Core histones of the amitochondriate protist, Giardia lamblia

Genes coding for the core histones H2a, H2b, H3, and H4 of Giardia lamblia were sequenced. A conserved organism- and gene-specific element, GRGCGCAGATTTVGG, was found upstream of the coding region in all core histone genes. The derived amino acid sequences of all four histones were similar to their homologs in other eukaryotes, although they were among the most divergent members of this protein family. Comparative protein structure modeling combined with energy evaluation of the resulting models indicated that the G. lamblia core histones individually and together can assume the same three-dimensional structures that were established by X-ray crystallography for Xenopus laevis histones and the nucleosome core particle. Since G. lamblia represents one of the earliest-diverging eukaryotes in many different molecular trees, the structure of its histones is potentially of relevance to understanding histone evolution. The G. lamblia proteins do not represent an intermediate stage between archaeal and eukaryotic histones.     

Trypanosoma cruzi histones. Further characterization and comparison with higher eukaryotes

Histones extracted from T. cruzi chromatin were analyzed in three electrophoretic systems. Our results show that a basic protein with some properties similar to those of histone H1 from higher eukaryotes is present in T. cruzi. However this protein presents different electrophoretic mobilities than H1 histone from higher eukaryotes in all three electrophoretic systems tested. Considering the marked differences observed in the electrophoretic mobilities of T. cruzi histones as compared with those from higher eukaryotes, it is proposed that histones are conservative proteins primarily with regard to their function.     

Histones of Neurospora crassa.

Neurospora crassa chromatin isolated by a rapid method minimizing proteolytic degradation contains approximately one weight of acid-extractable basic protein per weight of DNA. This basic protein consists of five major polypeptide species which are similar in size to the histone proteins of higher eukaryotes and are present in approximately the same molar ratios. These five polypeptides have been purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Their electrophoretic mobilities in polyacrylamide gels and their amino acid compositions indicate that they are histones homologous, although not identical, to the H1, H2A, H2B, H3, and H4 histones of mammals. The first 3 residues in the amino acid sequence of Neurospora H3 histone are identical to the first 3 residues in calf and pea H3; Neurospora H1, H2A, and H4 histones have blocked NH2 termini, like their mammalian counterparts. The finding of recognizable H1, H2A, H2B, H3, and H4 histones in Neurospora extends the range of eukaryotes now shown to contain a full complement of these strongly conserved chromosomal proteins, and supports the view that histones became involved in chromosome structure at a very early point in the evolution of eukaryotes.     

Evolution of Histone H4 and H3 Genes in Different Ciliate Lineages

The histones H4 are known as highly conserved proteins. However, in ciliates a high degree of variation was found compared both to other eukaryotes and between the ciliate species. To date, only H4 histones of species belonging to two distantly related classes have been investigated. In order to obtain more detailed information on histone H4 variation in ciliates we undertook a comprehensive sequence analysis of PCR-amplified internal H4 fragments from 12 species belonging to seven out of the nine currently recognized ciliate classes. In addition, we used PCR primers to amplify longer fragments of H3 and H4 genes including the intergenic region. The encoded amino acid sequences reveal a high number of differences when compared with those of other eukaryotes and the ciliate species investigated. Furthermore, in some species H4 gene variants were detected, which result in amino acid differences. The greatest number of substitutions and insertions found was in the amino terminal region of the H4 histones. However, all sequences possess a conserved region corresponding to those of all other eukaryotic H4 histones. The histone gene variations were used to reconstruct phylogenetic relationships. The tree from our data matches perfectly with the ribosomal RNA data: The heterotrichs, which were considered as a late branching lineage, diverge at the base of the ciliate tree and groups formerly thought to represent ancestral lineages now appear as highly derived ciliates. Received: 4 April 1997 / Accepted: 1 August 1997     

Codon usage in histone gene families of higher eukaryotes reflects functional rather than phylogenetic relationships

Summary The nucleic acid sequences coding for 23 H3 histone genes from a variety of species have been analyzed using a computer assisted alignment and analysis program. Although these histones are highly conserved within and between highly divergent species, they represent various classes of histones whose patterns of expression are distinctively regulated. Surprisingly, in dendrograms derived from these comparisons, H3 sequences cluster according to their modes of regulation rather than phylogenetically. These clusters are generated from highly distinctive patterns of codon usage within the functional gene classes. We suggest that one factor involved in specifying the differing codon usage patterns between functional classes is a difference in requirements for rapid translation of mRNA. In addition, the data presented here, together with structural and sequence information, suggest a heterodox evolutionary model in which genes related to the intron-bearing, basally expressed H3.3 vertebrate genes are the ancestors of the intronless H3. 1 class of genes of higher eukaryotes. The H3. 1 class must have arisen, therefore, following duplication of a primitive H3.3 gene, but prior to the plant-animal divergence. Implications of the data presented are discussed with regard to functional and evolutionary relationships.     

Suppression of histone H1 genes in Arabidopsis results in heritable developmental defects and stochastic changes in DNA methylation

Histone H1 is an abundant component of eukaryotic chromatin that is thought to stabilize higher-order chromatin structures. However, the complete knock-out of H1 genes in several lower eukaryotes has no discernible effect on their appearance or viability. In higher eukaryotes, the presence of many mutually compensating isoforms of this protein has made assessment of the global function of H1 more difficult. We have used double-stranded RNA (dsRNA) silencing to suppress all the H1 genes of Arabidopsis thaliana. Plants with a >90% reduction in H1 expression exhibited a spectrum of aberrant developmental phenotypes, some of them resembling those observed in DNA hypomethylation mutants. In subsequent generations these defects segregated independently of the anti-H1 dsRNA construct. Downregulation of H1 genes did not cause substantial genome-wide DNA hypo- or hypermethylation. However, it was correlated with minor but statistically significant changes in the methylation patterns of repetitive and single-copy sequences, occurring in a stochastic manner. These findings reveal an important and previously unrecognized link between linker histones and specific patterns of DNA methylation.     

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.     

Characterising the Binding Specificities of the Subunits Associated with the KMT2/Set1 Histone Lysine Methyltransferase

KMT2/Set1 is the catalytic subunit of the complex of proteins associated with Set1 (COMPASS) that is responsible for the methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. Whereas monomethylated H3K4 (H3K4me1) is found throughout the genome, di- (H3K4me2) and tri- (H3K4me3) methylated H3K4 are enriched at specific loci, which correlates with the promoter and 5′-ends of actively transcribed genes in the case of H3K4me3. The COMPASS subunits contain a number of domains that are conserved in homologous complexes in higher eukaryotes and are reported to interact with modified histones. However, the exact organization of these subunits and their role within the complex have not been elucidated. In this study we showed that: (1) subunits Swd1 and Swd3 form a stable heterodimer that dissociates upon binding to a modified H3K4me2 tail peptide, suggesting a regulatory role in COMPASS; (2) the affinity of the subunit Spp1 for modified histone H3 substrates is much higher than that of Swd1 and Swd3; (3) Spp1 has a preference for H3K4me2/3 methylation state; and (4) Spp1 contains a high-affinity DNA-binding domain in the previously uncharacterised C-terminal region. These data allow us to suggest a mechanism for the regulation of COMPASS activity at an actively transcribed gene.     

The lysine-rich H1 histones from the slime moulds, Physarum polycephalum and Dictyostelium discoideum lack phosphorylation sites recognised by cyclic AMP-dependent protein kinase in vitro.

Calcium chloride-extracted histones were prepared from nuclei of the slime moulds, Physarum polycephalum and Dictyostelium discoideum, and phosphorylation by purified preparations of cyclic AMP-dependent protein kinase (cAMP-d PK) and growth-associated H1 histone kinase (HKG) examined and compared. Among the major histone fractions and other proteins in the two preparations, the H1 histones from both organisms were found to be effective and exclusive substrates for HKG. cAMP-d PK, which phosphorylates mammalian H1 histone and certain, in particular H2B, of the mammalian core histones, phosphorylated several of the core histones from both slime moulds but did not phosphorylate H1 histone from either. The slime mould H1s remained ineffective substrates for cAMP-d PK even after extensive alkaline phosphatase treatment of the histone preparations. Additional studies demonstrated that the lack of slime mould H1 phosphorylation by cAMP-d PK was not due to competition of the H1 molecules with the core histones for the kinase. Our studies suggest that H1 histones from these organisms, whilst clearly containing sites for phosphorylation by HKG, apparently lack phosphorylation sites recognised by cAMP-d PK. Thus, the mediation of specific nuclear functions by cAMP-dependent phosphorylation of H1 in higher organisms may not occur or be required in these lower eukaryotes.     

The enhancers and promoters of the Xenopus laevis ribosomal spacer are associated with histones upon active transcription of the ribosomal genes

The presence of histones on the enhancer-promoter region of the X.laevis ribosomal spacer has been studied in embryos at stage 40, where the ribosomal genes are actively transcribed. Isolated tadpole nuclei were either fixed with formaldehyde or irradiated with UV laser to crosslink histones to DNA. The purified protein-DNA complexes were immunoprecipitated with antibodies to the histones H1, H2A and H4 and the DNA fragments carrying the respective histones were analyzed for the presence of spacer enhancer-promoter sequences by hybridization to specific DNA probe. The two independent crosslinking procedures revealed the presence of these DNA sequences in the precipitated DNA. The quantitative analysis of the UV laser-crosslinked complexes showed that histones H2A and H4 were associated with enhancer-promoter DNA in amounts similar to those found for bulk DNA, whilst the content of H1 was reduced.     

Trypanosome TOR as a major regulator of cell growth and autophagy

Trypanosomatid protozoa parasites are responsible for tropical diseases, and undergo complex life cycles involving developmental forms adapted to insect vectors and vertebrate hosts. During their life cycle these parasites proceed through different forms in response to dramatic environmental changes and/or developmentally regulated programs. Successful progression of the parasite through its life cycle is highly dependent on the capacity of adaptation to distinct stresses involving processes such as autophagy. In eukaryotes, Target Of Rapamycin (TOR) protein kinases act as a sensor, which integrates the nutritional and energetic status, adjusting cell metabolism and growth. Compromising cell viability in yeast and mammals leads to a reduction of TOR function, triggering processes aimed to overcome unfavourable conditions. This is partly achieved by TOR-mediated regulation of protein synthesis and recycling of cellular components by autophagy. In the last few years, autophagy has been described during developmental differentiation processes in Trypanosomatids. However, no link between TOR signalling, autophagy and differentiation has been described so far. This addendum is a commentary to the work published by our group,¹ where we discuss the possible role of TOR kinases, as a controller of cell growth and autophagy, in the regulation of differentiation processes during Trypanosomatids life cycles.     

Isolation and characterization of histones from Anopheles albimanus Weidemann

1. Histones from Anopheles albimanus adults were prepared by a combination of techniques including chromatin isolation and selective extractions. 2. The anopheline histones were identified on acid urea gels by comparing their electrophoretic profile with that of calf thymus histones and histones isolated from other tissue. 3. Excellent separation of histones was obtained after the extractions by a single electrophoretic run. 4. In addition to the five major classes of histones found in eukaryotes, a sixth class was detected and tentatively identified as histone H5. 5. This is the first report of histone H5 and its function in insects.     

Histone Synthesis inTrypanosoma cruzi

Trypanosoma cruziis an ancient, parasitic eukaryote which does not undergo chromatin condensation during cell division. This behavior may be explained if one considers the strong amino acid sequence divergence ofTrypanosomahistones compared to higher eukaryotes. In the latter organisms histone synthesis is coupled to DNA replication. Considering the nonconserved amino acid sequence ofT. cruzihistones, as well as the absence of chromatin condensation in this organism, we have studied histone synthesis in relation to DNA replication in this parasite. We have found that core histones and a fraction of histone H1 are synthesized concomitantly to DNA replication. However, another fraction of histone H1 is constitutively synthesized.     

Chromatin and histones from Giardia lamblia: a new puzzle in primitive eukaryotes

The three deepest eukaryote lineages in small subunit ribosomal RNA phylogenies are the amitochondriate Microsporidia, Metamonada, and Parabasalia. They are followed by either the Euglenozoa (e.g., Euglena and Trypanosoma) or the Percolozoa as the first mitochondria-containing eukaryotes. Considering the great divergence of histone proteins in protozoa we have extended our studies of histones from Trypanosomes (Trypanosoma cruzi, Crithidia fasciculata and Leishmania mexicana) to the Metamonada Giardia lamblia, since Giardia is thought to be one of the most primitive eukaryotes. In the present work, the structure of G. lamblia chromatin and the histone content of the soluble chromatin were investigated and compared with that of higher eukaryotes, represented by calf thymus. The chromatin is present as nucleosome filaments which resemble the calf thymus array in that they show a more regular arrangement than those described for Trypanosoma. SDS-polyacrylamide gel electrophoresis and protein characterization revealed that the four core histones described in Giardia are in the same range of divergence with the histones from other lower eukaryotes. In addition, G. lamblia presented an H1 histone with electrophoretic mobility resembling the H1 of higher eukaryotes, in spite of the fact that H1 has a different molecular mass in calf thymus. Giardia also presents a basic protein which was identified as an HU-like DNA-binding protein usually present in eubacteria, indicating a chimaeric composition for the DNA-binding protein set in this species. Finally, the phylogenetic analysis of selected core histone protein sequences place Giardia divergence before Trypanosoma, despite the fact that Trypanosoma branch shows an acceleration in the evolutionary rate pointing to an unusual evolutionary behavior in this lineage.