Abstract
Ustilago maydis is a haploid basidiomycete with single genes for two distinct histone H3 variants. The solitary U1 gene codes for H3.1, predicted to be a replication-independent replacement histone. The U2 gene is paired with histone H4 and produces a putative replication-coupled H3.2 variant. These predictions were evaluated experimentally. U2 was confirmed to be highly expressed in the S phase and had reduced expression in hydroxyurea, and H3.2 protein was not incorporated into transcribed chromatin of stationary phase cells. Constitutive expression of U1 during growth produced ~25% of H3 as H3.1 protein, more highly acetylated than H3.2. The level of H3.1 increased when cell proliferation slowed, a hallmark of replacement histones. Half of new H3.1 incorporated into highly acetylated chromatin was lost with a half-life of 2.5 h, the fastest rate of replacement H3 turnover reported to date. This response reflects the characteristic incorporation of replacement H3 into transcribed chromatin, subject to continued nucleosome displacement and a loss of H3 as in animals and plants. Although the two H3 variants are functionally distinct, neither appears to be essential for vegetative growth. KO gene disruption transformants of the U1 and U2 loci produced viable cell lines. The structural and functional similarities of the Ustilago replication-coupled and replication-independent H3 variants with those in animals, in plants, and in ciliates are remarkable because these distinct histone H3 pairs of variants arose independently in each of these clades and in basidiomycetes.
Highlights
In research going back decades, it was observed that the composition of nucleosomes across transcribed gene regions, identified in part by high levels of histone acetylation, changed over time
Study of the functions provided by RC and RI H3 variants in animals and in plants is limited because the multiplicity of genes of both types prevents effective use of gene knock-out and replacement approaches
Identification of Ustilago H3 Variants—Sequencing of the genome of the haploid basidiomycete U. maydis revealed the existence of two single-copy histone H3 genes [11]: one on chromosome 11 as a solitary histone, named U1 (GenBank AACP01000135.1, locus UM03916.1) and one, named U2, in a divergently transcribed gene pair with histone H4 on chromosome 6 (GenBank AACP01000090.1, locus UM02709.1) (Fig. 1A)
Summary
Culture of Ustilago—U. maydis 521, strain 9021 obtained from the Fungal Genetics Stock Center (University of MissouriKansas City, Kansas City, MO) and defined as WT, was grown in synthetic dextrose (SD) medium (6.7 g of Difco yeast nitrogen base without amino acids (Benton-Dickinson, Sparks, MI) with 20 g of glucose/liter) at 30 °C on 2% agar or in liquid culture at 150 rpm with typical experimental use at ϳ107 cells/ml. Cells from 0.25 to 2.0 liters of culture were collected by centrifugation (5 min, 800 ϫ g) and resuspended into two to four pellet volumes of 40% GuCl in KPi (40% guanidine HCl, 0.05 M KH2PO4, 0.05 M K2HPO4, adjusted by KOH to pH 6.8, with 1.4 mM 2-mercaptoethanol). Crude histones were dissolved in 0.25 ml of 8 M urea in 1 M acetic acid and injected in the column, equilibrated at 1 ml/min with 40% acetonitrile (Fisher) in 0.1% TFA (Sigma), washed for 2 min in this solvent, and eluted by a gradient from 40 to 55% acetonitrile in 0.1% TFA over 45 min. The two histone H3 variants co-eluted 43 min after injection, as monitored by absorbance at 214 nm They were identified by acid urea Triton (AUT) gel electrophoresis of lyophilized column fractions based on protein size and characteristic affinity for Triton X-100 [15, 16]. Specific radioactivity was determined by densitometry of fluorographic exposures exactly as described [17, 18]
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