Characterization of Lysine 56 of Histone H3 as an Acetylation Site in Saccharomyces cerevisiae

Abstract

Post-translational histone modifications abound and regulate multiple nuclear processes. Most modifications are targeted to the amino-terminal domains of histones. Here we report the identification and characterization of acetylation of lysine 56 within the core domain of histone H3. In the crystal structure of the nucleosome, lysine 56 contacts DNA. Phenotypic analysis suggests that lysine 56 is critical for histone function and that it modulates formamide resistance, ultraviolet radiation sensitivity, and sensitivity to hydroxyurea. We show that the acetylated form of histone H3 lysine 56 (H3-K56) is present during interphase, metaphase, and S phase. Finally, reverse genetic analysis indicates that none of the known histone acetyltransferases is solely responsible for H3-K56 acetylation in Saccharomyces cerevisiae.In eukaryotes, genetic information is packed in a higher order structure of histones and genomic DNA that is called chromatin. The fundamental unit of chromatin is the nucleosome and consists of 147 bp of DNA wrapped about twice around a histone octamer that contain a histone H3/H4 tetramer and two H2A/H2B dimers (1, 2). Post-translational modifications of the histone tails are linked to different states of chromatin that regulate processes like transcription, DNA repair, replication, and recombination (3-5). Overlapping actions of histone modifying enzymes on the very same or different histone residues generates a combinational complexity of modifications that is called the histone code (5). Hyperacetylation of lysines located in the amino-terminal tail of core histones correlates with transcriptional activation whereas hypoacetylation relates to transcriptional repression (3, 4). Histone acetylation is a dynamic process that is regulated by the opposing activities of histone acetyltransferases (HATs) 1 (6) and histone deacetylases (7). Metylation status of lysines in the aminoterminal tail, and the histone-fold domain of histone H3 plays an important role in the establishment of the active (and/or silenced) state of chromatin (5,8).In contrast, not much is known about histone core domain modifications and their functions. Recently, acetylation of histone H4 lysine 91 was shown to be important for chromatin assembly (9). It is also known that methylation of histone H3 lysine 79 impinges on transcription silencing (10, 11). Furthermore, a globular domain histone mutation, H3 leucine 61 to tryptophan, impaired association of SWI/SNF with chromatin (12). Here we identify and characterize acetylation of histone H3 lysine 56 as a novel core domain histone modification in S. cerevisiae. Histone Purification-Histones were purified according to Edmondson et al. (14) except that zymolyase (Seikagaku Corp. catalog no. 120493) was used at a final concentration of 0.1 mg/ml. MATERIALS AND METHODS Yeast SDS-PAGE and Western Blots-SDS-PAGEand Western blot analysis were performed according to standard procedures (15). Purified histones were separated on 15% SDS-PAGE gels and transferred to polyvinylidene difluori...

Histone H3 Lysine 56 Acetylation: A New Twist in the Chromosome Cycle

“…4 Using cell division cycle (cdc) mutants to arrest yeast cells at different stages of the cell cycle, high levels of K56 acetylation were also detected outside of S-phase. 5 All the studies of K56 acetylation thus far were performed with five distinct affinity-purified polyclonal antibodies raised against synthetic peptides. [4][5][6][7][8] The specificity of all these antibodies for K56 acetylation was rigorously demonstrated by the absence of signal in yeast strains where histone H3 K56 was mutated into a nonacetylatable residue.…”

“…1,2 Six independent research groups recently reported the discovery of lysine 56 as a novel site of histone H3 acetylation in the budding yeast Saccharomyces cerevisiae. [3][4][5][6][7][8] K56 acetylation has also been observed in the fission yeast Schizosaccharomyces pombe, 6 which is evolutionarily very distant from S. cerevisiae. 9 Based on mass spectrometry, K56 acetylation occurs in Plasmodium falciparum (A. Salcedo and H. Stunnenberg, in preparation) and the modification has also been reported in Drosophila.…”

“…7 However, K56 acetylation has thus far not been detected in mammalian cells. 5,7 Lysine 56 (K56) is the last residue of an a-helix, known as aN, that connects the N-terminal tail to the globular domain of H3 (Fig. 1).…”

“…For instance, K56 can be substituted by non-acetylatable residues, such as alanine or arginine. [3][4][5][6][7][8] Thus, the modification is not essential for cell viability or de novo nucleosome assembly. However, the introduction of a negatively charged glutamate residue at position 56 is lethal unless wild type histone H3 is also present in the same cells.…”

“…However, the introduction of a negatively charged glutamate residue at position 56 is lethal unless wild type histone H3 is also present in the same cells. 5 The reason why the H3 K56E mutation is lethal is not known. Nonetheless, this result reinforces the notion that lysine 56 plays a pivotal role in chromatin function.…”