Core Histones H2A Research Articles

Crystal structures of recombinant histones HMfA and HMfB from the hyperthermophilic archaeon Methanothermus fervidus

The hyperthermophilic archaeon Methanothermus fervidus contains two small basic proteins, HMfA (68 amino acid residues) and HMfB (69 residues) that share a common ancestry with the eukaryal nucleosome core histones H2A, H2B, H3, and H4. HMfA and HMfB have sequences that differ at 11 locations, they have different structural stabilities, and the complexes that they form with DNA have different electrophoretic mobilities. Here, crystal structures are documented for recombinant (r) HMfA at a resolution of 1.55 Å refined to a crystallographic R-value of 19.8 % (tetragonal form) and at 1.48 Å refined to a R-value of 18.8 % (orthorhombic form), and for rHMfB at 1.9 Å refined to a R-value of 18.0 %. The rHMfA and rHMfB monomers have structures that are just histone folds in which a long central α-helix (α2; 29 residues) is separated from shorter N-terminal (α1; 11 residues) and C-terminal (α3; 10 residues) α-helices by two loops (L1 and L2; both 6 residues). Within L1 and L2, three adjacent residues are in extended (β) conformation. rHMfA and rHMfB assemble into homodimers, with the α2 helices anti-parallel aligned and crossing at an angle of close to 35°, and with hydrogen bonds formed between the extended, parallel regions of L1 and L2 resulting in short β-ladders. Dimerization creates a novel N-terminal structure that contains four proline residues, two from each monomer. As prolines are present at these positions in all archaeal histone sequences, this proline-tetrad structure is likely to be a common feature of all archaeal histone dimers. Almost all residues that participate in monomer-monomer interactions are conserved in HMfA and HMfB, consistent with the ability of these monomers to form both homodimers and (HMfA+HMfB) heterodimers. Differences in side-chain interactions that result from non-conservative residue differences in HMfA and HMfB are identified, and the structure of a (rHMfA) 2-DNA complex is presented based on the structures documented here and modeled by homology to histone-DNA interactions in the eukaryal nucleosome.

Dynamic relocalization of histone MacroH2A1 from centrosomes to inactive X chromosomes during X inactivation.

Histone variant macroH2A1 (macroH2A1) contains an NH(2)-terminal domain that is highly similar to core histone H2A and a larger COOH-terminal domain of unknown function. MacroH2A1 is expressed at similar levels in male and female embryonic stem (ES) cells and adult tissues, but a portion of total macroH2A1 protein localizes to the inactive X chromosomes (Xi) of differentiated female cells in concentrations called macrochromatin bodies. Here, we show that centrosomes of undifferentiated male and female ES cells harbor a substantial store of macroH2A1 as a nonchromatin-associated pool. Greater than 95% of centrosomes from undifferentiated ES cells contain macroH2A1. Cell fractionation experiments confirmed that macroH2A1 resides at a pericentrosomal location in close proximity to the known centrosomal proteins gamma-tubulin and Skp1. Retention of macroH2A1 at centrosomes was partially labile in the presence of nocodazole suggesting that intact microtubules are necessary for accumulation of macroH2A1 at centrosomes. Upon differentiation of female ES cells, Xist RNA expression became upregulated and monoallelic as judged by fluorescent in situ hybridization, but early Xist signals lacked associated macroH2A1. Xi acquired macroH2A1 soon thereafter as indicated by the colocalization of Xist RNA and macroH2A1. Accumulation of macroH2A1 on X chromosomes occurred with a corresponding loss of centrosomal macroH2A1. Our results define a sequence for the loading of macroH2A1 on the Xi and place this event in the context of differentiation and Xist expression. Furthermore, these results suggest a role for the centrosome in the X inactivation process.

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.

Isolation of Histones and Nucleosome Cores from Mammalian Cells

In vitro analysis of DNA in chromatin is often important for understanding mechanisms of regulation of transcription and other processes that occur on DNA. The basic unit of chromatin is the nucleosome core, containing two copies each of the core histones H2A, H2B, H3, and H4 to form a histone octamer that wraps 145 base pairs of DNA in a left-handed superhelix. In vivo, chromatin is associated with linker histones (such as H1), which facilitate the ordered packing of nucleosomes. This linker histone-containing particle is properly termed the nucleosome (or chromatosome), while the linker histone-free particle is the nucleosome core. Pure polynucleosome cores and histones can be readily isolated from mammalian tissue culture cells. This unit describes procedures for isolation and purification of nuclei, isolation of polynucleosomes lacking linker histones from these nuclei, isolation of pure populations of mono- and dinucleosome cores from oligonucleosome fractions, and isolation of core histones from purified nuclei. The methods presented here do not denature the histones, and may yield histones that are more active for in vitro assemblies.

Interaction partners of Dlk/ZIP kinase: co-expression of Dlk/ZIP kinase and Par-4 results in cytoplasmic retention and apoptosis.

Dlk/ZIP kinase is a newly discovered serine/threonine kinase which, due to its homology to DAP kinase, was named DAP like kinase, Dlk. This kinase is tightly associated with nuclear structures, it undergoes extensive autophosphorylation and phosphorylates myosin light chain and core histones H3, H2A and H4 in vitro. Moreover, it possesses a leucine zipper which mediates interaction with transcription factor ATF-4, therefore it was called ZIP kinase. We employed the yeast two-hybrid system to identify interaction partners of Dlk that might serve as regulators or targets. Besides ATF-4 and others we found Par-4, a modulator of transcription factor WT1 and mediator of apoptosis. Complex formation between Dlk and Par-4 was confirmed by GST pull-down experiments and kinase reactions in vitro and coexpression experiments in vivo. The interaction domain within Dlk was mapped to an arginine-rich region between residues 338 - 417, rather than to the leucine zipper. Strikingly, coexpression of Dlk and Par-4 lead to relocation of Dlk from the nucleus to the cytoplasm, particularly to actin filaments. These interactions provoked a dramatic reorganization of the cytoskeleton and morphological symptoms of apoptosis, thus suggesting a functional relationship between Dlk and Par-4 in the control of apoptosis.

Sas3 Is a Histone Acetyltransferase and Requires a Zinc Finger Motif

SAS3 was originally isolated as a gene related to SAS2, which encodes a positive regulator of transcriptional silencing in yeast. The Sas3 protein possesses an evolutionally conserved domain that is shared by a group of SAS-like factors. This conserved domain contains an atypical zinc finger motif and a putative acetyl-CoA binding motif. We showed that recombinant Sas3 exhibits histone acetyltransferase (HAT) activity toward acetylate core histones H2A, H3, and H4. This substrate specificity is similar to those of Tip60 and Esa1. Analysis of a series of deletion mutants revealed that the minimum region required for HAT activity is located within amino acid residues 241–577, including the domain conserved in the MYST family proteins. Amino acid substitution mutant analysis showed that both the acetyl-CoA binding motif and the zinc finger motif are required for HAT activity. These results suggest that SAS3 and its family members require the zinc finger motif for their activity.

Preparation of Site-Specific Antibodies to Acetylated Histones

Synthetic peptides corresponding to regions within the amino-terminal domains of the core histones H2A, H2B, H3, and H4, in which ϵ-acetyllysine has been substituted for selected lysines, have been used to raise polyclonal antisera in rabbits. Such antisera can be specific not only for individual acetylated histones but also for histone isoforms acetylated at particular lysine residues. In this article, we describe procedures for the preparation, affinity purification, and initial characterization of site-specific antisera to acetylated histones.

Linker DNA and H1-dependent reorganization of histone-DNA interactions within the nucleosome.

We have employed a site-directed photochemical cross-linking procedure to precisely map interactions between nucleosomal DNA and the C-terminal tail of core histone H2A. We find that this tail has the potential to contact multiple sites within the nucleosome and that these contacts are dependent upon the configuration of the complex. This tail contacts DNA near the dyad axis within nucleosome core particles but rearranges to a site near the edge of the nucleosomal DNA when linker DNA is present. Moreover, in the presence of linker histone H1 the contacts near the edge of the nucleosome but not at the dyad are further rearranged. In addition, we present further evidence for the suggestion that the binding of linker histone causes a subtle but global change in core histone-DNA interactions within the nucleosome [Usachenko, S. I., Gavin, I. M., and Bavykin, S. G. (1996) J. Biol. Chem. 271, 3831-3836].

Mapping of linear histone regions exposed at the surface of the nucleosome in solution

Antibodies directed against defined regions of histone molecules represent one of the most specific probes for studying the topography and conformational changes of nucleosomes and chromatin. We have developed an assay involving a series of monoclonal and polyclonal antibody probes specifically reacting with a complete set of 40 overlapping synthetic peptides (6 to 28 residues long) covering the whole sequence of the four core histones H2A, H2B, H3 and H4. In this assay, mono-, di- and trinucleosomes, as well as a long chain of chromatin containing 20 to 35 nucleosomes, were used in solution as competitors of the antibody reaction. At least 11 surface-oriented linear regions were characterized on the mononucleosome; namely, the N-terminal domains of H2A (residues 1 to 20), H2B (residues 1 to 25) and H3 (residues 1 to 30), the C-terminal domains of H2A (residues 116 to 129) and H4 (residues 85 to 102), and six domains located in internal segments in the primary structures of core histones (33 to 49 H2A, 65 to 85 H2A, 60 to 78 H2B, 50 to 70 H3, 111 to 130 H3 and 42 to 59 H4). Only a few changes in the nucleosome topography were observed when free oligo- and polynucleosome structure were comparatively studied.

Nucleosome binding by the polymerase I transactivator upstream binding factor displaces linker histone H1.

Upstream binding factor (UBF) is a vertebrate RNA polymerase I transcription factor that can bend and wrap DNA. To investigate UBF's likely role as an architectural protein of rRNA genes organized in chromatin, we tested UBF's ability to bind rRNA gene enhancers assembled into nucleosome cores (DNA plus core histones) and nucleosomes (DNA plus core histones plus histone H1). UBF bound with low affinity to nucleosome cores formed with enhancer DNA probes of 162 bp. However, on nucleosome cores which contained approximately 60 bp of additional linker DNA, UBF bound with high affinity similar to its binding to naked DNA, forming a ternary DNA-core histone-UBF complex. UBF could be stripped from ternary complexes with competitor DNA to liberate nucleosome cores, rather than free DNA, suggesting that UBF binding to nucleosome cores does not displace the core histones H2A, H2B, H3, and H4. DNase I, micrococcal nuclease, and exonuclease III footprinting suggests that UBF and histone H1 interact with DNA on both sides flanking the histone octamer. Footprinting shows that UBF outcompetes histone H1 for binding to a nucleosome core and will displace, if not dissociate, H1 from its binding site on a preassembled nucleosome. These data suggest that UBF may act to prevent or reverse the assembly of transcriptionally inactive chromatin structures catalyzed by linker histone binding.

The chlamydial EUO gene encodes a histone H1-specific protease.

Chlamydia trachomatis is an obligate intracellular pathogen, long recognized as an agent of blinding eye disease and more recently as a common sexually transmitted infection. Recently, two eukaryotic histone H1-like proteins, designated Hc1 and Hc2, have been identified in Chlamydia. Expression of Hc1 in recombinant Escherichia coli produces chromatin condensation similar to nucleoid condensation observed late in the parasite's own life cycle. In contrast, chromatin decondensation, observed during the early life cycle, accompanies down-regulation and nondetection of Hc1 and Hc2 among internalized organisms. We reasoned that the early upstream open reading frame (EUO) gene product might play a role in Hc1 degradation and nucleoid decondensation since it is expressed very early in the chlamydial life cycle. To explore this possibility, we fused the EUO coding region between amino acids 4 and 177 from C. trachomatis serovar Lz with glutathione S-transferase (GST) and examined the effects of fusion protein on Hc1 in vitro. The purified fusion protein was able to digest Hc1 completely within 1 h at 37 degrees C. However, GST alone exhibited no Hc1-specific proteolytic activity. The chlamydial EUO-GST gene product also cleaves very-lysine-rich calf thymus histone H1 and chicken erythrocyte histone H5 but displays no measurable activity towards core histones H2A, H2B, H3, and H4 or chlamydial RNA polymerase alpha-subunit. This proteolytic activity appears sensitive to the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and aspartic protease inhibitor pepstatin but resistant to high temperature and other broad-spectrum protease inhibitors. The proteolytic activity specified by the EUO-GST fusion product selectively digested the C-terminal portion of chlamydial Hc1, the domain involved in DNA binding, while leaving the N terminus intact. At a molar equivalent ratio of 1:1 between Hc1 and DNA, the EUO gene product cleaves Hc1 complexed to DNA and this cleavage appears sufficient to initiate dissociation of DNA-Hc1 complexes. However, at a higher molar equivalent ratio of Hc1/DNA (10:1), there is partial protection conferred upon Hc1 to an extent that prevents dissociation of DNA-Hc1 complexes.

Characterization of nucleosome core particles containing histone proteins made in bacteria

The four core histone proteins, H2A, H2B, H3, and H4 of Xenopus laevis have been individually expressed in milligram quantities in Escherichia coli. The full-length proteins and the “trypsin-resistant” globular domains were purified under denaturing conditions and folded into histone octamers. Both intact and truncated recombinant octamers, as well as chicken erythrocyte octamer, were assembled into nucleosome core particles using a 146 bp defined-sequence DNA fragment from a 5 S RNA gene. The three types of core particles were characterized and compared by gel electrophoresis, DNase I cleavage, and tyrosine fluorescence emission during stepwise dissociation with increasing ionic strength. Nucleosome core particles containing native and mutant histones made in bacteria have facilitated its X-ray structure determination at 2.8 Å resolution.

The Response of Renal Tubular Epithelial Cells to Physiologically and Chemically Induced Growth Arrest

Cells respond to a variety of stresses by activating the transcription of a battery of "acute phase" or "stress response" genes. The nature of this response is tailored to the nature of the stress. The extent to which physiologically and pathophysiologically induced growth arrest share common genomic responses is unclear. We therefore compared the effects of a physiologically induced (serum and nutrient depletion) and a chemically induced (2-Br-bis-(GSyl)HQ and 2-Br-6-(GSyl)HQ) stress in renal tubular epithelial cells (LLC-PK1). The response to physiological stress, induced by serum depletion, involves growth arrest characterized by an inhibition of DNA synthesis that occurs in the absence of a decrease in histone mRNA or an increase in gadd153 mRNA, one of the growth arrest and DNA damage inducible genes. In contrast, the chemical-induced stress involves growth arrest accompanied by a decrease in histone mRNA, particularly core histone H2B and H2A mRNA, and the induction of gadd153. Chemical-induced changes in histone mRNA inversely correlate to changes in the expression of a stress gene, hsp70, whose expression is dependent upon the maintenance of appropriate nucleosomal structure.

2436 Angiotensinergic circuits associated noradrenaline release in the median preoptic area in rats

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.

Remodeling somatic nuclei in Xenopus laevis egg extracts: molecular mechanisms for the selective release of histones H1 and H1(0) from chromatin and the acquisition of transcriptional competence.

The molecular mechanisms responsible for the remodeling of entire somatic erythrocyte nuclei in Xenopus laevis egg cytoplasm have been examined. These transitions in chromosomal composition are associated with the capacity to activate new patterns of gene expression and the re-acquisition of replication competence. Somatic linker histone variants H1 and H1 (0) are released from chromatin in egg cytoplasm, whereas the oocyte-specific linker histone B4 and HMG1 are efficiently incorporated into remodeled chromatin. Histone H1 (0) is released from chromatin preferentially in comparison with histone H1. Core histones H2A and H4 in the somatic nucleus are phosphorylated during this remodeling process. These transitions recapitulate the chromosomal environment found within the nuclei of the early Xenopus embryo. Phosphorylation of somatic linker histone variants is demonstrated not to direct their release from chromatin, nor does direct competition with cytoplasmic stores of linker histone B4 determine their release. However, the molecular chaperone nucleoplasmin does have an important role in the selective removal of linker histones from somatic nuclei. For Xenopus erythrocyte nuclei, this disruption of chromatin structure leads to activation of the 5S rRNA genes. These results provide a molecular explanation for the remodeling of chromatin in Xenopus egg cytoplasm and indicate the capacity of molecular chaperones to disrupt a natural chromosomal environment, thereby facilitating transcription.

H2A.ZI, a new variant histone expressed during Xenopus early development exhibits several distinct features from the core histone H2A.

We have isolated from a subtractive cDNA library of Xenopus laevis a novel transcript, H2A.ZI, which belongs to the H2A.Z variant gene family. Characterization of its expression during oogenesis and development shows significant differences from the expression of the core histone H2A. First, H2A.ZI mRNA is mainly detected only during oogenesis and after the midblastula transition, whereas H2A is constitutively expressed, at much higher levels, throughout embryonic growth. Second, in contrast with H2A, the variant H2A.ZI is polyadenylated during development. Third, expression of H2A.ZI is uncoupled from the S phase after gastrula, whereas synthesis of the core histone H2A mRNA is tightly controlled to DNA replication. Interestingly, H2A.ZI is less charged in the N-terminal tail which is crucial for chromatin-mediated repression. The characteristics of H2A.ZI suggest that its incorporation into nucleosomes would lead to a chromatin structure more competent for gene expression during development.

The histone fold: evolutionary questions.

The histone fold: evolutionary questions.

The organization structure and regulatory elements of Chlamydomonas histone genes reveal features linking plant and animal genes.

The genome of the green alga Chlamydomonas reinhardtii contains approximately 15 gene clusters of the nucleosomal (or core) histone H2A, H2B, H3 and H4 genes and at least one histone H1 gene. Seven non-allelic histone gene loci were isolated from a genomic library, physically mapped, and the nucleotide sequences of three isotypes of each core histone gene species and one linked H1 gene determined. The core histone genes are organized in clusters of H2A-H2B and H3-H4 pairs, in which each gene pair shows outwardly divergent transcription from a short (< 300 bp) intercistronic region. These intercistronic regions contain typically conserved promoter elements, namely a TATA-box and the three motifs TGGCCAG-G(G/C)-CGAG, CGTTGACC and CGGTTG. Different from the genes of higher plants, but like those of animals and the related alga Volvox, the 3' untranslated regions contain no poly A signal, but a palindromic sequence (3' palindrome) essential for mRNA processing is present. One single H1 gene was found in close linkage to a H2A-H2B pair. The H1 upstream region contains the octameric promoter element GGTTGACC (also found upstream of the core histone genes) and two specific sequence motifs that are shared only with the Volvox H1 promoters. This suggests differential transcription of the H1 and the core histone genes. The H1 gene is interrupted by two introns. Unlike Volvox H3 genes, the three sequenced H3 isoforms are intron-free. Primer-directed PCR of genomic DNA demonstrated, however, that at least 8 of the about 15 H3 genes do contain one intron at a conserved position. In synchronized C. reinhardtii cells, H4 mRNA levels (representative of all core histone mRNAs) peak during cell division, suggesting strict replication-dependent gene control. The derived peptide sequences place C. reinhardtii core histones closer to plants than to animals, except that the H2A histones are more animal-like. The peptide sequence of histone H1 is closely related to the V. carteri VH1-II (66% identity). Organization of the core histone gene in pairs, and non-polyadenylation of mRNAs are features shared with animals, whereas peptide sequences and enhancer elements are shared with higher plants, assigning the volvocalean histone genes a position intermediate between animals and plants.

Evidence Indicating Proximity in the Nucleosome Between the Histone H4 N Termini and the Globular Domain of Histone H1

Proteolysis of rat liver chromatin by the Arg-C peptidase, clostripain, is characterized by a progressive fragmentation of the N-terminal segments of the four core histones H2A, H2B, H3 and H4, until a well-defined limit digest is reached. This work addresses the case of histone H4. Two intermediate proteolytic sites are identified for this histone, i.e. Arg3 and Arg17, before the limit digest is achieved through cleavage of the polypeptide chain after Arg19. The accessibility of these intermediate sites depends strongly on the presence or absence of histone H1. When H1 is absent, both intermediate sites of histone H4 are similarly accessible, whereas one of them, Arg3, becomes totally inaccessible in the presence of histone HI. Di- and trinucleosomes were used with the aim of avoiding any interference with superstructural effects which can occur with longer polynucleosomes in the presence of H1. We also investigated the accessibility of the Arg sites of H1 that are located primarily in the central globular domain of this histone. In free histone H1, all the centrally located Arg sites are accessible to clostripain. In contrast, in the chromatin-bound state none of these sites is accessible. Besides the arginyl sites in the central globular domain of H1, two Arg residues are observed with the most abundant H1d variant in rat chromatin, one in the N-terminal region and the other in the C-terminal region. The restricted number of proteolytic fragments observed with chromatin-bound H1 is accounted for by the cleavage of H1 after these Arg residues located on the outside of the globular domain. Our results suggest that mutual steric effects are at play between histones H1 and H4 and indicate that the N termini of both histones H4 in the nucleosome lie in close proximity to the globular domain of H1. Based on these observations and taking into account the known structural features of the nucleosome, we propose a model for positioning the N-terminal segments of both histones H4 at the periphery of the nucleoprotein structure. In this model both H4 segments are located within the expanded DNA minor grooves, at periods ±1, symmetrically disposed relatively to the nucleosome dyad axis. This arrangement brings the amino ends of both H4 molecules in close contact with the H1 globular domain thus accounting for the observed inaccessibility of the Arg3 site of H4 in the presence of H1.

Remodeling sperm chromatin in Xenopus laevis egg extracts: the role of core histone phosphorylation and linker histone B4 in chromatin assembly.

We find that the remodeling of the condensed Xenopus laevis sperm nucleus into the paternal pronucleus in egg extracts is associated with phosphorylation of the core histones H2A, H2A.X and H4, and uptake of a linker histone B4 and a HMG 2 protein. Histone B4 is required for the assembly of chromatosome structures in the pronucleus. However neither B4 nor core histone phosphorylation are required for the assembly of spaced nucleosomal arrays. We suggest that the spacing of nucleosomal arrays is determined by interaction between adjacent histone octamers under physiological assembly conditions.