Histone variants and acetylated species from the alfalfa plant Medicago sativa

The histones from the alfalfa plant Medicago sativa have been characterized in terms of type variants and levels of acetylation. Histones were isolated directly from total plant tissue (callus), eliminating the need to develop methods for nuclear isolation. An acid-urea-polyacrylamide gel with a transverse Triton X-100 gradient resolved and identified in a single gel at least one type of histone H4, two variant forms of histone H2B, two variant forms of histone H3, and four variant forms of histone H2A from a crude histone preparation. Histone H4 was present 25% in an unmodified state and 75% as monomodified, presumably as monoacetylated histone. Both histone H3 variants displayed five bands, consistent with up to four internal sites of acetylation. The two H3 variants differed in their steady-state level of acetylation, suggesting that they may reside in different chromatin environments. Several histone H1 species were identified by solubility and cross-reactivity with antiserum raised against the globular part of bovine H1(0), indicating conservation of epitopes between histone H1 of mammals and higher plants.     

A drought-stress-inducible histone gene in Arabidopsis thaliana is a member of a distinct class of plant linker histone variants

We have isolated and characterized a gene, His1-3, encoding a structurally divergent linker histone in Arabidopsis thaliana. Southern and northern hybridization data indicate that A. thaliana expresses three single-copy linker histone genes, each encoding a structurally distinct variant. H1-3 is a considerably smaller protein (167 amino acids with a mass of 19.0 kDa) than any other described linker histone from higher eukaryotes. We examined the expression of His1-3 at the RNA and protein levels and found that it is induced specifically by water stress. In contrast, expression of His1-1, His1-2 and His4 appear unaffected by water stress. Furthermore, the primary structure of the protein possesses distinct characteristics that are shared with another drought-inducible linker histone, H1-D, isolated from Lycopersicon pennellii. Based on structural characteristics of the deduced protein and its inducible expression, we hypothesize that H1-3 and H1-D are linker histone variants that have specialized roles in the structure and function of plant chromatin and therefore they can be considered to be members of a unique subclass of plant histones. Immunoblotting with an antibody produced against a short polypeptide in the conserved domain of this subtype indicates that similar proteins may exist in other plants.     

Evolution of histone H3: emergence of variants and conservation of post-translational modification sites

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.     

Characterization of sequence variability in nucleosome core histone folds

The three-helix, approximately 65-residue histone fold domain is the most structurally conserved part of the core histones H2A, H2B, H3, and H4. However, it evinces a notable degree of sequence variation within and between histone classes. We used two approaches to characterize sequence variation in these histone folds, toward elucidating their structure/function relationships and evolution. On the one hand we asked how much of the sequence variation seen in structure-based alignments of the folds maintains physicochemical properties at a position, and on the other, whether conservation correlates to structural importance, as measured by the number of residue-to-residue contacts a position makes. Strong physicochemical conservation or correlation of conservation to contacts would support the idea that functional constraints, rather than genetic drift, determines the observed range of variants at a given position. We used an 11-state table of physicochemical properties to classify each position in the core histone fold (CHF) alignments, and a public website (http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/valdar/scorecons_server.pl) to score conservation. We found that, depending on histone class, from 38 to 77% of CHF positions are maximally conserved physicochemically, and that for H2B, H3, and H4 the degree to which a position is conserved correlates positively to the number of contacts made by the residue at that position in the crystal structure of the nucleosome core particle. We also examined the correlation between conservation and the type of contact (e.g., inter- or intrachain, histone-histone, or histone-DNA, etc.). For H2B, H3, and H4 we found a positive correlation between conservation and number of interchain protein contacts. No such correlation or statistical significance was found for DNA or intrachain contacts. This suggests that variations in the CHF sequences could be functionally constrained by requirements to make sufficient interchain histone contacts. We also suggest that inventory of histone residue variants can augment functional studies of histones. An example is presented for histone H3.     

Histone phosphorylation during sea urchin development

Studies on histone phosphorylation during transitions in chromatin structure occurringin vivoduring spermatogenesis and early embryogenesis in sea urchins are reviewed and evaluated in the light of recent studies on histone phosphorylation occurring during chromatin synthesis in frog egg extractsin vitroand evidence that protein kinases and phosphatases play direct roles in the regulation of cellular structure. Sperm-specific histone variants Sp H1 and Sp H2B are maintained as phosphorylated derivatives N and O/P throughout spermatogenesis and early embryogenesis and egg specific histone variants CS H1 and CS H2A are phosphorylated during early embryogenesis. These developmental correlations provide clues about the roles of histone phosphorylation in control of chromatin structurein vivoand provide a basis for the interpretation of data obtained from in-vitro sperm chromatin remodeling in egg extracts and from biochemical studies on the effects of histone phosphorylation on DNA binding. The potential consequences for chromatin structure of the various histone phosphorylation events observed in sea urchins and frog egg extracts are discussed.     

A haploid affair: core histone transitions during spermatogenesis.

The process of meiosis reduces a diploid cell to four haploid gametes and is accompanied by extensive recombination. Thus, the dynamics of chromatin during meiosis are significantly different than in mitotic cells. As spermatogenesis progresses, there is a widespread reorganization of the haploid genome followed by extensive DNA compaction. It has become increasingly clear that the dynamic composition of chromatin plays a critical role in the activities of enzymes and processes that act upon it. Therefore, an analysis of the role of histone variants and modifications in these processes may shed light upon the mechanisms involved and the control of chromatin structure in general. Histone variants such as histone H3.3, H2AX, and macroH2A appear to play key roles in the various stages of spermiogenesis, in addition to the specifically modulated acetylation of histone H4 (acH4), ubiquitination of histones H2A and H2B (uH2A, uH2B), and phosphorylation of histone H3 (H3p). This review will examine recent discoveries concerning the role of histone modifications and variants during meiosis and spermatogenesis.     

Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth

At least six histone H1 variants exist in somatic mammalian cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition, H1 seems to be actively involved in the regulation of gene expression. However, it is not well known whether the different variants have distinct roles or if they regulate specific promoters. We have explored this by inducible shRNA-mediated knock-down of each of the H1 variants in a human breast cancer cell line. Rapid inhibition of each H1 variant was not compensated for by changes of expression of other variants. Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down. Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing. On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type. Thus, specific phenotypes are observed in breast cancer cells depleted of individual histone H1 variants, supporting the theory that distinct roles exist for the linker histone variants.     

Linker histones play a role in male meiosis and the development of pollen grains in tobacco

To examine the function of linker histone variants, we produced transgenic tobacco plants in which major somatic histone variants H1A and H1B were present at approximately 25% of their usual amounts in tobacco chromatin. The decrease in these major variants was accompanied by a compensatory increase in the four minor variants, namely, H1C to H1F. These minor variants are smaller and less highly charged than the major variants. This change offered a unique opportunity to examine the consequences to a plant of major remodeling of its chromatin set of linker histones. Plants with markedly altered proportions of H1 variants retained normal nucleosome spacing, but their chromosomes were less tightly packed than those of control plants. The transgenic plants grew normally but showed characteristic aberrations in flower development and were almost completely male sterile. These features correlated with changes in the temporal but not the spatial pattern of expression of developmental genes that could be linked to the abnormal flower phenotypes. Preceding these changes in flower morphology were strong aberrations in male gametogenesis. The earliest symptoms may have resulted from disturbances in correct pairing or segregation of homologous chromosomes during meiosis. No aberrations were observed during mitosis. We conclude that in plants, the physiological stoichiometry and distribution of linker histone variants are crucial for directing male meiosis and the subsequent development of functional pollen grains.     

Identification of a replication-independent replacement histone H3 in the basidiomycete Ustilago maydis

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.     

The human histone H3 complement anno 2011

Histones are highly basic, relatively small proteins that complex with DNA to form higher order structures that underlie chromosome topology. Of the four core histones H2A, H2B, H3 and H4, it is H3 that is most heavily modified at the post-translational level. The human genome harbours 16 annotated bona fide histone H3 genes which code for four H3 protein variants. In 2010, two novel histone H3.3 protein variants were reported, carrying over twenty amino acid substitutions. Nevertheless, they appear to be incorporated into chromatin. Interestingly, these new H3 genes are located on human chromosome 5 in a repetitive region that harbours an additional five H3 pseudogenes, but no other core histone ORFs. In addition, a human-specific novel putative histone H3.3 variant located at 12p11.21 was reported in 2011. These developments raised the question as to how many more human histone H3 ORFs there may be. Using homology searches, we detected 41 histone H3 pseudogenes in the current human genome assembly. The large majority are derived from the H3.3 gene H3F3A, and three of those may code for yet more histone H3.3 protein variants. We also identified one extra intact H3.2-type variant ORF in the vicinity of the canonical HIST2 gene cluster at chromosome 1p21.2. RNA polymerase II occupancy data revealed heterogeneity in H3 gene expression in human cell lines. None of the novel H3 genes were significantly occupied by RNA polymerase II in the data sets at hand, however. We discuss the implications of these recent developments.     

Proteomic analysis reveals the differential histone programs between male germline cells and vegetative cells in Lilium davidii

In flowering plants, male germline fate is determined after asymmetric division of the haploid microspore. Daughter cells have distinct fates: the generative cell (GC) undergoes further mitosis to generate sperm cells (SCs), and the vegetative cell (VC) terminally differentiates. However, our understanding of the mechanisms underlying germline development remains limited. Histone variants and modifications define chromatin states, and contribute to establishing and maintaining cell identities by affecting gene expression. Here, we constructed a lily protein database, then extracted and detailed histone entries into a comprehensive lily histone database. We isolated large amounts of nuclei from VCs, GCs and SCs from lily, and profiled histone variants of all five histone families in all three cell types using proteomics approaches. We revealed 92 identities representing 32 histone variants: six for H1, 11 for H2A, eight for H2B, five for H3 and two for H4. Nine variants, including five H1, two H2B, one H3 and one H4 variant, specifically accumulated in GCs and SCs. We also detected H3 modification patterns in the three cell types. GCs and SCs had almost identical histone profiles and similar H3 modification patterns, which were significantly different from those of VCs. Our study also revealed the presence of multiple isoforms, and differential expression patterns between isoforms of a variant. The results suggest that differential histone programs between the germline and companion VCs may be established following the asymmetric division, and are important for identity establishment and differentiation of the male germline as well as the VC.     

Distinctive core histone post-translational modification patterns in Arabidopsis thaliana

Post-translational modifications of histones play crucial roles in the genetic and epigenetic regulation of gene expression from chromatin. Studies in mammals and yeast have found conserved modifications at some residues of histones as well as non-conserved modifications at some other sites. Although plants have been excellent systems to study epigenetic regulation, and histone modifications are known to play critical roles, the histone modification sites and patterns in plants are poorly defined. In the present study we have used mass spectrometry in combination with high performance liquid chromatography (HPLC) separation and phospho-peptide enrichment to identify histone modification sites in the reference plant, Arabidopsis thaliana. We found not only modifications at many sites that are conserved in mammalian and yeast cells, but also modifications at many sites that are unique to plants. These unique modifications include H4 K20 acetylation (in contrast to H4 K20 methylation in non-plant systems), H2B K6, K11, K27 and K32 acetylation, S15 phosphorylation and K143 ubiquitination, and H2A K144 acetylation and S129, S141 and S145 phosphorylation, and H2A.X S138 phosphorylation. In addition, we found that lysine 79 of H3 which is highly conserved and modified by methylation and plays important roles in telomeric silencing in non-plant systems, is not modified in Arabidopsis. These results suggest distinctive histone modification patterns in plants and provide an invaluable foundation for future studies on histone modifications in plants.     

Dynamic acetylation of lysine-4-trimethylated histone H3 and H3 variant biology in a simple multicellular eukaryote

Dynamic acetylation of all lysine-4-trimethylated histone H3 is a complex phenomenon involved in Immediate-early gene induction in metazoan eukaryotes. Higher eukaryotes express repeated copies of three closely related H3 variants, inaccessible to genetic analysis. We demonstrate conservation of these phenomena in Dictyostelium which has three single-copy H3 variant genes. Although dynamic acetylation is targeted to two H3 variants which are K4-trimethylated, K9-acetylation is preferentially targeted to one. In cells lacking Set1 methyltransferase and any detectable K4-trimethylation, dynamic acetylation is lost demonstrating a direct link between the two. Gene replacement to express mutated H3 variants reveals a novel interaction between K4-trimethylation on different variants. Cells expressing only one variant show defects in growth, and in induction of a UV-inducible gene, demonstrating the functional importance of variant expression. These studies confirm that dynamic acetylation targeted to H3K4me3 arose early in evolution and reveal a very high level of specificity of histone variant utilization in a simple multicellular eukaryote.     

植物组蛋白变体生物学功能

组蛋白变体是重要的表观遗传调控因子,能够在染色质特定位置替换常规组蛋白,维持染色质结构进而保证转录激活或抑制的顺利进行.目前,组蛋白变体的调控功能已成为植物学研究领域的一个热点.近年来,随着植物组蛋白变体生物学功能研究的不断深入,发现组蛋白变体能够在植物生长发育和环境应答调控等多个生物学过程中发挥重要作用.该文简要介绍...