Synthetic Heterochromatin Bypasses RNAi and Centromeric Repeats to Establish Functional Centromeres
Alexander Kagansky,1,*
Hernan Diego Folco,1,*,
Ricardo Almeida,1,*
Alison L. Pidoux,1
Abdelhalim Boukaba,1,
Femke Simmer,1,
Takeshi Urano,2
Georgina L. Hamilton,1
Robin C. Allshire1,||
In the central domain of fission yeast centromeres, the kinetochore
is assembled on CENP-A
Cnp1 nucleosomes. Normally, small interfering
RNAs generated from flanking outer repeat transcripts direct
histone H3 lysine 9 methyltransferase Clr4 to homologous loci
to form heterochromatin. Outer repeats, RNA interference (RNAi),
and centromeric heterochromatin are required to establish CENP-A
Cnp1 chromatin. We demonstrated that tethering Clr4 via DNA-binding
sites at euchromatic loci induces heterochromatin assembly,
with or without active RNAi. This synthetic heterochromatin
completely substitutes for outer repeats on plasmid-based minichromosomes,
promoting de novo CENP-A
Cnp1 and kinetochore assembly, to allow
their mitotic segregation, even with RNAi inactive. Thus, the
role of outer repeats in centromere establishment is simply
the provision of RNAi substrates to direct heterochromatin formation;
H3K9 methylation-dependent heterochromatin is alone sufficient
to form functional centromeres.
2 Department of Biochemistry, Shimane University Faculty of Medicine, 89-1 Enya-cho, Izumo 693-8501, Japan.
* These authors contributed equally to this work.
Present Address: Ludwig Institute for Cancer Research, Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093–0660, USA.
Present Address: Laboratory of Cancer Epigenetics, Faculty of Medicine, Free University of Brussels, 808 route de Lennik, 1070 Brussels, Belgium.
Present Address: Department of Molecular Biology, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands.
|| To whom correspondence should be addressed. E-mail: robin.allshire{at}ed.ac.uk
