Displacement of histones by sperm-specific proteins at different stages of spermatogenesis of squid

Changes in the composition of the chromatin basic proteins during spermatogenesis of the squid Illex argentinus were studied. The core histones of I. argentinus slightly differ from those of calf thymus in the subfractional composition of histones H2A and H2B. A similar amino acid composition is revealed in the histones H1 of the squid I. argentinus and calf thymus. Histone H1 of the squid has a lower molecular mass and a special subfractional composition as compared to those of calf thymus, grass carp and carp studied formerly [Kadura et al. (1983) Comp. Biochem. Physiol. 743, 343-350]. Neither the fractional nor subfractional composition of histones changes during spermatogenesis. The two new proteins were revealed in the chromatin composition of squid testes and spermatozoa illexines I1 and I2. Illexine I2 is composed of two subfractions I2-1 and I2-2. Illexine I2 shows a high content of arginine (75 mol/100 mol). Serine (10 mol/100 mol), histidine (3,2 mol/100 mol) and tyrosine residues (2,9 mol/100 mol) are also present. Illexine I1 shows the presence of arginine (45,6 mol/100 mol), lysine (7.6 mol/100 mol), serine (11.4 mol/100 mol), hystidine (2.3 mol/100 mol) and tyrosine residues (2.8 mol/100 mol). Molecular masses of illexines I2 and I1 are approximately 7 kDa and 9 kDa respectively. It is supposed that during spermatogenesis the histones are displaced in two-stage order: histones----I1----I2.     

Marked differences in the ability of distinct protamines to disassemble nucleosomal core particles in vitro

In accordance with the results of classical experiments performed in vitro with calf thymus chromatin and the fish protamine salmine, we have observed that this highly basic, small molecular weight protamine cannot cause major displacement of histones from nucleosomal core particles at concentrations several times higher than physiological (arginine/nucleotide ratios 1-8) and that hyperacetylation of histones facilitates nucleosome disassembly. However, the avian protamine galline, with molecular weight and number of arginine residues almost twice those of common fish protamines, is able to displace the nucleosomal core histones from DNA in vitro at concentrations (arginine/nucleotide ratios 0.6-1.2) within the physiological range (0.8). Our results suggest that the binding of the avian protamine galline to chromatin could be directly involved in the rapid disassembly of nucleosomes that takes place during the nucleohistone nucleoprotamine transition in chicken spermiogenesis.     

Factors affecting nucleosome disassembly by protamines in vitro. Histone hyperacetylation and chromatin structure, time dependence, and the size of the sperm nuclear proteins

Histone displaced in vitro from nuclei by protamine competition display a higher degree of hyperacetylation than the residual histones. In addition, hyperacetylated core particle pools are disassembled in vitro with a higher efficiency than control or nonacetylated core particles and when analyzed by electron microscopy display an elongated shape (length/width ratio = 1.52 +/- 0.19) instead of the round compact shape of control nucleosomes (length/width ratio = 1.06 +/- 0.06). In the absence of histone hyperacetylation, the fish protamines, salmine and iridine (32-33 residues), are relatively inefficient in disassembling nucleosomal core particles in vitro as compared to the large (65-70 residues), tyrosine-containing protamines from rooster (galline), squid, and cuttlefish which disassemble nucleosomes in a range of protamine concentrations close to physiological. The fact that an artificially cross-linked salmine dimer acquires the ability of the large protamines from rooster, squid, and cuttlefish to disassemble core particles in vitro and also binds more tightly to the DNA, suggests that the size of the sperm nuclear protamines is a critical factor in this process. Even when the core histones of spermatid chromatin are hyperacetylated in the trout testis, the replacement process by iridine or salmine is slow and time-dependent in vitro. However, since spermiogenesis in trout occurs over several weeks, the slow in vitro nucleosome disassembly process by salmine is sufficient to allow complete displacement, thus supporting the hypothesis that a protamine-mediated displacement of the histones from DNA in vivo may take place in the salmonid fishes by a mechanism similar to that in the rooster, squid, and cuttlefish.     

Principles of nucleosome recognition by chromatin factors and enzymes

The recent torrent of structures of chromatin complexes determined by cryoelectron microscopy provides an opportunity to discern general principles for how chromatin factors and enzymes interact with their nucleosome substrate. We find that many chromatin proteins use a strikingly similar arginine anchor and variant arginine interactions to bind to the nucleosome acidic patch. We also observe that many chromatin proteins target the H3 and H2B histone fold α1-loop1 elbows and the H2B C-terminal helix on the nucleosomal histone face. These interactions with the histones can be complemented with interactions with and distortions of nucleosomal DNA.     

Structure of illexine I2 and its complexes with DNA

Conformational peculiarities of illexine I2 both in the solution and in the complexes with DNA were studied by circular dichroism, UV-spectroscopy and spectrophotometric melting. IIlexine I2 is shown to have an extended left-handed helical conformation of poly-L-proline II type, that are stable in a wide range of experimental conditions. Upon interaction of illexine I2 with DNA, the parameters of conformation are somewhat distorted but the main peculiarities remain. The DNA double helix changes from B- to the divection of C-form at its interaction with illexine I2. The interaction of illexine I2 with DNA at low ionic strength is non-cooperative and is characterized by some specificity to A--T sequences of DNA. Illexine I2 strongly affects the DNA stability by increasing the melting temperature of DNA.     

Quantitative analysis of HP1alpha binding to nucleosomal arrays

Elucidating how the metazoan genome is organised into distinct functional domains is fundamental to understanding all aspects of normal cellular growth and development. The "histone code" hypothesis predicts that post-translational modifications of specific histone residues regulate genomic function by selectively recruiting nuclear factors that modify chromatin structure. A paradigm supporting this hypothesis is the preferential binding of the silencing protein heterochromatin protein 1 (HP1) to histone H3 trimethylated at K9. However, a caveat to several in vitro studies is that they employed histone N-terminal tail peptides to determine dissociation constants, thus ignoring any potential role of DNA and/or the underlying chromatin structure in the recruitment of HP1. Using a well-defined in vitro chromatin assembly system (employing a 12-208 DNA template), we describe here, the use of a fluorescence spectroscopic method that enabled us to measure and quantify the relative binding affinities of HP1alpha to unmodified and variant nucleosomal arrays. Using this approach, we previously demonstrated that mouse HP1alpha (i) binds with high affinity to naked DNA, (ii) has an intrinsic affinity for highly folded chromatin, (iii) has a 2-fold higher affinity for nucleosomal arrays when H2A is replaced with H2A.Z, and (iv) binds to DNA or chromatin in a non-cooperative manner.     

Sperm histones and chromatin structure of the "primitive" sea urchin Eucidaris tribuloides

The "primitive" sea urchin Eucidaris tribuloides resembles the advanced sea urchins (euechinoids) in many respects, yet some features of its biochemistry and morphogenesis are more similar to other echinoderms such as starfish or sea cucumbers. Two unique characteristics of the sperm chromatin of all known euechinoids are an extremely long average nucleosomal repeat length and the presence of two male germ-line-specific histone variants, Sp H1 and Sp H2B. Histone composition and nucleosomal repeat length of the sperm chromatin of Eucidaris were compared to those of several euechinoids and a starfish. Eucidaris sperm chromatin contained large H1 and H2B histone variants typical of euechinoids. The H1 was about nine amino acids smaller than Sp H1 of the advanced urchin Strongylocentrotus purpuratus. Its Sp H2B molecules were the same size as in the euechinoids. Peptide maps showed that N-terminal regions of Sp H1 and Sp H2B contained repeating basic amino acid motifs characteristic of euechinoids. The smaller size of Eucidaris H1 is accounted for by a smaller C-terminal region. The repeat length of Eucidaris sperm chromatin was slightly shorter than that of two euechinoids, but significantly larger than starfish, which lacks a large H2B. The Sp H2B gene of Eucidaris was expressed during spermatogenesis in the same cell types as for S. purpuratus. Thus Sp histone subtype expression and chromatin structure in this distantly related echinoid closely resemble the euechinoids. The presence of an Sp H2B and a very long repeat length appear to be characteristic of the echinoids only.     

Features of the chromatin structure of erythrocytes depending on the properties of lysine-rich histones

A comparative analysis of chromatin from erythrocytes of frog, trout and hen has been performed in correlation with properties of the nucleosomal linker histones of H1 family. In the nucleosomes from frog erythrocytes the linker histone is represented by H1(0)-like variant with amino acid sequence highly homologous to that of the hen histone H5, however the arginine content in the proteins differs (3 mol% in the frog erythrocyte H1 and 12 mol% in the hen erythrocyte H5). On the other hand histone H5 from trout being significantly different in the primary structure from the hen histone H5 is at the same time rich in arginine (9 mol%). The nucleosomal repeat length, estimated by using agarose gel electrophoresis is 201, 213 and 213 b.p. in erythrocyte chromatin from frog, trout and hen, correspondingly. Chromatin packing density in fixed nuclei from erythrocytes of frog, trout and hen as determined using cytophotometric measurements is 0.144, 0.444 and 530 pg/mu 3, correspondingly. The data support the previously made suggestion that the increase in arginine content in nucleosomal linker proteins is connected with the increase of chromatin compaction in the nuclei and elongation of the linker in the nucleosome.     

Primary structure of the basic nuclear protein from spermatozoa of the mollusk Illex argentinus and its comparison with the structure of sperm proteins in other animals

The spermatic protein of chromatin I2 of squid Illex argentinus was separated by HPLC into two components I2-1 and I2-2. Amino acid sequences of the major portion of protein I2-1 (52 residues) and the N-terminal sequence of protein I2-2 (21 residues) were determined. Arginines in protein I2-1 are arranged in clusters typical of protamines; the first cluster is in the N-terminus, the longest heterogeneous basic cluster is in the central part of the protein chain, the C-terminal part of the molecule contains two clusters of three hydroxyamino acids each. The N-terminal sequences of illexins I2-1 and I2-2 (1-14 residues) are highly homologous. Homologous regions were found in illexin I2-1, tunnin of tuna fish and avian gallin thus defining the notion of proteins of an intermediate type from mollusc spermatozoa chromatin exemplified by the squid protamine-like protein.     

Properties of mouse spleen residual condensed chromatin associated with the nuclear matrix

Properties of condensed residual chromatin of mouse spleen, a component of residual nuclear structures, were studied. Extraction of the structures with buffers of different NaCl concentrations showed that the condensed chromatin consists of condensed nucleosomal chains. On increasing the ionic strength the complexes gradually fell apart into separate nucleosomal chains. DNA of condensed chromatin was accessible to staphylococcal nuclease and DNAase I, but digestion of this DNA was not accompanied by solubilization of the residual chromatin. Besides the essentially decreased total content of nonhistone chromosomal proteins the condensed chromatin practically did not contain HMG proteins. The nucleosome repeat length of this chromatin was shorter than that of chromatin solubilized by staphylococcal nuclease.     

Germ cell nucleosomes contain remodeled core protein complex

Electrophoretic analysis of the nucleosomal histones from MN1 and MN2 subpopulations of the seminiferous tubules in gels containing either 6.25 or 2.5 M urea revealed the presence of testis specific histone H2S, H1 and protein ‘A’ in addition to the somatic histones in the core protein complex. Size analysis indicated the presence of a 150–160 bp DNA segment in the MNI subpopulation, whereas, an approx 180 bp DNA fragment was present in the MN2 subpopulation of both liver and tubule nucleosomes. These data suggest an extensive remodeling of the nucleosomal core protein complex during mammalian spermatogenesis.     

Differential association of linker histones H1 and H5 with telomeric nucleosomes in chicken erythrocytes.

Rat liver telomeric DNA is organised into nucleosomes characterised by a shorter and more homogeneous average nucleosomal repeat than bulk chromatin as shown by Makarov et al. (1). The latter authors were unable to detect the association of any linker histone with the telomeric DNA. We have confirmed these observations but show that in sharp contrast chicken erythrocyte telomeric DNA is organised into nucleosomes whose spacing length and heterogeneity are indistinguishable from those of bulk chromatin. We further show that chicken erythrocyte telomeric chromatin contains chromatosomes which are preferentially associated with histone H1 relative to histone H5. This contrasts with bulk chromatin where histone H5 is the more abundant species. This observation strongly suggests that telomeric DNA condensed into nucleosome core particles has a higher affinity for H1 than H5. We discuss the origin of the discrimination of the lysine rich histones in terms of DNA sequence preferences, telomere nucleosome preferences and particular constraints of the higher order chromatin structure of telomeres.     

Nucleosome DNA repeat length and histone complement in a fungus exhibiting condensed chromatin

Fungal chromatins are reported to exhibit unusually short nucleosomal DNA repeat lengths. To test whether this is a phylogenetic feature of fungi or rather is correlated with an apparent absence of condensed chromatin in the organisms studied, we have examined the chromatin organization and the complement of basic nuclear proteins in the fungus Entomophthora, an organism which exhibits marked chromatin condensation. Micrococcal nuclease digestion of Entomophthora chromatin revealed a nucleosomal DNA repeat length of 197 +/- 1.2 base pairs (bp). This repeat length is 20-40 bp longer than that reported for any fungus. Entomophthora nucleosomes exhibited an HI-like protein which was much less basic than the HI histones reported for higher eukaryotes but which was similar in basicity to the HI histone reported for the fungus Neurospora. However, the nucleosomal DNA repeat length of Neurospora chromatin is reported to be unusually short, whereas that of Entomophthora was found to be typical of the repeat lengths observed for chromatins of higher eukaryotes. Thus, repeat length, at least in fungi, would not appear to be directly determined by the basicity of the fungal cognate of histone HI.     

BAPTA blocks DNA fragmentation and chromatin condensation downstream of caspase-3 and DFF activation in HT-induced apoptosis in HL-60 cells

DFF ((DNA Fragmentation Factor) is a heterodimer composed of 40 kDa (DFF40, CAD) and 45 kDa (DFF45, ICAD) subunits. During apoptosis, activated caspase-3 cleaves DFF45 and activates DFF40, a DNase that targets nucleosomal linker region and cleaves chromatin DNA into nucleosomal fragments. We have previously reported that HT induced apoptosis in HL-60 cells, and intracellular Ca²⁺ chelator BAPTA blocked apoptosis-associated DNA fragmentation induced by HT. We report here that HT also induced activation of caspase-3 and cleavage of DFF45. BAPTA prevented neither the caspase-3 activation nor the cleavage of DFF45. Mitochondrial membrane potential was disrupted in BAPTA-AM treated cells. However, BAPTA did prevent DNA fragmentation and chromatin condensation in HT-treated cells. These data suggest a novel role for intracellular calcium in regulating apoptotic nuclease that causes DNA fragmentation and chromatin condensation.     

Structural repeat units of Chinese hamster ovary chromatin. Evidence for variations in repeat unit DNA size in higher eukaryotes.

DNA lengths in the structural repeat units of Chinese hamster ovary (CHO) and chicken erythrocyte chromatin were compared by analyzing the sizes of DNA fragments produced after treatment of nuclei with staphylococcal nuclease. The repeat length of CHO chromatin (173 +- 4 BP) is about 20 base pairs (BP) smaller than that of chicken erythrocyte chromatin (194 +- 8 BP). Repeat lengths of rat liver and calf thymus chromatin were found to be about 10 BP shorter than that of chicken erythrocyte chromatin. Thus significant variations occur in repeat units of chromatin of higher eukaryotes. These variations occur in the lengths of "spacer" (or "internucleosomal") DNA segments, not in "core particle" (or "nucleosomal") DNA lengths. The concept of spacer regions and the possible influence of H1 histones is discussed.