Testis-specific histone H1t is truly a testis-specific variant and not a meiotic-specific variant

Immunoblotting studies using highly specific polyclonal anti-histone H1t-IgG, which was extensively characterized by us previously, did not produce a signal with any of the histone H1 subtypes of either 1-day-old or adult rat ovarian nuclei. The absence of histone H1t in ovarian nuclei was also confirmed by indirect immunofluorescence studies. It is concluded, therefore, that histone H1t is truly a testis-specific histone variant and not a meiotic-specific variant.     

Isolation of the gene for the testis-specific H1 histone variant H1t

H1t is a testis-specific H1 variant found in pachytene spermatocytes and round spermatids of mammals. The H1t gene was isolated from the Sargent-Bonner library of recombinant lambda bacteriophage containing EcoRI fragments of rat liver DNA using a hybridization probe derived from a chicken H1 variant. The rat H1t gene encodes a 207-amino acid protein (ignoring the initiating methionine) that matches perfectly what is known of the sequence and composition of H1t isolated from rat testes. The gene lacks introns and has good matches to all the consensus sequences known to lie upstream from a variety of H1 genes from diverse organisms. It also has the standard downstream palindromic sequence that specifies the 3'-end of most histone messages. Accordingly, the features of the gene or its environs that restrict its expression to a particular phase of spermatogenesis are not yet evident.     

Mapping the binding of monoclonal antibodies to histone H5

The binding sites of nine monoclonal antibodies along the polypeptide chain of histone H5 were mapped. Immunoblotting experiments with peptides generated from H5 by trypsin digestion, N-bromosuccinimide cleavage, and cyanogen bromide cleavage revealed that all of the monoclonal antibodies reacted with the globular region of H5 which is encompassed by amino acid residues 22-98. Within this globular segment, the epitopes could be subdivided into three regions. Monoclonals 1G11, 2E5, and 2H5 bind to residues 28-31. The close proximity of the epitopes was verified by a competitive enzyme-linked immunosorbent assay and by their binding pattern to a tryptic digest of H5. Monoclonals 4C6, 6E12, and 2E12 bind to a region encompassed by amino acids 28-53 while monoclonals 4H7, 1C3, and 3H9 bind to a region encompassed by residues 53-98. Precise localization of the epitopes in the primary sequence of H5 will allow detailed studies on the mode of binding of H5 to core particles in chromatin.     

C-terminal phosphorylation of murine testis-specific histone H1t in elongating spermatids

Previous studies gave differing results as to whether the testis-specific histone H1t was phosphorylated during rodent spermatogenesis. We show here that histones extracted from germ cell populations enriched with spermatids at different stages of development in rat testes reveal an electrophoretic shift in the position of H1t to slower mobilities in elongating spermatids as compared to that from preceding stages. Alkaline phosphatase treatment and radioactive labeling with (32)P demonstrated that the electrophoretic shift is due to phosphorylation. Mass spectrometric analysis of histone H1t purified from sexually mature mice and rat testes confirmed the occurrence of singly, doubly, and triply phosphorylated species, with phosphorylation sites predominantly found at the C-terminal end of the molecule. Furthermore, using collision-activated dissociation (CAD) and electron transfer dissociation (ETD), we have been able to identify the major phosphorylation sites. These include a new, previously unidentified putative H1t-specific cdc2 phosphorylation site in linker histones. The presence of phosphorylation at the C-terminal end of H1t and the timing of its appearance suggest that this post-translational modification is involved in the reduction of H1t binding strength to DNA. It is proposed that this could participate in the opening of the chromatin fiber in preparation for histone displacement by transition proteins in the next phase of spermiogenesis.     

Detection and characterization of IgG-and sIgA-abzymes capable of hydrolyzing histone H1

Immunoglobulins IgG and sIgA actively hydrolyzing histone H1 have been detected on analyzing proteolytic activity of antibodies isolated by chromatography on Protein A-agarose from blood serum of patients with multiple sclerosis and from colostrum of healthy mothers. These antibodies hydrolyze other histones less actively and virtually failed to cleave lysozyme of chicken egg. By gel filtration at acidic pH and subsequent analysis of protease activity of chromatographic fractions, it was shown that IgG and sIgA molecules were responsible for hydrolysis of histone H1. Anti-histone H1 antibodies of IgG and sIgA classes were purified by affinity chromatography on histone H1-Sepharose from catalytically active antibody preparations. The protease activity of anti-histone H1 IgG antibodies was inhibited by serine proteinase inhibitors, whereas anti-histone H1 sIgA antibodies were insensitive to inhibitors of serine, asparagine, and cysteine proteases.     

Changes in nucleosome structure and histone H3 accessibility : Iodoacetamidofluorescein labelling after treatment with phosphatidylserine vesicles

The accessibility of the sulfhydryl-specific dye 6-iodoacetamidofluorescein (IAF) to H3 histone has been studied in isolated rat liver nuclei and mononucleosomal core particles after treatment with phosphatidylserine (PS) multilamellar vesicles (MLV). In isolated nuclei, despite the enhancement of total RNA synthesis and the massive chromatin decondensation produced by liposomes, the amount of histone H3 which can be labelled with the dye remains essentially the same in PS-treated as in control nuclei. However, when mononucleosomal core particles, treated with PS vesicles, are reacted with IAF, H3 becomes derivatized by the dye, while controls do not. These data provide additional evidence that the metabolic and structural changes observed in isolated nuclei treated with PS MLV, are due mainly to the reported removal of histone H1. Moreover, the experiments reported confirm the usefulness of IAF in studying the changes of nucleosome organization, since PS is able to affect the nucleosomal core configuration in isolated nucleosome particles, derivatizing the buried cysteine groups of H3 histone.     

Histone H3 is aberrantly phosphorylated in glutamine-repeat diseases

Double-labeling immunohistochemical studies staining with anti-ubiquitin and anti-phosphoserine antibodies and application of an enzymatic dephosphorylation technique reveal neuronal inclusions and affected nuclei to be aberrantly phosphorylated in brain tissues with patients with glutamine-repeat diseases. Regional distribution of the phosphorylated nuclei in neurons correlates with the pathology. To identify the target nuclear protein, transient expression of Huntington's disease exon 1 gene containing an expanded glutamine repeat was generated in a cell culture and nuclear inclusions were isolated with a fluorescence-activated cell sorting system. Immunoblotting studies of the aggregated nuclear proteins using anti-phosphoserine antibody demonstrate the protein of the aberrant phosphorylation as histone H3. The immunoblots of control and diseased brain tissues demonstrate that the phosphorylation of histone H3 is commonly increased in the diseased brains. Aberrant phosphorylation of histone H3 is surmised to be a shared pathological process in glutamine-repeat diseases.     

The localization of histone H3.3 in germ line chromatin of Drosophila males as established with a histone H3.3-specific antiserum

A rabbit antiserum, specific for the histone H3.3 replacement variant, was raised with the aid of a histone H3.3-specific peptide. Immuno blot experiments demonstrated the specificity of this polyclonal antiserum. In addition, we showed on immuno blots that two monoclonal antibodies isolated from mice with systemic lupus erythematosus (SLE) display strong reactivity with the H3.3 histone, but not with its replication-dependent counterparts. Our observations indicate that histone H3.3 might play a role as autoantigen in SLE. We used the histone H3.3-specific antiserum to characterize the germ line chromatin in cytological preparations of Drosophila testes, because our previous studies had shown that a histone H3.3-encoding gene is strongly expressed in the germ line of Drosophila males. The antiserum reacted with some of the lampbrush loops in spermatocytes and with chromatin of the postmeiotic germ cells of males. Our data indicate that histone H3.3 is not evenly distributed throughout the chromatin of germ cells, but is concentrated in distinct regions. Histone H3.3 disappears from the spermatid nuclei, along with the other core histones, during the late stages of spermatogenesis. In Drosophila polytene chromosomes, however, a rather uniform distribution of the histone H3.3 was observed. The possible role of histone H3.3 is discussed. Received: 12 May 1997 / Accepted: 4 July 1997     

Histone H1--DNA interaction. On the mechanism of DNA strands crosslinking by histone H1.

Crosslinking of DNA fibers by histone H1 or phosphorylated on Ser-37 histone H1, and by the individual fragments of the H1 polypeptide chain was studied by the method of turbidimetry. The dependence of the turbidity of DNA-protein complexes on the ionic strength in solution suggests that the condensation of H1.DNA complexes in vitro is apparently due to both specific histone-DNA interactions with the contribution of hydrogen and/or hydrophobic bonds and the formation of polycationic "bridges" fastening the DNA fibers. The effectiveness of the condensation is postulated to be a function of a proportion between the two mechanisms which in turn can be controlled by slight changes in ionic surroundings. The sharp dependence of shrinkage of H1.DNA complexes on ionic strength at "physiological" salt concentrations could provide a mechanism to regulate density and consequently the total activity of chromatin in the cell nuclei. The phosphorylation of histone H1 on Ser-37 by a specific histone kinase does not noticeably affect the pattern of DNA crosslinking by the H1.     

Antibacterial peptides in stimulated human granulocytes: characterization of ubiquitinated histone H1A.

Antibacterial peptides were isolated from human peripheral granulocytes of a healthy donor who had been treated with granulocyte-colony stimulating factor (G-CSF) and cortisol. Peptides were solubilized in acidified chloroform/methanol, and partitioned in chloroform/methanol/water. Water- soluble polypeptides were separated by cation-exchange and reversed-phase chromatography. Several previously characterized antibacterial polypeptides were identified; defensins 1-3, defensin 4, lysozyme, eosinophil cationic protein, and calgranulin A. In addition, several histone fragments were isolated and exhibited activity against the Gram- positive bacterium Bacillus megaterium strain Bm11. These fragments included two C-terminal fragments of histone H1A, three C-terminal fragments of histone H1D, one fragment of histone H1B, and two fragments of histone H4. The molecular masses of both histone H1A fragments, as determined by electrospray (ES) MS, were 270 Da higher than those calculated from their amino acid sequences. The two histone H1A fragments corresponded to Lys152-Lys222 (7527 +/- 1 Da) and Lys167-Lys222 (6023 +/- 1 Da). Tandem MS (MS/MS) of the 7.5 kDa and 6.0 kDa fragments indicated that the post-translational modification is on Lys222, the epsilon-amino group of which was conjugated with the alpha-carboxyl group of the tripeptide Arg-Gly-Gly. This finding was substantiated by digestion of the 7.5-kDa polypeptide with trypsin and analysis of the resulting peptides by ES MS and MS/MS. The tripeptide Arg-Gly-Gly corresponded uniquely to the three C-terminal residues of ubiquitin, demonstrating the presence of ubiquitinated histone H1A.     

Accessibility of histone H1° and its structural domains to antibody binding in extended and folded chromatin

The aim of this work was to study the accessibility of histone H1° and its structural domains to antibody binding in high molecular mass chromatin fragments of different conformations. Three types of specific antibody populations were used: (1) anti-H1° which reacted with antigenic determinants situated along the whole polypeptide chain, (2) anti-GH5 or anti-GH1° which recognized epitopes located in the globular region of H1° and (3) anti-C-tail antibodies reacting specifically with fragment 99–193 of the protein molecule. The immunoreactivity of the chromatin-bound antigen was investigated by solid-phase ELISA performed on glutaraldehyde-cross-linked chromatin and by an inhibition assay carried out with native chromatin in solution. The results of both methods were unidirectional and showed that: (1) the accessibility of H1° did not change with the compaction of the fiber; (2) the G-domain was not accessible to antibodies either in the relaxed or in the condensed state of the fragments, (3) the binding of the C-terminus-specific antibodies was different for isolated monosomes and for the chromatin fiber and (4) the degree of exposure of the epitopes of H1° in chromatin was much less than that of histone H1.Abbreviations ELISA Enzyme-Linked Immunosorbent Assay - G-domain Globular domain - IgG Immunoglobulin G - SDS Sodium Dodecylsulphate     

Histone H1 binding at the 5' end of the rat albumin gene

Cloned DNA containing the first nine exons of the rat albumin gene was digested with EcoRI and HindIII, and the resulting fragments were used to screen for regions with relatively high affinity for protein. Of three restriction fragments preferentially bound, the fragment containing the first two exons of the albumin gene was consistently bound over others by heat-stable protein extracted from liver nuclei with 0.35-1.0 M NaCl. Proteins extracted with lower and higher ionic strength buffers bound the DNA fragments, but with little specificity. The DNA fragment that was preferentially bound consistently by the 1.0 M nuclear extract was subcloned into pBR325 and was used to isolate the specific DNA-binding activity. After purification, histone H1 was the polypeptide with preferential DNA-binding activity. Histone H1 has a high-affinity binding site in the 5' end of the rat albumin gene within 440 5'-flanking base pairs and the first two exons of the gene.     

Histones H1(0) and H5 share common epitopes with RNA polymerase II

We report here the cross-reaction of RNA polymerase II antiserum with histones H1(0) and H5 and the complementary cross-reactions of antisera to the globular domain of histone H1(0) (GH1(0)) and histone H5 (GH5) with RNA polymerase II. Immunoblotting of RNA polymerase II antiserum with fragments of histone H1(0) localized the cross-reaction at the junction of the globular and C-terminal domains of histone H1(0). The structural homology implied by these cross-reactions is interesting in light of reports that suggest H1(0) may play a role in differentiation and development.     

Antigenic structure of histone H1°

In order to study the antigenic structure of histone Hl° the purified protein from mouse liver was subjected to different chemical and enzymatic treatments (CNBr, acetic acid, trypsin, chymotrypsin). The resulting peptides were fractionated in SDS-containing or acid-urea polyacrylamide gels, transferred by electroblotting onto nitrocellulose paper and probed with specific rabbit anti-H1° antiserum. The C-terminal fragments 99–193 (obtained following acetic acid hydrolysis) and 107–193 (obtained by chymotrypsin digestion) also exhibited strong immunoreactivity. Fragment 1–30 (CNBr cleavage) contained antigenic determinants while the shorter fragments 1–22 and 1–28 (acetic acid hydrolysis) failed to show any detectable reactivity. It was concluded that, in contrast to histone H5 whose reactivity is mainly concentrated to the globular domain of the molecule, the antigenic determinants in histone H1° are more or less evenly distributed along the polypeptide chain with the possible exception of the short unstructured N-nose.     

H1 histone variants in Xenopus laevis

The H1 histones from erythrocytes, livers, intestines, testes, and embryos of Xenopus laevis have been examined electrophoretically. This species has been found to contain at least five electrophoretically resolvable lysine-rich histones in addition to the presumptive H5 histone of erythrocytes. Quantitative and qualitative distinctions between the H1 histones from each source were readily observed. Three H1 histones (H1A, H1B, and H1C) were found in both embryos and adult tissues, although in varying amounts. Two other H1 histones (H1D and H1E) were found only in adult tissues. Comparative SDS gel V8 protease cleavage maps of the lysine-rich histones from testes and erythrocytes have demonstrated that the “adult-specific” H1D and H1E are not artifacts of proteolysis and may be closely related to the presumptive H5 histone. Spermatogenic cells were found to be similar to embryonic cells in being deficient in H1D and H1E. These observations suggest that H1D and H1E are enriched in cell types with low rates of cell division similar to the mammalian H1° histone. The results presented here demonstrate a previously unrecognized degree of developmental and cell-specific variance in the H1 histones of Xenopus laevis.     

A rat histone H4 gene closely associated with the testis-specific H1t gene

A rat histone H4 gene closely associated with the testis-specific H1t gene was isolated by screening the Sargent-Bonner rat genomic library using cloned human histone genes as probes. Both the H4 gene and the H1t gene are located on a 7-kb EcoRI genomic DNA fragment. Although the deduced amino acid sequence of the rat H4 histone is identical to that of the sequence of human histone H4, the nucleotide sequence of the coding region differs significantly from the coding region of the human H4 gene. Moreover, the relative spacing between the 5'-consensus sequence elements is unique for an H4 gene. S1-nuclease protection analyses reveal that both the H4 and H1t mRNA species are present in a fraction of rat testis cells highly enriched in pachytene spermatocytes, while only the H4 mRNA species is present in a rat myeloma cell line (Y3-Ag1.2.3). During a 1-h hydroxyurea treatment of the Y3 cells, which produces a 99% inhibition of DNA synthesis, the level of this H4 mRNA drops by only 50%, indicating that the stability of this mRNA is only partially coupled with DNA synthesis.