence of RdRPs in D. melanogaster and vertebrates (Fukagawa et al 2004;Kanellopoulou et al 2005;Kavi et al 2005;Pal-Bhadra et al 2004;Ting et al 2005;Weinberg et al 2006). Adding to this complexity, cytosine DNA methylation and histone modifications seem to be interconnected in self-reinforcing feedback loops in higher eukaryotes (Fuks 2005), although the role (if any) of the RNAi machinery in this cycle is not clear.…”

“…Adding to this complexity, cytosine DNA methylation and histone modifications seem to be interconnected in self-reinforcing feedback loops in higher eukaryotes (Fuks 2005), although the role (if any) of the RNAi machinery in this cycle is not clear. Further, in S. pombe and chicken DT40 cells RNAi-mediated (hetero)chromatin formation may now play a critical role in determining chromosome structure and function during mitosis and/or meiosis (Fukagawa et al 2004;Martienssen et al 2005;Wong and Choo 2004). Conversely, in other organisms such as N. crassa the RNAi machinery appears to be dispensable for the methylation of both DNA and H3K9 associated with repetitive sequences (Chicas et al 2004;Freitag et al 2004), whereas in mouse there might be cell-type-specific differences in the mechanism(s) of (hetero)chromatin formation (Kanellopoulou et al 2005;Murchison et al 2005).…”

Heterochromatin and its Relationship to Cell Senescence and Cancer Therapy

“…2 Second, heterochromatin plays a structural role. For example, the heterochromatin at repetitive pericentromeric DNA that flanks the chromosome centromeres and kinetochores is necessary for sister chromatid cohesion, [5][6][7][8] correct assembly of kinetochores 9 and, perhaps, for proper orientation of sister kinetochores to promote biorientation of chromosome attachments in mitosis. 10 For all these reasons, pericentromeric heterochromatin is crucial for equal segregation of chromosomes between daughter cells in mitosis.…”

L’hétérochromatine constitutive dans tous ses états

“…Les centromères chez les eucaryotes supérieurs produisent aussi des ARN non-codants ( Figure 1A) [10][11][12] qui ne sont apparemment pas associés à la production de siARN. Pourtant, la machinerie d'interférence elle-même semble jouer un rôle dans la structuration des centromères puisque l'inactivation de la RNAse III dicer conduit à des altérations de l'architecture hétérochromatinienne centromérique [13]. Par ailleurs, des expériences de traitement de cellules vivantes par la RNase ont montré qu'un composant ribonucléique est nécessaire au maintien de l'organisation des centromères et à la présence de HP1 à l'hétérochro-matine péricentrique [14], mais la nature de l'ARN mis en jeu reste inconnue.…”