Juan Ausió

A unified phylogeny-based nomenclature for histone variants

Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.

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 Hl‐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.—Kasinsky, H. E., Lewis, J. D., Dacks, J. B., Ausió, J. Origin of H1 linker histones. FASEB J. 15, 34–42 (2001)

A walk though vertebrate and invertebrate protamines.

An updated comparative analysis of protamines and their corresponding genes is presented, including representative organisms from each of the vertebrate classes and one invertebrate (squid, Loligo opalescens). Special emphasis is placed on the implications for sperm chromatin organization and the evolutionary significance. The review is based on some of the most recent publications in the field and builds upon previously published reviews on this topic.

Persistent Interactions of Core Histone Tails with Nucleosomal DNA following Acetylation and Transcription Factor Binding

ABSTRACT In this study, we examined the effect of acetylation of the NH2 tails of core histones on their binding to nucleosomal DNA in the absence or presence of bound transcription factors. To do this, we used a novel UV laser-induced protein-DNA cross-linking technique, combined with immunochemical and molecular biology approaches. Nucleosomes containing one or five GAL4 binding sites were reconstituted with hypoacetylated or hyperacetylated core histones. Within these reconstituted particles, UV laser-induced histone-DNA cross-linking was found to occur only via the nonstructured histone tails and thus presented a unique tool for studying histone tail interactions with nucleosomal DNA. Importantly, these studies demonstrated that the NH2 tails were not released from nucleosomal DNA upon histone acetylation, although some weakening of their interactions was observed at elevated ionic strengths. Moreover, the binding of up to five GAL4-AH dimers to nucleosomes occupying the central 90 bp occurred without displacement of the histone NH2 tails from DNA. GAL4-AH binding perturbed the interaction of each histone tail with nucleosomal DNA to different degrees. However, in all cases, greater than 50% of the interactions between the histone tails and DNA was retained upon GAL4-AH binding, even if the tails were highly acetylated. These data illustrate an interaction of acetylated or nonacetylated histone tails with DNA that persists in the presence of simultaneously bound transcription factors.

Acetylation increases the alpha-helical content of the histone tails of the nucleosome

The nature of the structural changes induced by histone acetylation at the different levels of chromatin organization has been very elusive. At the histone level, it has been proposed on several occasions that acetylation may induce an α-helical conformation of their acetylated N-terminal domains (tails). In an attempt to provide experimental support for this hypothesis, we have purified and characterized the tail of histone H4 in its native and mono-, di-, tri-, and tetra- acetylated form. The circular dichroism analysis of these peptides shows conclusively that acetylation does increase their α-helical content. Furthermore, the same spectroscopic analysis shows that this is also true for both the acetylated nucleosome core particle and the whole histone octamer in solution. In contrast to the native tails in which the α-helical organization appears to be dependent upon interaction of these histone regions with DNA, the acetylated tails show an increase in α-helical content that does not depend on such an interaction.

Histone variant H2ABbd confers lower stability to the nucleosome.

The histone H2ABbd is a novel histone variant of H2A with a totally unknown function. We have investigated the behaviour of the H2ABbd nucleosomes. Nucleosomes were reconstituted with recombinant histone H2ABbd and changes in their conformations at different salt concentrations were studied by analytical centrifugation. The data are in agreement with H2ABbd being less tightly bound compared with conventional H2A in the nucleosome. In addition, stable cell lines expressing either green fluorescent protein (GFP)–H2A or GFP–H2ABbd were established and the mobility of both fusions was measured by fluorescence recovery after photobleaching. We show that GFP–H2ABbd exchanges much more rapidly than GFP–H2A within the nucleosome. The reported data are compatible with a lower stability of the variant H2ABbd nucleosome compared with the conventional H2A particle.

H2A.Z and H3.3 histone variants affect nucleosome structure: biochemical and biophysical studies.

Histone variants play important roles in regulation of chromatin structure and function. To understand the structural role played by histone variants H2A.Z and H3.3, both of which are implicated in transcription regulation, we conducted extensive biochemical and biophysical analysis on mononucleosomes reconstituted from either random-sequence DNA derived from native nucleosomes or a defined DNA nucleosome positioning sequence and recombinant human histones. Using established electrophoretic and sedimentation analysis methods, we compared the properties of nucleosomes containing canonical histones and histone variants H2A.Z and H3.3 (in isolation or in combination). We find only subtle differences in the compaction and stability of the particles. Interestingly, both H2A.Z and H3.3 affect nucleosome positioning, either creating new positions or altering the relative occupancy of the existing nucleosome position space. On the other hand, only H2A.Z-containing nucleosomes exhibit altered linker histone binding. These properties could be physiologically significant as nucleosome positions and linker histone binding partly determine factor binding accessibility.

H2A.Z Stabilizes Chromatin in a Way That Is Dependent on Core Histone Acetylation

The functional and structural chromatin roles of H2A.Z are still controversial. This work represents a further attempt to resolve the current functional and structural dichotomy by characterizing chromatin structures containing native H2A.Z. We have analyzed the role of this variant in mediating the stability of the histone octamer in solution using gel-filtration chromatography at different pH. It was found that decreasing the pH from neutral to acidic conditions destabilized the histone complex. Furthermore, it was shown that the H2A.Z-H2B dimer had a reduced stability. Sedimentation velocity analysis of nucleosome core particles (NCPs) reconstituted from native H2A.Z-containing octamers indicated that these particles exhibit a very similar behavior to that of native NCPs consisting of canonical H2A. Sucrose gradient fractionation of native NCPs under different ionic strengths indicated that H2A.Z had a subtle tendency to fractionate with more stabilized populations. An extensive analysis of the salt-dependent dissociation of histones from hydroxyapatite-adsorbed chromatin revealed that, whereas H2A.Z co-elutes with H3–H4, hyperacetylation of histones (by treatment of chicken MSB cells with sodium butyrate) resulted in a significant fraction of this variant eluting with the canonical H2A. These studies also showed that the late elution of this variant (correlated to enhanced binding stability) was independent of the chromatin size and of the presence or absence of linker histones.

Acetylation of vertebrate H2A.Z and its effect on the structure of the nucleosome.

Purified histone H2A.Z from chicken erythrocytes and a sodium butyrate-treated chicken erythroleukemic cell line was used as a model system to identify the acetylation sites (K4, K7, K11, K13, and K15) and quantify their distribution in this vertebrate histone variant. To understand the role played by acetylation in the modulation of the H2A.Z nucleosome core particle (NCP) stability and conformation, an extensive analysis was conducted on NCPs reconstituted from acetylated forms of histones, including H2A.Z and recombinant H2A.Z (K/Q) acetylation mimic mutants. Although the overall global acetylation of core histones destabilizes the NCP, we found that H2A.Z stabilizes the NCP regardless of its state of acetylation. Interestingly and quite unexpectedly, we found that the change in NCP conformation induced by global histone acetylation is dependent on H2A/H2A.Z acetylation. This suggests that acetylated H2A variants act synergistically with the acetylated forms of the core histone complement to alter the particle conformation. Furthermore, the simultaneous occurrence of H2A.Z and H2A in heteromorphic NCPs that most likely occurs in vivo slightly destabilizes the NCP, but only in the presence of acetylation.

Protamines, in the Footsteps of Linker Histone Evolution

It was perhaps a lucky coincidence that the early attempts to establish the chemical composition of the cell nucleus were initially carried out on such diverse biological systems as salmon sperm heads (1) and geese and chicken erythrocytes (2). Examination of sperm and erythrocyte systems, respectively, lead to the protamine and histone concepts (3). We know with certainty that, with the exception of the male gametes, all somatic cells exclusively contain histones. Hence, in metazoans, protamines (4, 5) are confined to the sperm nuclear chromatin, and even among sperm, protamines are not always present. Indeed, a large number of metazoans contain somatic-like histones in their sperm, and some crustaceans (order Decapoda) lack any chromosomal proteins in their sperm (6, 7). Therefore, in contrast to the somatic nucleus, sperm chromatin may have a much more diverse protein composition. It was not until the first attempt of classification of the sperm nuclear basic proteins (SNBPs)2 by David Bloch (6, 8), an effort later on extended by Harold Kasinsky (7), that a clearer picture started to emerge in this regard. More recently, an enormous effort has been carried out in several laboratories, including our own (9–15), to extend this analysis to a large number of representative organisms from the different phylogenetic groups. With a broader perspective now available, SNBP heterogeneity can be restricted to three major groups or types: histone (H), protamine (P), and protamine-like (PL) (16). Histones consist of core histones (histones H2A, H2B, H3, and H4) and linker histones (histone H1 family). The names refer to the structural role of these proteins. Core histones are responsible for constraining DNA wrapped about a histone core to produce a nucleoprotein complex (chromatin subunit) known as a nucleosome core particle. Linker histones bind to the linker DNA regions connecting adjacent nucleosome core particles and assist in the folding of the chromatin fiber (17). Protamines are a highly compositionally and structurally heterogeneous group of proteins (9). They exhibit a high charge density and a prevalence of arginine in their composition (13), a fact that it is most likely related to the higher affinity with which this basic amino acid binds to DNA (18). They lack any secondary structure in solution butmay adopt a folded conformation upon interactionwithDNA. Protamine-like proteins share compositional and structural similarities between histones and protamines (9, 16). Hence they represent a structurally intermediate group that will be discussed more extensively in the following sections of this review. To a certain extent, all three types of SNBP can be considered structurally analogous as all of them produce folded chromatin fibers of 30–50 nm (19) regardless of the particular structure of the individual nucleoprotamine complexes. At the functional level, somatic histones bind to DNA in a highly dynamic way that not only helps in the folding of the genome but also has an important role in the epigenetic regulation of gene expression (20, 21). In contrast, protamine and protamine-like SNBPs of the spermatozoa bind very tightly to the genome to produce a maximal genome compaction, which completely abolishes the epigenetic information of the paternal histones (9) in this terminally differentiated system. This epigenetic silencing can be reverted only with the assistance of highly specialized protamine-removing proteins such as nucleoplasmin during the molecular events involved in nuclear metabolism during early fertilization (22). The analogous structural DNA condensation potential of the three types of SNBPs raises a question as to the extent of structural homology between them. In the next sections we are going to discuss a series of recent papers that suggest that protamines and protamine-like proteins are evolutionarily related to linker histones.