The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells.

Eukaryotes have long been thought to have arisen by evolving a nucleus, endomembrane, and cytoskeleton. In contrast, it was recently proposed that the first complex cells, which were actually proto-eukaryotes, arose simultaneously with the acquisition of mitochondria. This so-called symbiotic association hypothesis states that eukaryotes emerged when some ancient anaerobic archaebacteria (hosts) engulfed respiring alpha-proteobacteria (symbionts), which evolved into the first energy-producing organelles. Therefore, the intracellular compartmentalization of the energy-converting metabolism that was bound originally to the plasma membrane appears to be the key innovation towards eukaryotic genome and cellular organization. The novel energy metabolism made it possible for the nucleotide synthetic apparatus of cells to be no longer limited by subsaturation with substrates and catalytic components. As a consequence, a considerable increase has occurred in the size and complexity of eukaryotic genomes, providing the genetic basis for most of the further evolutionary changes in cellular complexity. On the other hand, the active uptake of exogenous DNA, which is general in bacteria, was no longer essential in the genome organization of eukaryotes. The mitochondrion-driven scenario for the first eukaryotes explains the chimera-like composition of eukaryotic genomes as well as the metabolic and cellular organization of eukaryotes.     

Evidence for the nuclear import of histones H3.1 and H4 as monomers

Newly synthesised histones are thought to dimerise in the cytosol and undergo nuclear import in complex with histone chaperones. Here, we provide evidence that human H3.1 and H4 are imported into the nucleus as monomers. Using a tether‐and‐release system to study the import dynamics of newly synthesised histones, we find that cytosolic H3.1 and H4 can be maintained as stable monomeric units. Cytosolically tethered histones are bound to importin‐alpha proteins (predominantly IPO4), but not to histone‐specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, IPO4 dissociation and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones reveals an excess of H3 specifically associated with sNASP, suggesting that NASP maintains a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol.     

Distribution of the core histones H2A.H2B.H3 and H4 during cell replication.

The distribution of newly synthesized core histones H2A, H2B, H3 and H4 relative to the DNA strand synthesized in the same generation has been examined in replicating Chinese Hamster ovary cells. Cells are grown for one generation in [14C]-lysine and thymidine, and then for one generation in [3H]-lysine and 5-bromodeoxyuridine (BrUdRib) and a further generation in unlabeled lysine and thymidine. This protocol produces equal amounts of unifilarly substituted and unsubstituted DNA. Monomer nucleosomes isolated from chromatin containing these two types of DNA can be distinguished by crosslinking with formaldehyde and banding to equilibrium in CsCl density gradients. The results indicate that the core histones are equally distributed between the two types of DNA. These findings are discussed in terms of current models for chromatin replication; they do not support any long term association of newly replicated histones with either the leading or lagging side of the replication fork.     

Phylogenetic analysis of the core histones H2A, H2B, H3, and H4.

Despite the ubiquity of histones in eukaryotes and their important role in determining the structure and function of chromatin, no detailed studies of the evolution of the histones have been reported. We have constructed phylogenetic trees for the core histones H2A, H2B, H3, and H4. Histones which form dimers (H2A/H2B and H3/H4) have very similar trees and appear to have co-evolved, with the exception of the divergent sea urchin testis H2Bs, for which no corresponding divergent H2As have been identified. The trees for H2A and H2B also support the theory that animals and fungi have a common ancestor. H3 and H4 are 10-fold less divergent than H2A and H2B. Three evolutionary histories are observed for histone variants. H2A.F/Z-type variants arose once early in evolution, while H2A.X variants arose separately, during the evolution of multicellular animals. H3.3-type variants have arisen in multiple independent events.     

Plant histones. Relevance to the evolution of prokaryotes to eukaryotes

There are many procaryotic and eucaryotic organisms in plant kingdom. It is hoped that the study of plant histones will be useful in evolutionary studies. The histones of great variety of animal species have been studied and well characterized. Less information is available concerning plant histones. The general conclusion drawn from these investigations is that most organisms of eucaryotic plant and animal species contain the same five major histone fractions. Recently the histone-like proteins were found in some primitive eucaryotes and procaryotes. Data on histones from higher and lower eucaryotes and histone-like proteins of procaryotes are reviewed. Evolution of histones and their appearance prior to that of eucaryotic cell is postulated. The role of histones in evolution of nucleosomes is discussed.     

Deposition of newly synthesized histones: new histones H2A and H2B do not deposit in the same nucleosome with new histones H3 and H4

We have developed procedures to study histone-histone interactions during the deposition of histones in replicating cells. Cells are labeled for 60 min with dense amino acids, and subsequently, the histones within the nucleosomes are cross-linked into an octameric complex with formaldehyde. These complexes are sedimented to equilibrium in density gradients and octamer and dioctamer complexes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With reversal of the cross-link, the distribution of the individual density-labeled histones in the octamer is determined. Newly synthesized H3 and H4 deposit as a tetramer and are associated with old H2A and H2B. Newly synthesized H2A and H2B deposit as a dimer associated with old H2A, H2B, H3, and H4. The significance of these results with respect to the dynamics of histone interactions in the nucleus is discussed. Control experiments are presented to test for artifactual formation of these complexes during preparative procedures. In addition, reconstitution experiments were performed to demonstrate that the composition of these octameric complexes can be determined from their distribution on density gradients.     

An Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A

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.     

Ribosomal protein-sequence block structure suggests complex prokaryotic evolution with implications for the origin of eukaryotes

Amino acid sequence alignments of orthologous ribosomal proteins found in Bacteria, Archaea, and Eukaryota display, relative to one another, an unusual segment or block structure, with major evolutionary implications. Within each of the prokaryotic phylodomains the sequences exhibit substantial similarity, but cross-domain alignments break up into (a) universal blocks (conserved in both phylodomains), (b) bacterial blocks (unalignable with any archaeal counterparts), and (c) archaeal blocks (unalignable with any bacterial counterparts). Sequences of those eukaryotic cytoplasmic riboproteins that have orthologs in both Bacteria and Archaea, exclusively match the archaeal block structure. The distinct blocks do not correlate consistently with any identifiable functional or structural feature including RNA and protein contacts. This phylodomain-specific block pattern also exists in a number of other proteins associated with protein synthesis, but not among enzymes of intermediary metabolism. While the universal blocks imply that modern Bacteria and Archaea (as defined by their translational machinery) clearly have had a common ancestor, the phylodomain-specific blocks imply that these two groups derive from single, phylodomain-specific types that came into existence at some point long after that common ancestor. The simplest explanation for this pattern would be a major evolutionary bottleneck, or other scenario that drastically limited the progenitors of modern prokaryotic diversity at a time considerably after the evolution of a fully functional translation apparatus. The vast range of habitats and metabolisms that prokaryotes occupy today would thus reflect divergent evolution after such a restricting event. Interestingly, phylogenetic analysis places the origin of eukaryotes at about the same time and shows a closer relationship of the eukaryotic ribosome-associated proteins to crenarchaeal rather than euryarchaeal counterparts.     

A nucleosome-like structure containing DNA and the arginine-rich histones H3 and H4.

The low-angle X-ray diffraction pattern from fibres of reconstituted H3/H4/DNA complexes is very similar to that of chromatin and has well defined maxima at 10.6, 5.4, 3.4 and 2.6 nm. Staphyloccal nuclease digestion of reconstituted H3/H4/DNA yields DNA fragments of length 49, 69, 100, 128, 193 and 255 b.p. as principal components. Comparison of the relative amounts of DNA fragments shows that the larger components (100 and 128 b.p.) increase with respect to the smaller (49 and 69 b.p.) as the histone to DNA ratio increases. A structural unit containing intergral of 65 b.p. of DNA and tetrameric (H3/H4)2 is proposed such that longer DNA fragments result from multiples of this unit. The principal nucleo-protein particle resulting from nuclease digestion contains 128/139 b.p. of DNA and has electrophoretic mobility very close to that of 'core' nucleosome. It probably represents a dimer of the basic structural unit.     

A genomic timescale for the origin of eukaryotes

Background  

Genomic sequence analyses have shown that horizontal gene transfer occurred during the origin of eukaryotes as a consequence of symbiosis. However, details of the timing and number of symbiotic events are unclear. A timescale for the early evolution of eukaryotes would help to better understand the relationship between these biological events and changes in Earth's environment, such as the rise in oxygen. We used refined methods of sequence alignment, site selection, and time estimation to address these questions with protein sequences from complete genomes of prokaryotes and eukaryotes.     

Contribution of histones H2A and H2B to the folding of nucleosomal DNA

We have studied the structural properties of nucleosomal particles deficient in histones H2A and H2B produced by modification of histone amino groups with dimethylmaleic anhydride [Jordano, J., Montero, F., & Palacián, E. (1984) Biochemistry (preceding paper in this issue)]. Digestion with DNase I of residual particles containing only 15% of the original H2A . H2B complement produces only discrete DNA fragments no longer than 70 nucleotides. As compared with the original nucleosomes, thermal denaturation of the residual particles shows a decrease from 140 to about 90 in the number of nucleotide base pairs per particle that melt at the highest temperature transition as well as a drop in the temperature of this transition. Circular dichroism spectra of the residual particles give ellipticity values around 275 nm, much higher than those corresponding to the control nucleosomes, which appears to indicate a loss in the compact DNA tertiary structure. When regeneration of the modified amino groups of the residual particles takes place in the presence of the complementary fraction containing histones H2A and H2B, but not in its absence, nucleosomal particles with the structural properties of the original nucleosomes are reconstituted. Therefore, the structural change observed in the residual particles can be assigned to the lack of histones H2A and H2B and not to the modified amino groups of the histones present in the residual particles. The results are consistent with the stabilization by histones H2A and H2B of a DNA length of 50-70 base pairs per nucleosome.     

DNA instructed displacement of histones H2A and H2B at an inducible promoter

Regulation of gene expression requires dynamic changes in chromatin, but the nature of these changes is not well understood. Here, we show that progesterone treatment of cultured cells leads to recruitment of progesterone receptor (PR) and SWI/SNF-related complexes to Mouse Mammary Tumor Virus (MMTV) promoter, accompanied by displacement of histones H2A and H2B from the nucleosome containing the receptor binding sites, but not from adjacent nucleosomes. PR recruits SWI/SNF to MMTV nucleosomes in vitro and facilitates synergistic binding of receptors and nuclear factor 1 to the promoter. In nucleosomes assembled on MMTV or mouse rDNA promoter sequences, SWI/SNF catalyzes ATP-dependent sliding of the histone octamer followed only on the MMTV promoter by displacement of histones H2A and H2B. In MMTV nucleosome arrays, SWI/SNF displaces H2A and H2B from nucleosome B and not from the adjacent nucleosome. Thus, the outcome of nucleosome remodeling by SWI/SNF depends on DNA sequence.     

Proteome analysis of protein partners to nucleosomes containing canonical H2A or the variant histones H2A.Z or H2A.X

Although the existence of histone variants has been known for quite some time, only recently are we grasping the breadth and diversity of the cellular processes in which they are involved. Of particular interest are the two variants of histone H2A, H2A.Z and H2A.X because of their roles in regulation of gene expression and in DNA double-strand break repair, respectively. We hypothesize that nucleosomes containing these variants may perform their distinct functions by interacting with different sets of proteins. Here, we present our proteome analysis aimed at identifying protein partners that interact with nucleosomes containing H2A.Z, H2A.X or their canonical H2A counterpart. Our development of a nucleosome-pull down assay and analysis of the recovered nucleosome-interacting proteins by mass spectrometry allowed us to directly compare nuclear partners of these variant-containing nucleosomes to those containing canonical H2A. To our knowledge, our data represent the first systematic analysis of the H2A.Z and H2A.X interactome in the context of nucleosome structure.     

A molecular timeline for the origin of photosynthetic eukaryotes

The appearance of photosynthetic eukaryotes (algae and plants) dramatically altered the Earth's ecosystem, making possible all vertebrate life on land, including humans. Dating algal origin is, however, frustrated by a meager fossil record. We generated a plastid multi-gene phylogeny with Bayesian inference and then used maximum likelihood molecular clock methods to estimate algal divergence times. The plastid tree was used as a surrogate for algal host evolution because of recent phylogenetic evidence supporting the vertical ancestry of the plastid in the red, green, and glaucophyte algae. Nodes in the plastid tree were constrained with six reliable fossil dates and a maximum age of 3,500 MYA based on the earliest known eubacterial fossil. Our analyses support an ancient (late Paleoproterozoic) origin of photosynthetic eukaryotes with the primary endosymbiosis that gave rise to the first alga having occurred after the split of the Plantae (i.e., red, green, and glaucophyte algae plus land plants) from the opisthokonts sometime before 1,558 MYA. The split of the red and green algae is calculated to have occurred about 1,500 MYA, and the putative single red algal secondary endosymbiosis that gave rise to the plastid in the cryptophyte, haptophyte, and stramenopile algae (chromists) occurred about 1,300 MYA. These dates, which are consistent with fossil evidence for putative marine algae (i.e., acritarchs) from the early Mesoproterozoic (1,500 MYA) and with a major eukaryotic diversification in the very late Mesoproterozoic and Neoproterozoic, provide a molecular timeline for understanding algal evolution.     

Studies on the prolactin-releasing mechanism of histones H2A and H2B

In previous studies we demonstrated that histone preparations possess multiple effects in vivo on pituitary hormone secretion. We have now studied the specificity and signal transduction pathways involved in the prolactin (PRL)-releasing activity of histones H2A and H2B on perifused and incubated rat pituitary cells. In the perifusion experiments, freshly dispersed pituitary cells were packed into short columns and were continuously perifused with serum-free medium. The substances to be tested (stimuli) were pumped through the perifusion circuit, at the end of which perifusate fractions were collected and PRL measured by specific RIA. In the incubation studies, freshly dispersed pituitary cells were incubated in a metabolic incubator with different stimuli at different doses and for varying times. Perifusion of cells with median eminence extract (1/30), histone H2A (30 microM) or histone H2B (30 microM), generated clear PRL release responses. Cells incubated with histone H2A and H2B showed a dose- and time-dependent stimulatory effect on PRL release which, for H2A, was blocked by peptide MB-35, an 86-120 amino acid synthetic fragment of histone H2A. The polycation, poly-lys was unable to mimic the action of histones. To detect the possible signal transduction pathways involved in the response of lactotrophs to histones, cells were incubated with the calcium ionophore A23187, the calcium chelator EGTA, the intracellular phosphoinositide enhancer LiCl, the intracellular cAMP enhancers caffeine, NaF and forskolin, and the protein kinase C inhibitor, trifluoperazine (TFP). Both EGTA (or EGTA plus A23187 ionophore) and TFP were able to reduce significantly the response of lactotrophs to histones. Our results confirm previous evidence that histones may act as hypophysotropic signals. The data also suggest that calcium- and diacylglycerol-associated pathways participate in these effects.     

A chromatin-associated protein from pea seeds preferentially binds histones H3 and H4.

Pisum sativum p16 is a protein present in the chromatin of ungerminated embryonic axes. The purification of p16 and the isolation of a cDNA clone of psp54, the gene encoding its precursor have been recently reported [Castillo, J., Rodrigo, M. I., Márquez, J. A., Zú?iga, A and Franco, L. (2000) Eur. J. Biochem.267, 2156-2165]. In the present paper, we present data showing that p16 is a nuclear protein. First, after subcellular fractionation, p16 was clearly found in nuclei, although the protein is also present in other organelles. Immunocytochemical methods also confirm the above results. p16 seems to be firmly anchored to chromatin, as only extensive DNase I digestion of nuclei allows its release. Far Western and pull-down experiments demonstrate a strong in vitro interaction between p16 and histones, especially H3 and H4, suggesting that p16 is tethered to chromatin through histones. Because the psp54 gene is specifically expressed during the late development of seed, the role of p16 might be related to the changes that occur in chromatin during the processes of seed maturation and germination.     

The origin and early evolution of eukaryotes in the light of phylogenomics

Phylogenomics of eukaryote supergroups suggest a highly complex last common ancestor of eukaryotes and a key role of mitochondrial endosymbiosis in the origin of eukaryotes.