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

Abstract

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 (cac⌬ 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.

E2F RegulatesFASCIATA1, a Chromatin Assembly Gene Whose Loss Switches on the Endocycle and Activates Gene Expression by Changing the Epigenetic Status

“…In human cells, loss of CAF-1 activity by eliminating the function of the p150 or the p60 subunits inhibits nucleosome assembly, induces S-phase arrest, and causes cell death (Hoek and Stillman, 2003;Ye et al, 2003;Nabatiyan and Krude, 2004;Nabatiyan et al, 2006). In addition, CAF-1 has been involved in transcriptional silencing (Tchenio et al, 2001). In plants, mutations in the FAS1 or the FAS2 genes cause pleiotropic developmental defects, including fasciation, dwarfism, flower abnormalities and reduced fertility, Figure 8.…”

Chromatin assembly factor CAF-1 is required for cellular differentiation during plant development

“…As CAF-1 facilitates replicationcoupled chromatin assembly, it may be needed to faithfully duplicate epigenetic information in chromatin during mitosis (Enomoto and Berman, 1998;Ridgway and Almouzni, 2000;van Nocker, 2003;Henikoff et al, 2004). Indeed, CAF-1 is needed for the maintenance of gene silencing in yeast, mammals and plants (Enomoto and Berman, 1998;Tchenio et al, 2001;Takeda et al, 2004;Ono et al, 2006;Schönrock et al, 2006b). In Arabidopsis, CAF-1 contributes to the compaction of heterochromatin but is not essential for the silencing of most heterochromatic genes (Schönrock et al, 2006b).…”

“…Transgenic approaches were therefore used to analyze CAF-1 function during the development of model organisms. For example, expression of a truncated version of human p150 interfered with the maintenance of transcriptional gene silencing in mammalian cells (Tchenio et al, 2001), and expression of a truncated version of p150 in Xenopus caused severe defects in embryo development, suggesting that CAF-1 is essential in vertebrates (Quivy et al, 2001). This hypothesis was strongly supported by RNAi knock-down experiments of p150 in human cell lines.…”

Histone Deposition Proteins: Links between the DNA Replication Machinery and Epigenetic Gene Silencing

“…Likewise, Arabidopsis mutants lacking CAF-1 display loss of transcriptional gene silencing and developmental defects in meristematic tissue (Kaya et al 2001;Takeda et al 2004). Additionally, human cells overexpressing a dominant-negative fragment of the large subunit of CAF-1 display reduced transcriptional silencing of a reporter gene (Tchenio et al 2001). Together, these data demonstrate that CAF-1 plays a role in regulating chromatin-mediated transcriptional states, and that this function is conserved throughout eukaryotic organisms.…”