Dominant Mutants of the Saccharomyces cerevisiae ASF1 Histone Chaperone Bypass the Need for CAF-1 in Transcriptional Silencing by Altering Histone and Sir Protein Recruitment

Department of Biology Graduate Program, University of Colorado Health Sciences Center, Aurora, Colorado 80045, USA.
Genetics (Impact Factor: 5.96). 07/2006; 173(2):599-610. DOI: 10.1534/genetics.105.054783
Source: PubMed


Transcriptional silencing involves the formation of specialized repressive chromatin structures. Previous studies have shown that the histone H3-H4 chaperone known as chromatin assembly factor 1 (CAF-1) contributes to transcriptional silencing in yeast, although the molecular basis for this was unknown. In this work we have identified mutations in the nonconserved C terminus of antisilencing function 1 (Asf1) that result in enhanced silencing of HMR and telomere-proximal reporters, overcoming the requirement for CAF-1 in transcriptional silencing. We show that CAF-1 mutants have a drastic reduction in DNA-bound histone H3 levels, resulting in reduced recruitment of Sir2 and Sir4 to the silent loci. C-terminal mutants of another histone H3-H4 chaperone Asf1 restore the H3 levels and Sir protein recruitment to the silent loci in CAF-1 mutants, probably as a consequence of the weakened interaction between these Asf1 mutants and histone H3. As such, these studies have identified the nature of the molecular defect in the silent chromatin structure that results from inactivation of the histone chaperone CAF-1.

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    • "Indeed, CAF-1 plays an essential role in maintaining constitutive heterochromatin in yeast (Huang et al. 2007). Despite the established role of CAF-1 in replication-coupled nucleosome assembly, deletion of any of the three CAF-1 genes has minimal adverse effect on normal growth in yeast (Kaufman et al. 1997), suggesting that other histone chaperones such as Asf1 (anti-silencing factor 1) and HIR/HIRA (histone regulation) may function in H3/H4 assembly cooperatively with CAF-1 (Tamburini et al. 2006; Greenall et al. 2006). The DNA replication checkpoint has a surveillance function that regulates origin firing, maintains the integrity of the stalled replication fork, and prevents cells from proceeding to mitosis before completion of the DNA replication (McNeely et al. 2013). "
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    ABSTRACT: Highly conserved chromatin assembly factor 1 (CAF-1) is required for histone deposition onto newly synthesized DNA to maintain genome stability. This study shows that the fission yeast Pcf1, the large subunit in CAF-1, is crucial for maintaining checkpoint kinase Cds1. Chromatin recruitment of Cds1 is enhanced by Pcf1 overproduction but is attenuated by the Δpcf1 mutation. Mutation of acetylation sites in the histone H4 tail abrogates the chromatin recruitment of Pcf1, which resembles that of Cds1 as reported previously. The present results provide evidence that chromatin recruitment of Pcf1, moderated by Clr6-HDAC activity, is essential for inactivating Cds1.
    Full-text · Article · Jan 2014 · SpringerPlus
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    • "It is not required for chaperone function but it is associated with regulatory roles of the protein (18). For example, it is necessary for interaction with the chromatin fiber (19), transcriptional silencing and it mediates binding of histones to Rad53 (20). Heterogeneity of the C-terminal tails most likely accounts for different functions of Asf1 proteins in eukaryotes (21,22). "
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    ABSTRACT: The anti-silencing function protein 1 (Asf1) is a chaperone that forms a complex with histones H3 and H4 facilitating dimer deposition and removal from chromatin. Most eukaryotes possess two different Asf1 chaperones but their specific functions are still unknown. Trypanosomes, a group of early-diverged eukaryotes, also have two, but more divergent Asf1 paralogs than Asf1 of higher eukaryotes. To unravel possible different functions, we characterized the two Asf1 proteins in Trypanosoma brucei. Asf1A is mainly localized in the cytosol but translocates to the nucleus in S phase. In contrast, Asf1B is predominantly localized in the nucleus, as described for other organisms. Cytosolic Asf1 knockdown results in accumulation of cells in early S phase of the cell cycle, whereas nuclear Asf1 knockdown arrests cells in S/G2 phase. Overexpression of cytosolic Asf1 increases the levels of histone H3 and H4 acetylation. In contrast to cytosolic Asf1, overexpression of nuclear Asf1 causes less pronounced growth defects in parasites exposed to genotoxic agents, prompting a function in chromatin remodeling in response to DNA damage. Only the cytosolic Asf1 interacts with recombinant H3/H4 dimers in vitro. These findings denote the early appearance in evolution of distinguishable functions for the two Asf1 chaperons in trypanosomes.
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    • "Interestingly, as previously observed with cac1Δ mutants (Tamburini et al., 2006), ChIP analysis for pol30-8 cells showed less histone H3 bound in all chromosomal regions tested, including an intergenic region on chromosome VIII-R (Figure 3F). Therefore, the global upregulation of poorly expressed genes could be possibly due to lower histone density on chromatin in pol30-8 mutants. "
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    ABSTRACT: Telomere-associated position-effect variegation (TPEV) in budding yeast has been used as a model for understanding epigenetic inheritance and gene silencing. A widely used assay to identify mutants with improper TPEV employs the URA3 gene at the telomere of chromosome VII-L that can be counterselected with 5-fluoroorotic acid (5-FOA). 5-FOA resistance has been inferred to represent lack of transcription of URA3 and therefore to represent heterochromatin-induced gene silencing. For two genes implicated in telomere silencing, POL30 and DOT1, we show that the URA3 telomere reporter assay does not reflect their role in heterochromatin formation. Rather, an imbalance in ribonucleotide reductase (RNR), which is induced by 5-FOA, and the specific promoter of URA3 fused to ADH4 at telomere VII-L are jointly responsible for the variegated phenotype. We conclude that metabolic changes caused by the drug employed and certain mutants being studied are incompatible with the use of certain prototrophic markers for TPEV.
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