RESUMO
Histone H3 proteins are highly conserved across all eukaryotes and are dynamically modified by many post-translational modifications (PTMs). Here we describe a method that defines the evolution of the family of histone H3 proteins, including the emergence of functionally distinct variants. It combines information from histone H3 protein sequences in eukaryotic species with the evolution of these species as described by the tree of life (TOL) project. This so-called TOL analysis identified the time when the few observed protein sequence changes occurred and when distinct, co-existing H3 protein variants arose. Four distinct ancient duplication events were identified where replication-coupled (RC) H3 variants diverged from replication-independent (RI) forms, like histone H3.3 in animals. These independent events occurred in ancestral lineages leading to the clades of metazoa, viridiplantae, basidiomycota, and alveolata. The proto-H3 sequence in the last eukaryotic common ancestor (LECA) was expanded to at least 133 of its 135 residues. Extreme conservation of known acetylation and methylation sites of lysines and arginines predicts that these PTMs will exist across the eukaryotic crown phyla and in protists with canonical chromatin structures. Less complete conservation was found for most serine and threonine phosphorylation sites. This study demonstrates that TOL analysis can determine the evolution of slowly evolving proteins in sequence-saturated datasets.
Assuntos
Evolução Molecular , Variação Genética/genética , Histonas/genética , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Histonas/química , Humanos , Processamento de Proteína Pós-Traducional/genéticaRESUMO
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.
Assuntos
Replicação do DNA , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Histonas/genética , Histonas/metabolismo , Ustilago/genética , Ustilago/metabolismo , Acetilação , Sequência de Aminoácidos , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Técnicas de Inativação de Genes , Loci Gênicos/genética , Histonas/biossíntese , Histonas/química , Dados de Sequência Molecular , Mutação , Nucleossomos/metabolismo , Transcrição Gênica , Ustilago/citologiaRESUMO
The study of histone acetylation in plants started with protein purification and sequencing, with gel analysis and the use of radioactive tracers. In alfalfa, acid urea Triton gel electrophoresis and in vivo labeling with tritated acetate and lysine quantified dynamic acetylation of core histones and identified the replication-coupled and -independent expression patterns of the histone H3.1 and H3.2 variants. Pulse-chase analyses demonstrated protein turnover of newly synthesized histone H3.2 and thereby identified the replacement H3 histones of plants which maintain the nucleosome density of transcribed chromatin. Sequence analysis of histone H4 revealed acetylation of lysine 20, a site typically methylated in animals and yeasts. Histone deacetylase inhibitors butyrate and trichostatin A are metabolized in alfalfa, but loss of TSA is slow, allowing its use to induce transient hyperacetylation of histones H2B, H4 and H3. This article is part of a Special Issue entitled: Epigenetic Control of cellular and developmental processes in plants.
Assuntos
Histonas/história , Plantas/genética , Plantas/metabolismo , Acetilação , Sequência de Aminoácidos , Sequência Conservada , Epigênese Genética , Histonas/genética , Histonas/metabolismo , História do Século XX , Medicago sativa/genética , Medicago sativa/metabolismo , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Pesquisa/história , Homologia de Sequência de AminoácidosRESUMO
Histone Lys methylation plays an important role in determining chromatin states and is mostly catalyzed by SET domain-containing proteins. The outcome, transcriptional repression or activation, depends on the methylated histone residue, the degree of methylation, and the chromatin context. Dimethylation or trimethylation of histone H3 Lys 4 (H3K4me2 or H3K4me3) has been correlated with transcriptionally competent/active genes. However, H3K4 methylation has also been implicated in gene silencing. This dualistic nature of the H3K4 methyl mark has thus far remained unresolved. In the green alga Chlamydomonas reinhardtii, Mut11p, related to a subunit of trithorax-like methyltransferase complexes, is required for transcriptional silencing. Here, we show that Mut11p interacts with conserved components of H3K4 methyltransferase machineries, and an affinity-purified Mut11p complex(es) methylates histones H3, H2A, and H4. Moreover, a Mut11 mutant showed global loss of monomethylated H3K4 (H3K4me1) and an increase in dimethylated H3K4. By chromatin immunoprecipitation analysis, this strain also displayed substantial reduction in H3K4me1 and enrichment in H3K4me2 associated with transcriptionally derepressed genes, transgenes, and retrotransposons. RNA interference-mediated suppression of Set1, encoding an H3K4 methyltransferase, induced similar phenotypes, but of lower magnitude, and no detectable increase in H3K4me2. Together, our results suggest functional differentiation between dimethyl H3K4 and monomethyl H3K4, with the latter operating as an epigenetic mark for repressed euchromatin.
Assuntos
Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/genética , Eucromatina/metabolismo , Inativação Gênica , Histonas/metabolismo , Lisina/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Algas/classificação , Proteínas de Algas/genética , Sequência de Aminoácidos , Animais , Chlamydomonas reinhardtii/metabolismo , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Histonas/genética , Substâncias Macromoleculares , Metilação , Dados de Sequência Molecular , Filogenia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Interferência de RNA , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
In yeast, the establishment and maintenance of a transcriptionally silent chromatin state are dependent upon the acetylation state of the N terminus of histone proteins. Histone H4 proteins that contain mutations in N-terminal lysines disrupt heterochromatin and result in yeast that cannot mate. Introduction of a wild-type copy of histone H4 restores mating, despite the presence of the mutant protein, suggesting that mutant H4 protein is either excluded from, or tolerated in, chromatin. To understand how the cell differentiates wild-type histone and mutant histone in which the four N-terminal lysines were replaced with alanine (H4-4A), we analyzed silencing, growth phenotypes, and the histone composition of chromatin in yeast strains coexpressing equal amounts of wild-type and mutant H4 proteins (histone H4 heterozygote). We found that histone H4 heterozygotes have defects in heterochromatin silencing and growth, implying that mutations in H4 are not completely recessive. Nuclear preparations from histone H4 heterozygotes contained less mutant H4 than wild-type H4, consistent with the idea that cells exclude some of the mutant histone. Surprisingly, the N-terminal nuclear localization signal of H4-4A fused to green fluorescent protein was defective in nuclear localization, while a mutant in which the four lysines were replaced with arginine (H4-4R) appeared to have normal nuclear import, implying a role for the charged state of the acetylatable lysines in the nuclear import of histones. The biased partial exclusion of H4-4A was dependent upon Cac1p, the largest subunit of yeast chromatin assembly factor 1 (CAF-1), as well as upon the karyopherin Kap123p, but was independent of Cac2p, another CAF-1 component, and other chromatin assembly proteins (Hir3p, Nap1p, and Asf1p). We conclude that N-terminal lysines of histone H4 are important for efficient histone nuclear import. In addition, our data support a model whereby Cac1p and Kap123 cooperate to ensure that only appropriately acetylated histone H4 proteins are imported into the nucleus.
Assuntos
Núcleo Celular/metabolismo , Cromatina/química , Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Ligação a DNA/fisiologia , Histonas/química , Acetilação , Alanina/química , Western Blotting , Proteínas de Transporte/química , Proteínas de Ciclo Celular/química , Cromatina/metabolismo , Fator 1 de Modelagem da Cromatina , Imunoprecipitação da Cromatina , Inativação Gênica , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Heterozigoto , Immunoblotting , Imunoprecipitação , Chaperonas Moleculares , Mutação , Fenótipo , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Transcrição Gênica , beta CarioferinasRESUMO
A yeast gene has been identified that encodes a novel, evolutionarily conserved Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A. The gene has been named NAT4. Recombinant Nat4 protein acetylated a peptide corresponding to the N-terminal tail of H4, but not an H3 peptide nor the peptide adrenocorticotropin. H4 and H2A are N-terminally acetylated in all species from yeast to mammals and hence blocked from sequencing by Edman degradation. In contrast, H4 and H2A purified from a nat4 mutant were unacetylated and could be sequenced. Analysis of yeast histones by acid-urea gel electrophoresis showed that all the H4 and H2A from the mutant migrated more rapidly than the same histones from a wild type strain, consistent with the histones from the mutant having one extra positive charge due to one less acetylated amino group. A comparison of yeast proteins from wild type and a nat4 mutant by two-dimensional gel electrophoresis showed no evidence that other yeast proteins are substrates of this acetyltransferase. Thus, Nat4 may be dedicated specifically to the N-terminal acetylation of histones H4 and H2A. Surprisingly, nat4 mutants grow at a normal rate and have no readily observable phenotypes.
Assuntos
Acetiltransferases/química , Acetiltransferases/metabolismo , Acetiltransferases/fisiologia , Histonas/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Hormônio Adrenocorticotrópico/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Histona Acetiltransferases , Histonas/metabolismo , Dados de Sequência Molecular , Mutação , Acetiltransferase N-Terminal D , Peptídeos/química , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Saccharomyces cerevisiae/metabolismo , Ureia/farmacologiaRESUMO
Dynamic histone acetylation is a characteristic of chromatin transcription. The first estimates for the rate of acetylation turnover of plants are reported, measured in alfalfa cells by pulse, pulse-chase, and steady-state acetylation labeling. Acetylation turnover half-lives of about 0.5 h were observed by all methods used for histones H3, H4, and H2B. This is consistent with the rate at which changes in gene expression occur in plants. Treatment with histone deacetylase inhibitor Trichostatin A (TSA) induced hyperacetylation at a similar rate. Replacement histone variant H3.2, preferentially localized in highly acetylated chromatin, displayed faster acetyl turnover. Histone H2A with a low level of acetylation was not subject to rapid turnover or hyperacetylation. Patterns of acetate labeling revealed fundamental differences between histone H3 versus histones H4 and H2B. In H3, acetylation of all molecules, limited by lysine methylation, had similar rates, independent of the level of lysine acetylation. Acetylation of histones H4 and H2B was seen in only a fraction of all molecules and involved multiacetylation. Acetylation turnover rates increased from mono- to penta- and hexaacetylated forms, respectively. TSA was an effective inhibitor of alfalfa histone deacetylases in vivo and caused a doubling in steady-state acetylation levels by 4-6 h after addition. However, hyperacetylation was transient due to loss of TSA inhibition. TSA-induced overexpression of cellular deacetylase activity produced hypoacetylation by 18 h treatment with enhanced acetate turnover labeling of alfalfa histones. Thus, application of TSA to change gene expression in vivo in plants may have unexpected consequences.
Assuntos
Inibidores Enzimáticos/farmacologia , Histona Desacetilases/metabolismo , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Acetatos/metabolismo , Acetilação , Inibidores de Histona Desacetilases , Cinética , Medicago sativa/metabolismo , Coloração e Rotulagem/métodosRESUMO
Histone acetylation, discovered more than 40 years ago, is a reversible modification of lysines within the amino-terminal domain of core histones. Amino-terminal histone domains contribute to the compaction of genes into repressed chromatin fibers. It is thought that their acetylation causes localized relaxation of chromatin as a necessary but not sufficient condition for processes that repackage DNA such as transcription, replication, repair, recombination, and sperm formation. While increased histone acetylation enhances gene transcription and loss of acetylation represses and silences genes, the function of the rapid continuous or repetitive acetylation and deacetylation reactions with half-lives of just a few minutes remains unknown. Thirty years of in vivo measurements of acetylation turnover and rates of change in histone modification levels have been reviewed to identify common chromatin characteristics measured by distinct protocols. It has now become possible to look across a wider spectrum of organisms than ever before and identify common features. The rapid turnover rates in transcriptionally active and competent chromatin are one such feature. While ubiquitously observed, we still do not know whether turnover itself is linked to chromatin transcription beyond its contribution to rapid changes towards hyper- or hypoacetylation of nucleosomes. However, recent experiments suggest that turnover may be linked directly to steps in gene transcription, interacting with nucleosome remodeling complexes.
Assuntos
Acetiltransferases/metabolismo , Histonas/química , Acetilação , Animais , Inibidores de Histona Desacetilases , Humanos , Cinética , Medicago sativa , Radioisótopos de Fósforo/metabolismo , RNA Mensageiro/metabolismo , Coloração e Rotulagem , Tetrahymena , Transcrição Gênica/fisiologiaRESUMO
This study explored the possibility of using non-viral, plant-based gene sequences to create strong and constitutive expression vectors. Replacement histone H3 genes are highly and constitutively expressed in all plants. Sequences of the cloned alfalfa histone H3.2 gene MsH3gl were tested. Constructs of the beta-glucuronidase (GUS) reporter gene were produced with H3.2 gene promoter and intron sequences. Their efficiency was compared with that of the commonly used strong 35S cauliflower mosaic virus promoter in transgenic tobacco plants. Combination of the H3.2 promoter and intron produced significantly higher GUS expression than the strong viral 35S promoter. Histochemical GUS analysis revealed a constitutive pattern of expression. Thus, alfalfa replacement H3 gene sequences can be used instead of viral promoters to drive heterologous gene expression in plants, avoiding perceived risks of viral sequences.