PL-I of Spisula solidissima, a highly elongated sperm-specific histone H1

The major chromosomal protein of the mature sperm of the surf clam, Spisula solidissima, is a histone H1-related protamine-like (PL-I) protein of low electrophoretic mobility. We report here the complete sequence of two isoforms of its encoding genes. These genes encode a protein of 453 and 454 amino acids, respectively. The predicted mass of the larger isoforms (51,437 Da) was confirmed using electrospray ionization mass spectrometry. The amino-terminal tail of the S. solidissima PL-I is greatly elongated because of the presence of 39 tandem hexapeptide repeats of the motif (K/R)KRSAS with a few semiconservative amino acid substitutions. These repeats are very closely mirrored by their encoding DNA sequence, which indicates that an expansion because of sequence duplication most likely occurred. The C-terminal domain consists of a histone H1-related core with a predicted winged-helix tertiary structure, which is followed by an unstructured lysine-rich tail. This information provides additional molecular support for the classification and underlying evolution of sperm nuclear basic proteins in bivalve molluscs.     

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.     

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.     

An unusual cysteine-containing histone H1-like protein and two protamine-like proteins are the major nuclear proteins of the sperm of the bivalve mollusc Macoma nasuta

The sperm-specific protamine-like (PL) components PL-I, PL-II, and PL-III from the sperm of the bent-nose clam Macoma nasuta have been isolated and characterized for the first time. These proteins coexist in the sperm nuclei with a small percentage of a full histone complement. All of them have a very similar amino acid composition, following what seems to be the general composition prototype for the class Bivalvia (Ausió, J. (1986) Comp. Biochem. Physiol. B Comp. Biochem. 85, 439-449). Nevertheless, they have different molecular weights (PL-I = 23,500, PL-II = 15,600, and PL-III = 7,900) as measured by sedimentation equilibrium in the analytical ultracentrifuge. Furthermore, the PL-I component shares common features with the proteins of the histone H1 family. Yet, it is very unusual, for it contains 2 cysteine residues that are located in the trypsin-resistant core of this protein. The protamine-like fraction PL-III exhibits intraspecific microheterogeneity which is reflected by the presence of two protein variants which most probably are the result of an allelic polymorphism.     

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.     

Calculated flexibility of histone proteins correlate with mammalian histone H1 subtype.

We have calculated the polypeptide flexibility index for mammalian histone H1 sequences obtained from the National Center for Biotechnology Information Histone Sequence Database. This database contains over 1000 histone protein entries, from various species, compiled from SWISS_PROT, PIR, the Protein Data Bank (PDB), and CDS translations from GenBank. Histone H1 proteins were analyzed because of their critical role in chromatin structure and gene expression. Flexibility calculations revealed that histone subtype H1.0, which accumulates during terminal differentiation, has the highest flexibility index of all mammalian H1 subtypes. Other mammalian H1 subtypes had lower flexibility indices, including the human H1.2 subtype whose mRNA contains both a hairpin loop sequence and a poly(A) addition sequence. Histone mRNAs containing both of these structures have been shown to be expressed prior to and after terminal differentiation, yet these proteins do not necessarily accumulate in the chromatin of terminally differentiated cells. H1.2 and the H1.t have the lowest flexibility index (most ridged) of all human H1 subtypes. All human H1 proteins of the replication dependent subtypes have intermediate values for their flexibility indices.     

Replacement of nucleosomal histones by histone H1-like proteins during spermiogenesis in Cnidaria: Evolutionary implications

We have analyzed the chromosomal protein composition of the sperm from several species belonging to three different classes (Hydrozoa, Scyphozoa, Anthozoa) of the phylum Cnidaria. In every instance, the sperm nuclear basic proteins (SNBPs) were found to consist of one to two major protein fractions that belong to the histone H1 family, as can be deduced from their amino acid composition and solubility in dilute perchloric acid, and the presence of a trypsin-resistant core. In those species where mature spawned sperm could be obtained, we were able to show that these proteins completely replace the somatic histones from the stem cells that are present at the onset of spermatogenesis. The presence of a highly specialized histone H1 molecule in the sperm of this phylum provides support for the idea that the protamine-like proteins (PL) from higher groups in the phylogenetic tree (and possibly protamines as well) may all have evolved from a primitive histone H1 ancestor.     

Expression of histone 1 (H1) and testis-specific histone 1 (H1t) genes during stallion spermatogenesis

In eukaryotic cells, the major protein constituents of the chromatin are histones, which can be divided into five classes, identified as H1, H2A, H2B, H3 and H4. During normal spermatogenesis, a testis-specific H1t is expressed in primary spermatocytes and believed to facilitate histone to protamine exchanges during spermiogenesis. In equine testes we detected the H1 protein at 22kDa by western blot analysis while H1t was detected at 29kDa. H1 protein was found to be expressed in all germ cells up to elongating spermatids (Sc) at stage IV. In peripubertal animals, there was a prolonged expression up to elongating spermatids (Sd1) at stage V. A fragment of the equine H1t gene was cloned (GenBank Accession No. AJ865320). The mRNA expression of H1t was found at the level in spermatogonia and in primary spermatocytes up to mid-pachytene at stage VIII/I, whereas H1t protein was found to be expressed up to round spermatides (Sa/Sb1) at stage VIII/I. In peripubertal animals, the H1t protein expression was detected up to elongating spermatids (Sb2) at stage II. Analysis of testes of different ages (< or =2 years) and (> or =3 years) by real-time RT-PCR revealed an increase of H1t mRNA expression, with a wide range of individual variety between 2 and 4 years old animals indicating a stable expression in animals older than 4 years old. This is the first study to show the testis-specific H1t in the stallion and gives evidence that the well-known peripubertal infertility in the stallion may be related to an insufficient histone to protamine exchange. The pattern of protamine gene expression, however, has still to be elucidated.     

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.     

Identification of nonhistone chromatin proteins in chromatin subunits (or mononucleosomes) devoid of histone H1.

Rat liver chromatin was digested by micrococcal nuclease. Chromatin subunits (or mononucleosomes) were isolated by sucrose density gradient and subsequently fractionated by 6% polyacrylamide gel electrophoresis into two major components. One component (MN1) of the mononucleosomes had a higher mobility, contained histones H2A, H2B, H3, H4, and shorter DNA fragments (140 base pairs) while the other (MN2) contained all five histones and longer DNA fragments (180 base pairs). Both submononucleosomes (MN1 and MN2) were found to contain nonhistone chromatin proteins (NHCP). By electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel, 9 and 11 major fractions of NHCP were identified in the submononucleosomes MN1 and MN2, respectively. It was also observed that treatment of mononucleosomes with 0.6 M NaCl removes most of these NHCP and histone H1 except for two major NHCP which remain in the core particles.     

Xenopus laevis sperm proteins, previously identified as surface proteins with egg coat binding capability, are indeed histone H4, histone H3, and sperm specific protein SP2.

Recently, four Xenopus sperm proteins thought to be involved in binding to the egg envelope were identified (Lindsay and Hedrick, J. Exp. Zool., 245:286-293, '88). We have studied the three more abundant ones of apparent molecular weight of 14, 19, and 25 kd in SDS-PAGE. We have shown that these proteins are indeed nuclear basic proteins: the 14 kd is the histone H4, the 19 kd is the histone H3, and the 25 kd is the sperm-specific protein SP2.     

Immunological evidence for the existence of H1-like histone in yeast

In view of the controversies about the existence of histone H1 in yeast we have reinvestigated the problem by studying yeast proteins extracted with perchloric acid and salt. Perchloric-acid-extracted proteins from whole cells contain only two fractions which comigrate with 'authentic' yeast high-mobility-group proteins (HMG) in both SDS and acid urea gels. These extracts show a considerable cross-reaction with anti-(calf thymus HMG) antiserum and do not react with antiserum to mouse liver H1. The isolation of 'authentic' yeast HMG by the standard salt/trichloroacetic acid procedure gives two types of preparations containing different numbers of protein bands. The poorer preparation reacts only with the anti-HMG antiserum whereas the richer preparation also gives considerable cross-reaction with the anti-H1 antiserum. Immunoblotting analysis performed on the salt-extracted proteins reveals the presence of three protein bands giving positive immunoreaction with the anti-H1 antiserum. The immunoreactive bands have electrophoretic mobilities close to that of the marker calf thymus H1 and similar to the mobilities of the presumptive yeast H1 fractions found by other authors.     

Hyper(ADP-ribosyl)ation of histone H1

Nucleosomal chains of various repeat unit lengths were generated by a mild micrococcal nuclease digestion of purified pancreatic nuclei. Maximal nucleosome associated poly(ADP-ribose) polymerase activity was recovered in trimeric to tetrameric chromatin fragments, after which the enzyme activity gradually decreased and stabilized towards oligomeric periodicities of 11 to 16 nucleosomes. Electrophoresis of [32P]ADP-ribosylated histones on first-dimension acid-urea or acid-urea-Triton gels and on second-dimension acid--urea--cetyltriammonium bromide gels revealed that, of all histones, only histone H1 could be significantly poly(ADP-ribosyl)ated while only minimal modification could be recovered with histone H1(0). Furthermore, the extent of ADP-ribosylation present on pancreatic histone H1 is shown to proportionally retard this protein's electrophoretic mobility in all gel systems and to consist of a distinct series of at least 12 modification intermediates which can be evidenced, in nuclei or nucleosomes, and fully recovered along with histone H1 upon its selective extraction with 5% perchloric acid. The generation of these increasingly ADP-ribosylated forms of histone H1 is also demonstrated to be time dependent and the more complex ADP-ribosylated forms of this histone are favored at high NAD+ concentrations. Moreover, the electrophoretic mobilities of all intermediates are unaffected by the presence of the nonionic detergent Triton X-100.     

An H1-like protein from the sperm chromatin of Mytilus galloprovincialis

We have isolated and purified a sperm-specific protein (S3) from the mussel M. galloprovincialis. Antibodies against S3 were raised in rabbits and used for its immunological comparison to somatic histones. The results showed that S3 did not share common immunological determinants with H2b or any other core histone-contrary to the suggestion that it was an H2b-like protein (Ausio and Subirana, 1982). With H1 there was a crossreaction between S3 and anti-H1 as well as with H1 and anti-S3. Although similar to somatic H1, S3 is not identical with it. This fact makes S3 an interesting example of another protein of the H1-H5 type, present in a completely inactive chromatin.