Immunological evidence for an H1° type of histone protein in chicken liver

We prepared monoclonal antibodies against chicken histone H5. These antibodies could be divided into two classes, and we present the results obtained with one representative antibody of each class. One class reacted exclusively with chicken H5, whereas the other additionally cross-reacted with rat H1(0) and with material present in adult but not embryonic chicken liver. The cross-reacting material in adult liver was identified by Western blotting as representing a minor band in histone preparations. The protein was not present in histone extracts from chicken erythrocytes. It is likely that this newly identified protein is a chicken H1(0) histone.     

Sequence specific binding of chlamydial histone H1-like protein.

Chlamydia trachomatis is one of the few prokaryotic organisms known to contain proteins that bear homology to eukaryotic histone H1. Changes in macromolecular conformation of DNA mediated by the histone H1-like protein (Hc1) appear to regulate stage specific differentiation. We have developed a cross-linking immunoprecipitation protocol to examine in vivo protein-DNA interaction by immune precipitating chlamydial Hc1 cross linked to DNA. Our results strongly support the presence of sequence specific binding sites on the chlamydial plasmid and hc1 gene upstream of its open reading frame. The preferential binding sites were mapped to 520 bp BamHI-XhoI and 547 bp BamHI-DraI DNA fragments on the plasmid and hc1 respectively. Comparison of these two DNA sequences using Bestfit program has identified a 24 bp region with >75% identity that is unique to the chlamydial genome. Double-stranded DNA prepared by annealing complementary oligonucleotides corresponding to the conserved 24 bp region bind Hc1, in contrast to control sequences with similar A+T ratios. Further, Hc1 binds to DNA in a strand specific fashion, with preferential binding for only one strand. The site specific affinity to plasmid DNA was also demonstrated by atomic force microscopy data images. Binding was always followed by coiling, shrinking and aggregation of the affected DNA. Very low protein-DNA ratio was required if incubations were carried out in solution. However, if DNA was partially immobilized on mica substrate individual strands with dark foci were still visible even after the addition of excess Hc1.     

Cysteine-containing histone H1-like (PL-I) proteins of sperm.

We have determined the presence of cysteine in the protein PL-I from the sperm of the surf clam Spisula solidissima. The existence of cysteine in this histone H1-related protein is responsible for its previously described aggregation behavior. The location of this residue, within the trypsin-resistant domain of the protein, has been established. We have also shown that cysteine is ubiquitously present in the PL-I proteins from the sperm of other bivalve mollusks but is absent from other PL of smaller molecular mass (PL-II, PL-III, PL-IV). We have also found cysteine to be present in the PL-I from a tunicate (Chelysoma productum) but absent in a PL-I from a fish (Mullus barbatus). The possible significance of the unusual occurrence of cysteine in these histone-H1-related proteins is discussed.     

Decreased expression of specific genes in yeast cells lacking histone H1

Chromatin plays an important role in regulating eukaryotic gene expression. Chromatin is composed of DNA wrapped around a nucleosome core (consisting of two copies of the well conserved histones H2A, H2B, H3, and H4) and a more variable linker histone H1. Various in vitro and in vivo studies have implicated histone H1 as a repressor of gene expression or as an activator, but its exact role is still unclear. Sequencing of the yeast genome has led to the identification of a putative histone H1 gene. Biochemical studies demonstrated that yeast does indeed possess a bona fide histone H1. However, deletion of the unique yeast H1 gene is not associated with any phenotypes, and it was questioned whether it plays any role. To address this issue, we performed whole-genome microarray analysis to identify genes that are affected by H1 removal. Surprisingly, deletion of the gene encoding histone H1 does not result in increased gene expression but rather in a modest reduction. Northern blot analysis of selected genes confirmed the results obtained with the microarray analysis. A similar effect was observed with an integrated lacZ reporter. Thus, our data demonstrate that removal of yeast histone H1 only results in decreased gene expression.     

A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena.

Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis. It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation. However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear. In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity. Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth-associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes. Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)-coated beads and closely follows H1 kinase activity. Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation. The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur. These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells.     

Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1

Using the method of salt dialysis, we have reconstituted histone octamers onto DNA templates consisting of 12 tandem repeats, each containing a fragment of the sea urchin 5S rRNA gene [Simpson, R.T., Thoma, F., & Brubaker, J.M. (1985) Cell 42, 799-808]. In these templates, each sea urchin repeat contains a sequence for preferred nucleosome positioning. Sedimentation velocity and sedimentation equilibrium studies in the analytical ultracentrifuge indicate that at molar histone/DNA ratios of 1.0-1.1 extremely homogeneous preparations of fully loaded oligonucleosomes (12 nucleosomes/template) can be regularly obtained. Digestion of the oligonucleosomes with micrococcal nuclease, followed by restriction mapping of purified nucleosome-bound DNA sequences, yields a complicated but consistent pattern of nucleosome positioning. Roughly 50% of the nucleosomes appear to be phased at positions 1-146 of each repeat, while the remainder of the nucleosomes occupy a number of other minor discrete positions along the template that differ by multiples of 10 bp. From sedimentation velocity studies of the oligonucleosomes in 0-0.2 M NaCl, we observe a reversible increase in mean sedimentation coefficient by almost 30%, accompanied by development of heterogeneity in sedimentation. These results, in combination with theoretical predictions, indicate that linear stretches of chromatin in the absence of lysine-rich histones exist in solution in a salt-dependent equilibrium between an extended (low salt) conformation and one or more folded (high salt) structures. In addition, by 100 mM NaCl, salt-dependent dissociation of histone octamers from these linear oligonucleosomes is observed.     

The activity of the histone chaperone yeast Asf1 in the assembly and disassembly of histone H3/H4-DNA complexes

The deposition of the histones H3/H4 onto DNA to give the tetrasome intermediate and the displacement of H3/H4 from DNA are thought to be the first and the last steps in nucleosome assembly and disassembly, respectively. Anti-silencing function 1 (Asf1) is a chaperone of the H3/H4 dimer that functions in both of these processes. However, little is known about the thermodynamics of chaperone–histone interactions or the direct role of Asf1 in the formation or disassembly of histone–DNA complexes. Here, we show that Saccharomyces cerevisiae Asf1 shields H3/H4 from unfavorable DNA interactions and aids the formation of favorable histone–DNA interactions through the formation of disomes. However, Asf1 was unable to disengage histones from DNA for tetrasomes formed with H3/H4 and strong nucleosome positioning DNA sequences or tetrasomes weakened by mutant (H3K56Q/H4) histones or non-positioning DNA sequences. Furthermore, Asf1 did not associate with preformed tetrasomes. These results are consistent with the measured affinity of Asf1 for H3/H4 dimers of 2.5 nM, which is weaker than the association of H3/H4 for DNA. These studies support a mechanism by which Asf1 aids H3/H4 deposition onto DNA but suggest that additional factors or post-translational modifications are required for Asf1 to remove H3/H4 from tetrasome intermediates in chromatin.     

Interactions of a replication initiator with histone H1-like proteins remodel the condensed mitochondrial genome

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomatids, consists of several thousand topologically interlocked DNA circles. Mitochondrial histone H1-like proteins were implicated in the condensation of kDNA into a nucleoid structure in the mitochondrial matrix. However, the mechanism that remodels kDNA, promoting its accessibility to the replication machinery, has not yet been described. Analyses, using yeast two hybrid system, co-immunoprecipitation, and protein-protein cross-linking, revealed specific protein-protein interactions between the kDNA replication initiator protein universal minicircle sequence-binding protein (UMSBP) and two mitochondrial histone H1-like proteins. Fluorescence and electron microscopy, as well as biochemical analyses, demonstrated that these protein-protein interactions result in the decondensation of kDNA. UMSBP-mediated decondensation rendered the kDNA network accessible to topological decatenation by topoisomerase II, yielding free kDNA minicircle monomers. Hence, UMSBP has the potential capacity to function in vivo in the activation of the prereplication release of minicircles from the network, a key step in kDNA replication, which precedes and enables its replication initiation. These observations demonstrate the prereplication remodeling of a condensed mitochondrial DNA, which is mediated via specific interactions of histone-like proteins with a replication initiator, rather than through their posttranslational covalent modifications.     

Analysis of a histone H2A variant from fission yeast: evidence for a role in chromosome stability

We have isolated and characterised the pht1 gene from the fission yeast Schizosaccharomyces pombe. The sequence of the predicted translation product has revealed a striking similarity to the family of H2A.F/Z histone variant proteins, which have been found in a variety of different organisms. Cells deleted for the pht1 gene locus grow slowly, exhibit an altered colony morphology, increased resistance to heat shock and show a significant decrease in the fidelity of segregation of an S. pombe minichromosome. We propose that the histone H2A variant encoded by the pht1 gene is important for chromosomal structure and function, possibly including a role in controlling the fidelity of chromosomal segregation during mitosis.     

Nucleus-encoded histone H1-like proteins are associated with kinetoplast DNA in the trypanosomatid Crithidia fasciculata.

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomatids, consists of thousands of minicircles and 20 to 30 maxicircles catenated into a single large network and exists in the cell as a highly organized compact disc structure. To investigate the role of kinetoplast-associated proteins in organizing and condensing kDNA networks into this disc structure, we have cloned three genes encoding kinetoplast-associated proteins. The KAP2, KAP3, and KAP4 genes encode proteins p18, p17, and p16, respectively. These proteins are small basic proteins rich in lysine and alanine residues and contain 9-amino-acid cleavable presequences. Proteins p17 and p18 are closely related to each other, with 48% identical residues and carboxyl tails containing almost exclusively lysine, alanine, and serine or threonine residues. These proteins have been expressed as Met-His6-tagged recombinant proteins and purified by metal chelate chromatography. Each of the recombinant proteins is capable of compacting kDNA networks in vitro and was shown to bind preferentially to a specific fragment of minicircle DNA. Expression of each of these proteins in an Escherichia coli mutant lacking the HU protein rescued a defect in chromosome condensation and segregation in the mutant cells and restored a near-normal morphological appearance. Proteins p16, p17, and p18 have been localized within the cell by immunofluorescence methods and appear to be present throughout the kDNA. Electron-microscopic immunolocalization of p16 shows that p16 is present both within the kDNA disc and in the mitochondrial matrix at opposite edges of the kDNA disc. Our results suggest that nucleus-encoded H1-like proteins may be involved in the organization and segregation of kDNA networks in trypanosomatids.     

Immunofluorescence evidence for the absence of histone H1 in a mitotically dividing, genetically inactive nucleus

Antibodies directed against whole histone and purified lysine-rich histone H1 extracted from isolated macronuclei of the ciliate Tetrahymena were obtained and conjugated to fluorescein isothiocyanate. The fluorescein-antibody conjugates were used to directly label Tetrahymena cells. Both macro- and micronuclei were visibly fluorescent in cells stained with anti-whole histone conjugate. However, the anti-H1 conjugate only labeled macronuclei. This in situ demonstration of the lack of positive immunofluorescent staining of micronuclei with anti-H1 conjugate provide further evidence for the absence of H1 in the genetically inactive, mitotically dividing Tetrahymena micronucleus.     

Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene

Glycogen synthase preparations from Saccharomyces cerevisiae contained two polypeptides of molecular weights 85,000 and 77,000. Oligonucleotides based on protein sequence were utilized to clone a S. cerevisiae glycogen synthase gene, GSY1. The gene would encode a protein of 707 residues, molecular mass 80,501 daltons, with 50% overall identity to mammalian muscle glycogen synthases. The amino-terminal sequence obtained from the 85,000-dalton species matched the NH2 terminus predicted by the GSY1 sequence. Disruption of the GSY1 gene resulted in a viable haploid with glycogen synthase activity, and purification of glycogen synthase from this mutant strain resulted in an enzyme that contained the 77,000-dalton polypeptide. Southern hybridization of genomic DNA using the GSY1 coding sequence as a probe revealed a second weakly hybridizing fragment, present also in the strain with the GSY1 gene disrupted. However, the sequences of several tryptic peptides derived from the 77,000-dalton polypeptide were identical or similar to the sequence predicted by the GSY1 gene. The data are explained if S. cerevisiae has two glycogen synthase genes encoding proteins with significant sequence similarity The protein sequence predicted by the GSY1 gene lacks the extreme NH2-terminal phosphorylation sites of the mammalian enzymes. The COOH-terminal phosphorylated region of the mammalian enzyme over-all displayed low identity to the yeast COOH terminus, but there was homology in the region of the mammalian phosphorylation sites 3 and 4. Three potential cyclic AMP-dependent protein kinase sites are located in this region of the yeast enzyme. The region of glycogen synthase likely to be involved in covalent regulation are thus more variable than the catalytic center of the molecule.     

Presence of a highly specific histone H1-like protein in the chromatin of the sperm of the bivalve mollusks

Chromatin organization in the sperm of the bivalve mollusks results from the interaction between a discrete number of protamine-like proteins (PL) and DNA. A small variable amount of histones is also present. An extensive study carried out on a relatively large number of species, within the class Bivalvia, has shown that it is possible to arrange these mollusks into five major categories on the basis of their PL composition (Ausio, J. Comp. Biochem. Physiol. 85, 439–449, (1986) [1]). In the present work, we have extended this analysis to a larger number of species and found that in spite of the inter- and intra-specific similarity of all PL proteins in their chemical composition, they exhibit different degrees of structural variability. Moreover one of these PL proteins is present in all the species analyzed, and bears an enormous resemblance to histones of the H1 family. The evolutionary significance of this finding is discussed.