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.     

Origin of H1 linker histones.

In which taxa did H1 linker histones appear in the course of evolution? Detailed comparative analysis of the histone H1 and histone H1-related sequences available to date suggests that the origin of histone H1 can be traced to bacteria. The data also reveal that the sequence corresponding to the 'winged helix' motif of the globular structural domain, a domain characteristic of all metazoan histone H1 molecules, is evolutionarily conserved and appears separately in several divergent lines of protists. Some protists, however, appear to have only a lysine-rich basic protein, which has compositional similarity to some of the histone H1-like proteins from eubacteria and to the carboxy-terminal domain of the H1 linker histones from animals and plants. No lysine-rich basic proteins have been described in archaebacteria. The data presented in this review provide the surprising conclusion that whereas DNA-condensing H1-related histones may have arisen early in evolution in eubacteria, the appearance of the sequence motif corresponding to the globular domain of metazoan H1s occurred much later in the protists, after and independently of the appearance of the chromosomal core histones in archaebacteria.     

Nucleotide sequence and expression of a maize H1 histone cDNA.

The first complete amino acid sequence of a H1 histone of a monocotyledonous plant was deduced from a cDNA isolated from a maize library. The encoded H1 protein is 245 amino acid-long and shows the classical tripartite organization of this class of histones. The central globular region of 76 residues shows 60% sequence homology with H1 proteins from dicots but only 20% with the animal H1 proteins. However, several of the amino acids considered as being important in the structure of the nucleosome are conserved between this protein and its animal counterparts. The N-terminal region contains an equal number of acidic and basic residues which appears as a general feature of plant H1 proteins. The 124 residue long and highly basic C-terminal region contains a 7-fold repeated element KA/PKXA/PAKA/PK. Southern-blot hybridization showed that the H1 protein is encoded by a small multigene family. Highly homologous H1 gene families were also detected in the genomes of several more or less closely related plant species. The general expression pattern of these genes was not significantly different from that of these genes encoding the core-histones neither during germination nor in the different tissues of adult maize.     

Genes encoding a histone H3.3-like variant in Arabidopsis contain intervening sequences.

Two genes encoding a particular H3 histone variant were isolated from Arabidopsis thaliana. These genes differ from the H3 genes previously cloned from Arabidopsis and other plants by several interesting properties: (1) the two genes are located close to each other; (2) their coding regions are interrupted by two or three small introns, the two closest to the initiation codon being located at the same place in the two genes; (3) another, long intron is located in the 5'-untranslated region just before the initiation codon of gene I as deduced from the sequence of several corresponding cDNAs, and very likely also of gene II; (4) these genes do not show preferential expression in organs containing meristematic tissues contrary to the classical intronless replication-dependent histone genes, thus suggesting that their expression is not replication-dependent; (5) the protein encoded by both genes is the same and corresponds to a minor H3 variant highly conserved among all the plant species studied up to now. All these characteristics are common with the animal replication-independent H3.3 histone genes and it is assumed that the genes described here are the first example of the equivalent H3.3 gene family in plants. Interestingly, the promoter regions of the two genes have the same general structure as the Arabidopsis intronless genes. Possible implications on the regulation of H3 genes expression are discussed.     

N- and C-terminal domains determine differential nucleosomal binding geometry and affinity of linker histone isotypes H1(0) and H1c

Eukaryotic linker or H1 histones modulate DNA compaction and gene expression in vivo. In mammals, these proteins exist as multiple isotypes with distinct properties, suggesting a functional significance to the heterogeneity. Linker histones typically have a tripartite structure composed of a conserved central globular domain flanked by a highly variable short N-terminal domain and a longer highly basic C-terminal domain. We hypothesized that the variable terminal domains of individual subtypes contribute to their functional heterogeneity by influencing chromatin binding interactions. We developed a novel dual color fluorescence recovery after photobleaching assay system in which two H1 proteins fused to spectrally separable fluorescent proteins can be co-expressed and their independent binding kinetics simultaneously monitored in a single cell. This approach was combined with domain swap and point mutagenesis to determine the roles of the terminal domains in the differential binding characteristics of the linker histone isotypes, mouse H1(0) and H1c. Exchanging the N-terminal domains between H1(0) and H1c changed their overall binding affinity to that of the other variant. In contrast, switching the C-terminal domains altered the chromatin interaction surface of the globular domain. These results indicate that linker histone subtypes bind to chromatin in an intrinsically specific manner and that the highly variable terminal domains contribute to differences between subtypes. The methods developed in this study will have broad applications in studying dynamic properties of additional histone subtypes and other mobile proteins.     

The maize Single myb histone 1 gene, Smh1, belongs to a novel gene family and encodes a protein that binds telomere DNA repeats in vitro

We screened maize (Zea mays) cDNAs for sequences similar to the single myb-like DNA-binding domain of known telomeric complex proteins. We identified, cloned, and sequenced five full-length cDNAs representing a novel gene family, and we describe the analysis of one of them, the gene Single myb histone 1 (Smh1). The Smh1 gene encodes a small, basic protein with a unique triple motif structure of (a) an N-terminal SANT/myb-like domain of the homeodomain-like superfamily of 3-helical-bundle-fold proteins, (b) a central region with homology to the conserved H1 globular domain found in the linker histones H1/H5, and (c) a coiled-coil domain near the C terminus. The Smh-type genes are plant specific and include a gene family in Arabidopsis and the PcMYB1 gene of parsley (Petroselinum crispum) but are distinct from those (AtTRP1, AtTBP1, and OsRTBP1) recently shown to encode in vitro telomere-repeat DNA-binding activity. The Smh1 gene is expressed in leaf tissue and maps to chromosome 8 (bin 8.05), with a duplicate locus on chromosome 3 (bin 3.09). A recombinant full-length SMH1, rSMH1, was found by band-shift assays to bind double-stranded oligonucleotide probes with at least two internal tandem copies of the maize telomere repeat, TTTAGGG. Point mutations in the telomere repeat residues reduced or abolished the binding, whereas rSMH1 bound nonspecifically to single-stranded DNA probes. The two DNA-binding motifs in SMH proteins may provide a link between sequence recognition and chromatin dynamics and may function at telomeres or other sites in the nucleus.     

Histone genes of Volvox carteri: DNA sequence and organization of two H3-H4 gene loci.

Two Volvox genomic clones each containing a pair of histone H3-H4 genes were sequenced. In both loci the H3 and H4 genes show outwardly divergent polarity, their coding regions being separated by short intercistronic sequences containing TATA boxes and a conserved 14-bp element. The 3' untranslated regions contain a characteristic motif with hyphenated dyad symmetry otherwise only found associated with animal histone genes. Derived amino acid sequences of histones H3 and H4 are highly conserved and identical between the two sets. The Volvox H3 genes both contain one intron whose relative position is shifted by one basepair. Sequence comparisons led to a new interpretation of intron sliding. The Volvox H3 gene structure combines the exon-intron organization of fungal H3 and vertebrate H3.3 genes with a termination signal typical for animal H3.1 genes. These features are discussed in view of histone gene evolution.     

The requirement of H1 histones for a heterodimeric nuclear import receptor

After synthesis in the cytoplasm, H1 histones are imported into the nucleus through an energy-dependent process that can be mediated by an importin beta-importin 7 (Impbeta-Imp7) heterodimer. H1 histones contain two structurally different types of nuclear localization signals (NLS). The first type of NLS resides within the unstructured C-terminal domain and is rich in basic amino acids. In contrast, the highly conserved central domain of the H1 histone contains comparatively few basic amino acids but also represents a functional NLS. The competence for the nuclear import of this globular domain seems to be based on its secondary structure. Here, we show that the Impbeta-Imp7 heterodimer is the only receptor for H1 import. Furthermore, we identified the import receptors mediating the in vitro transport of different NLS of the H1 histone. Using the digitonin-permeabilized cell import assay we show that Impbeta is the most efficient import receptor for the globular domain of H1 histones, whereas the heterodimer of Impbeta and Imp7 is the functional receptor for the entire C-terminal domain. However, short fragments of the C-terminal domain are imported in vitro by at least four different importins, which resembles the import pathway of ribosomal proteins and core histones. In addition, we show that heterodimerization of Impbeta with Imp7 is absolutely necessary for their proper function as an import receptor for H1 histones. These findings point to a chaperone-like function of the heterodimeric complex in addition to its function as an import receptor. It appears that the Impbeta-Imp7 heterodimer is specialized for NLS consisting of extended basic domains.     

The vertebrate linker histones H10, H5, and H1M are descendants of invertebrate “orphon” histone H1 genes

We investigated the evolutionary history of the divergent vertebrate linker histones H1⁰, H5, and HIM. We observed that the sequence of the central conserved domain of these vertebrate proteins shares characteristic features with histone H1 proteins of plants and invertebrate animals which otherwise never appear in any vertebrate histone H1 protein. A quantitative analysis of 58 linker histone sequences also reveals that these proteins are more similar to invertebrate and plant histone H1 than to histone H1 of vertebrates. A phylogenetic tree deduced from an alignment of the central domain of all known linker histones places H1⁰, H5, and HIM in close vicinity to invertebrate sperm histone H1 proteins and to invertebrate histone H1 proteins encoded by polyadenylated mRNAs. We therefore conclude that the ancestors of the vertebrate linker histones H1⁰, H5, and HIM diverged from the main group of histone H1 proteins before the vertebrate type of histone H1 was established in evolution. We discuss this observation in the general context of linker histone evolution. Correspondence to: B. and E. Schulze     

Nucleotide sequence and expression of two cDNA coding for two histone H2B variants of maize

The complete amino acid sequences of two variants of histone H2B of maize were deduced from the cDNAs isolated from a maize cDNA library. The two encoded proteins are 150 (H2B(1)) and 149 (H2B(2)) amino acids long and shows the classical organization of H2B histones. The hydrophobic C-terminal region is highly conserved as compared to that of the animal counterparts with only 21 changes (13 conservative) among the 90 residues. Between the N-terminal part and the C-terminal region we note the presence of a basic cluster (9 residues) characteristic of histones H2B. The N-terminal third is extended as compared to the animal consensus H2B and has the same size as the H2B histone of wheat. Up to 9 acidic residues and a five time repeated pentapeptide PA/KXE/KK are present in this region. Southern-blot hybrization showed that the H2B histones are encoded by a multigenic family like the other core histones (H3 and H4) of plants. The general expression pattern of these genes was not significantly different from that of the H3 and H4 genes neither in germinating seeds nor in different tissues of adult maize.     

A highly conserved sequence in H1 histone genes as an oligonucleotide hybridization probe: Isolation and sequence of a duck H1 gene

A 3.5-kb HindIII fragment of a histone gene cluster was isolated from a recombinant phage out of a duck genomic library. This DNA contains a duck H1 gene and its flanking sequences. The hybridization probe, which was used to screen for the H1 gene, had been designed on the basis of a comparative analysis of available H1 gene and protein data. Most H1 histones contain repeated motifs in their C-terminal domain, and these form part of an octapeptide (ser pro lys lys ala lys lys pro) that is highly conserved in many H1 histone proteins. A comparison of the duck H1 described here with two different published chicken H1 histone sequences reveals conservative amino acid exchanges at 22 (of 217 and 218, respectively) positions. The homology is maintained at the flanking sequences, and includes the putative H1 histone gene-specific signal structures and the established 3' stem and loop structures and the CAAGA box. The duck H1 gene and its flanking sequence have been found in identical arrangements in two recombinant bacteriophages, but minor sequence variations and genomic Southern blotting after HindIII digestion suggest that we have either isolated alleles of this genome segment or that the gene described may occur twice per haploid duck genome.     

Isolation and characterization of two human H1 histone genes within clusters of core histone genes

Two human H1 histone genes, termed H1.3 and H1.4, were isolated from two cosmid clones. The H1.4 gene is associated with an H2B gene, whereas genes coding for all four core histones are located in the vicinity of the H1.3 gene. This cluster arrangement was found both in the two cosmid clones and on overlapping bacteriophage clones isolated from an EMBL3 library. In continuation of our previous analysis of two human H1 genes, this analysis raises the number of completely sequenced H1 histone genes within clusters of core histone genes to four.     

The organization of the histone genes in the genome of Xenopus laevis.

We have studied the organization of the histone genes in the DNA from several individuals of Xenopus laevis. For that purpose, Southern blots of genomic DNA, that was digested with several restriction enzymes, were hybridized with radioactively labeled DNA fragments from clone X1-hi-1 (14), containing genes for Xenopus histones H2A, H2B, H3 and H4. In the DNA of all animals that were screened we found a major repeating unit of 14 kilobasepairs, which contains genes for histones H2A, H2B, H3 and H4 (H1 not tested) and is represented up to 30 times in the genome. The order of the genes in this major repeating unit is H4 - H3 - H2A - H2B. This order is different from that in the histone DNA of clone X1-hi-1, i.e. H3 - H4 - H2A - H2B. In addition to the genes in the major repeating unit, histone genes are present in unique restriction fragments in numbers that vary from one animal to another. The restriction patterns for the histone genes in these unique fragments were found to be different for all eight Xenopus individuals that were screened. The cloned Xenopus histone gene fragment X1-hi-1 represents such a unique fragment and is not present in the DNA of each single individual. The total number of genes coding for each of the nucleosomal histones is 45-50 per haploid genome.     

Histone H2A.Z has a conserved function that is distinct from that of the major H2A sequence variants

Saccharomyces cerevisiae contains three genes that encode members of the histone H2A gene family. The last of these to be discovered, HTZ1 (also known as HTA3), encodes a member of the highly conserved H2A.Z class of histones. Little is known about how its in vivo function compares with that of the better studied genes (HTA1 and HTA2) encoding the two major H2As. We show here that, while the HTZ1 gene encoding H2A.Z is not essential in budding yeast, its disruption results in slow growth and formamide sensitivity. Using plasmid shuffle experiments, we show that the major H2A genes cannot provide the function of HTZ1 and the HTZ1 gene cannot provide the essential function of the genes encoding the major H2As. We also demonstrate for the first time that H2A.Z genes are functionally conserved by showing that the gene encoding the H2A.Z variant of the ciliated protozoan TETRAHYMENA: thermophila is able to rescue the phenotypes associated with disruption of the yeast HTZ1 gene. Thus, the functions of H2A.Z are distinct from those of the major H2As and are highly conserved.     

Molecular evolution of the nontandemly repeated genes of the histone 3 multigene family.

In some species, histone gene clusters consist of tandem arrays of each type of histone gene, whereas in other species the genes may be clustered but not arranged in tandem. In certain species, however, histone genes are found scattered across several different chromosomes. This study examines the evolution of histone 3 (H3) genes that are not arranged in large clusters of tandem repeats. Although H3 amino acid sequences are highly conserved both within and between species, we found that the nucleotide sequence divergence at synonymous sites is high, indicating that purifying selection is the major force for maintaining H3 amino acid sequence homogeneity over long-term evolution. In cases where synonymous-site divergence was low, recent gene duplication appeared to be a better explanation than gene conversion. These results, and other observations on gene inactivation, organization, and phylogeny, indicated that these H3 genes evolve according to a birth-and-death process under strong purifying selection. Thus, we found little evidence to support previous claims that all H3 proteins, regardless of their genome organization, undergo concerted evolution. Further analyses of the structure of H3 proteins revealed that the histones of higher eukaryotes might have evolved from a replication-independent-like H3 gene.     

Water-deficit induction of a tomato H1 histone requires abscisic acid

Many genes are induced by periods of water deficit, and a subset of these are dependent on elevated ABA content for expression. A number of drought-induced genes are not induced in leaves of the ABA-deficient mutant flacca from tomato (Lycopersicon esculentum) but are induced in detached, wilted wild-type leaves and ABA-treated leaves of both genotypes. The nucleotide sequence of the cDNA and corresponding genomic DNA fragment of one of these genes, his1-s (formerly called le20), encodes an amino acid sequence that is rich in Lys, Ala, and Ser. The predicted protein contains the tripartite structure of H1 histone and is similar to other H1 histones, especially in the globular domain. Since, his1-s is more closely related to a stress-induced gene from Lycopersicon pennellii than to another H1 histone in the tomato genome it is considered a stress-induced variant of H1 histone. his1-s mRNA accumulated in vegetative plants in response to other abiotic stress treatments, including application of polyethylene glycol, and salt. The mRNA preferentially accumulated in leaves as compared to roots. his1-s mRNA accumulation was controlled during development; the level was higher in developing seeds of mature green fruit than in detached wilted leaves. H1 histones have been implicated in the general repression of gene expression and in the regulation of specific genes. The rapid accumulation of his1-s mRNA during stress may indicate that this unique, stress-induced H1 histone is involved in controlling gene expression during plant stress.     

Mapping the interaction surface of linker histone H1(0) with the nucleosome of native chromatin in vivo

H1 linker histones stabilize the nucleosome, limit nucleosome mobility and facilitate the condensation of metazoan chromatin. Here, we have combined systematic mutagenesis, measurement of in vivo binding by photobleaching microscopy, and structural modeling to determine the binding geometry of the globular domain of the H1(0) linker histone variant within the nucleosome in unperturbed, native chromatin in vivo. We demonstrate the existence of two distinct DNA-binding sites within the globular domain that are formed by spatial clustering of multiple residues. The globular domain is positioned via interaction of one binding site with the major groove near the nucleosome dyad. The second site interacts with linker DNA adjacent to the nucleosome core. Multiple residues bind cooperatively to form a highly specific chromatosome structure that provides a mechanism by which individual domains of linker histones interact to facilitate chromatin condensation.     

Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid-urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, and H4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid-urea or SDS-polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid-urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.