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Molecular and Cellular Biology, March 2001, p. 1953-1961, Vol. 21, No. 6
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.6.1953-1961.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

A Truncated Form of the Human CAF-1 p150 Subunit Impairs the Maintenance of Transcriptional Gene Silencing in Mammalian Cells

Thierry Tchénio, Jean-François Casella, and Thierry Heidmann

Unité des Rétrovirus Endogènes et Eléments Rétroïdes des Eucaryotes Supérieurs, CNRS UMR 1573, Institut Gustave Roussy, 94805 Villejuif, France

Received 21 August 2000/Returned for modification 19 September 2000/Accepted 15 December 2000

Chromatin assembly factor 1 (CAF-1) is a protein complex formed of three subunits, p150, p60, and p48, conserved from the yeast Saccharomyces cerevisiae to humans, which can promote nucleosome assembly onto newly replicated DNA. In S. cerevisiae, deletion of the genes encoding any of the three CAF-1 subunits (cacDelta mutants), although nonlethal, results in a silencing defect of genes packaged into heterochromatin. Here we report on a mammalian cell model that we devised to monitor gene silencing and its reversal in a quantitative manner. This model relies on the use of a cell line stably transfected with a reporter gene in a silenced state. Reversal of reporter gene silencing was achieved upon treatment of the cells with 5-azacytidine, which resulted in the demethylation of the reporter gene copies. We show that expression of a cDNA for the human p150 CAF-1 subunit harboring 5' truncations, but not that of a cDNA encoding the full-length p150 CAF-1 subunit, increases by more than 500-fold the frequency at which transcriptional silencing of the reporter gene copies is reversed in these cells. Reversal of gene silencing is dependent upon expression of a truncated protein, possibly acting as a dominant negative mutant of the wild-type CAF-1, is associated with alterations in chromatin structure as measured by an endonuclease sensitivity assay and is not associated with detectable changes in the methylation status of the silenced genes. These results suggest that the role of CAF-1 in the epigenetic control of gene expression has been conserved between yeast and mammals, despite the lack of DNA methylation in yeast chromatin.

Molecular and Cellular Biology, March 2001, p. 1953-1961, Vol. 21, No. 6
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.6.1953-1961.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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