Hypoacetylated H4 is present at regional centromeres; however, its role in kinetochore function is poorly understood. We characterized H4 acetylation at point centromeres in Saccharomyces cerevisiae and determined the consequences of altered H4 acetylation on chromosome segregation. We observed low levels of tetra-acetylated and K16 acetylated histone H4 (H4K16Ac) at centromeres. Low levels of H4K16Ac were also observed at noncentromeric regions associated with Cse4p. Inhibition of histone deacetylases (HDAC) using nicotinamide (NAM) caused lethality in cse4 and hhf1-20 kinetochore mutants and increased centromeric H4K16Ac. Overexpression of Sas2-mediated H4K16 acetylation activity in wild-type cells led to increased rates of chromosome loss and synthetic dosage lethality in kinetochore mutants. Consistent with increased H4K16 acetylation as a cause of the phenotypes, deletion of the H4K16 deacetylase SIR2 or a sir2-H364Y catalytic mutant resulted in higher rates of chromosome loss compared to wild-type cells. Moreover, H4K16Q acetylmimic mutants displayed increased rates of chromosome loss compared to H4K16R nonacetylatable mutants and wild-type cells. Our work shows that hypoacetylated centromeric H4 is conserved across eukaryotic centromeres and hypoacetylation of H4K16 at centromeres plays an important role in accurate chromosome segregation.C ENTROMERES serve as the chromosomal regions on which the kinetochores (i.e., centromeric DNA and associated proteins) are assembled, multisubunit complexes essential for mediating chromosome transmission fidelity (ctf) during cell division. Eukaryotes, such as budding yeast, have "point" centromeres, whereas fission yeast, fruit flies, and humans have "regional" centromeres. The point centromeres are small (125 bp) and consist of CDE I-III, DNA elements that are highly conserved between each centromere. By contrast, regional centromeres are composed of repeated arrays of a-satellite DNA that range in size from 0.1 to 5 Mb (Karpen and Allshire 1997). Despite the lack of sequence conservation, centromeres in all eukaryotes are assembled into a specialized chromatin that contains the centromere specific histone H3 variant (CenH3; Cse4p in budding yeast) (Ekwall 2007).Several reports indicate that a unique chromatin structure is assembled at the centromere and pericentromeric regions in budding yeast. Work by Bloom and Carbon (1982) reported a specialized chromatin that extended several kilobases beyond the centromere sequence on both sides. Studies using fluorescence microscopy and chromosome conformation capture (3C) assays, revealed that the pericentromeric chromatin can form an intramolecular cruciform structure (pericentromeric loop) that has been proposed to be an important component of the mitotic spindle, balancing the forces between the kinetochore and spindle pole body (Bouck et al. 2008;Yeh et al. 2008;Anderson et al. 2009). In turn, disruption of nucleosomes by histone depletion (Saunders et al. 1990;Bouck and Bloom 2007), deletion of both CAC1 and HIR...

Phosducin-like proteins are conserved regulatory components of G-protein signalling pathways, which mediate many physiological processes. Identified throughout eukaryotic genomes, they are thought to serve as regulators of Gβγ assembly. Cryphonectria parasitica, a plant pathogen and causative agent of chestnut blight, contains three Gα, one Gβ, one Gγ subunits and phosducin-like protein BDM-1 that have important roles in pigmentation, sporulation and virulence. Deletion of either Gβ subunit or BDM-1 produces identical phenotypes. Additionally, we report that the Gβ subunit is not detectable in absence of BDM-1. Given that the regulatory role of phosducin-like proteins may be influenced by protein kinase 2 (CK2), we confirmed that BDM-1 is a phosphoprotein that can be targeted by CK2 in vitro. Mutagenesis of the five putative CK2 sites revealed that native phosphorylation likely occurs at two locations. Strains bearing a single or double serine to alanine substitutions at those sites were significantly less virulent with only minor phenotypic changes from vegetative colonies. Therefore, CK2 activity appears to mediate key signals that are required for virulence, but not for vegetative growth. Expression of selected CK2 mutants resulted in reduced accumulation of the Gβ subunit, suggesting that phosphorylation of BDM-1 influences Gβ stability.