Site-specific Loss of Acetylation upon Phosphorylation of Histone H3

Abstract

Post-translational modification of histones is a central aspect of gene regulation. Emerging data indicate that modification at one site can influence modification of a second site. As one example, histone H3 phosphorylation at serine 10 (Ser 10 ) facilitates acetylation of lysine 14 (Lys 14 ) by Gcn5 in vitro (1, 2). In vivo, phosphorylation of H3 precedes acetylation at certain promoters. Whether H3 phosphorylation globally affects acetylation, or whether it affects all acetylation sites in H3 equally, is not known. We have taken a genetic approach to this question by mutating Ser 10 in H3 to fix either a negative or a neutral charge at this position, followed by analysis of the acetylation states of the mutant histones using site-specific antibodies. Surprisingly, we find that conversion of Ser 10 to glutamate (S10E) or aspartate (S10D) causes almost complete loss of H3 acetylation at lysine 9 (Lys 9 ) in vivo. Acetylation of Lys 9 is also significantly reduced in cells bearing mutations in the Glc7 phosphatase that increase H3 phosphorylation levels. Mutation of Ser 10 in H3 and the concomitant loss of Lys 9 acetylation has minimal effects on expression of a Gcn5-dependent reporter gene. However, synergistic growth defects are observed upon loss of GCN5 in cells bearing H3 Ser 10 mutations that are reminiscent of delays in G 2 /M progression caused by combined loss of GCN5 and acetylation site mutations. Together these results demonstrate that H3 phosphorylation directly causes sitespecific and opposite changes in acetylation levels of two residues within this histone, Lys 9 and Lys 14 , and they highlight the importance of these histone modifications to normal cell functions.

Valosin-containing Protein (VCP) in Novel Feedback Machinery between Abnormal Protein Accumulation and Transcriptional Suppression

“…VCP(DEA) and VCP (AAA). Although these substitutions may not perfectly represent modified or non-modified protein configurations, this method has been well-used in elucidating biological functions of the modified or non-modified proteins, respectively, such as kinases, kinase substrates, tubulin, histones etc (45)(46)(47).…”

Liquid chromatography mass spectrometry profiling of histones

“…Traditionally, immunoassay methodologies such as immunoprecipitation, Western-Blotting, and immunofluorescence have dominated the biologists' toolbox for the characterization of histones and their PTMs [4][5][6]. However, these approaches have disadvantages that limit their application.…”

Impact of nucleosome dynamics and histone modifications on cell proliferation during Arabidopsis development

“…Recent evidence in animal cells supports the notion that H3 phosphorylation is not only involved in chromosome compaction but also in transcriptional regulation. Thus, H3S10ph can increase H3K14ac (Lo et al, 2000) and suppress H3K9ac (Edmondson et al, 2002) and H3K9me (Rea et al, 2000), but information on these functional links in Arabidopsis is lacking.…”