Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase

Abstract

The Gcn5p histone acetyltransferase exhibits a limited substrate specificity in vitro. However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined. To probe these questions, we mutated specific lysines in the N-termini of histones H3 and H4 and examined the effects of these mutations in yeast strains with and without functional GCN5. We found that in vivo, GCN5 is required either directly or indirectly for the acetylation of several sites in H3 and H4 in addition to those recognized by the recombinant enzyme in vitro. Moreover, in the absence of GCN5, cells accumulate in G 2 /M indicating that Gcn5p functions are important for normal cell-cycle progression. Mutation of K14 in H3, which serves as the major target of recombinant Gcn5p acetylation in vitro, confers a strong, synthetic growth defect in gcn5 cells. Synergistic growth defects were also observed in gcn5 cells carrying mutations in lysine pairs (K8/K16 or K5/K12) in histone H4. Strikingly, simultaneous mutation of K14 in H3 and K8 and K16 in H4 to arginine, or deletion of either the H3 or the H4 N-terminal tail, results in the death of gcn5 cells. Mutation of these same three sites to glutamine is not lethal. Indeed, this combination of mutations largely bypasses the need for GCN5 for transcriptional activation by Gal4-VP16, supporting an important role for histone acetylation in Gcn5p-mediated regulation of transcription. Our data indicate that acetylation of particular lysines in histones H3 and H4 serves both unique and overlapping functions important for normal cell growth, and that a critical overall level of histone acetylation is essential for cell viability.

Developmental potential of Gcn5−/− embryonic stem cells in vivo and in vitro

“…4D). Of interest, we previously determined that gcn5⌬ yeast cells accumulate in G2/M phase (Zhang et al, 1998). Together, these results indicate that Gcn5 is required for efficient mitotic progression.…”

Splitting of H3–H4 tetramers at transcriptionally active genes undergoing dynamic histone exchange

“…YKY64 was generated by first transforming pHHF2-HHT2VSVx3 into the histone shuffle strain WZY42 (38) and selecting on 5-FOA-containing plates for cells that lost the original URA3 plasmid encoding H3 and H4. A second plasmid p316H4H3-HAx2 was then transformed to produce the final strain.…”

Proper expression of the Gcn5 histone acetyltransferase is required for neural tube closure in mouse embryos

“…Gcn5 Ϫ/Ϫ embryos exhibit a large increase in apoptotic cells as early as E7.5 (Xu et al, 2000), and deletion of Gcn5 in chicken cells (Kikuchi et al, 2005) or yeast (Zhang et al, 1998) leads to defective cell cycle progression. Therefore, we next determined whether neural tube closure defects in the Gcn5 flox(neo)/⌬ mutants might be due to changes in cell proliferation or survival.…”

“…Immunoblots were performed as described (Lin et al, 2007) using a Gcn5-specific antisera (Abcam) at 1:400 dilution, a H3 K9,18Ac-specific antisera (Zhang et al, 1998) at 1:1,000 dilution, an H3-secific antisera (Abcam) at 1:5,000 dilution, and an actin-specific antibody (Sigma) at 1:4,000 dilution.…”