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Background Protamines are small basic proteins that condense the DNA in mature spermatozoa. Typical protamines are of simple composition and very arginine-rich, usually in the range of 60-80%. Arginine residues are distributed in a number of stretches separated by neutral amino acids. We have used Fourier transform infrared spectroscopy (FTIR) to gain access for the first time to the secondary structure of protamines in sperm nuclei. This technique is particularly well suited to the study of DNA-bound protamine in whole nuclei since it is not affected by turbidity. Results We show that DNA -bound salmon (salmine) and squid protamines contain α-helix, β-turns and a proportion of other structures not stabilized by intramolecular hydrogen bonding. No β-sheet was observed. In salmine, the α-helix amounted to ~20%, while in squid protamine it reached ~40%. In contrast, the structure not stabilized by intermolecular hydrogen bonding was more abundant in salmine (~40%) than in squid protamine (~20%). Both protamines contained ~40% β-turns. The different helical potential of salmine and squid protamine was confirmed by structure predictions and CD in the presence of trifluoroethanol. Conclusion DNA-bound protamine in sperm nuclei contains large amounts of defined secondary structure stabilized by intramolecular hydrogen bonding. Both salmine and squid protamine contain similar amounts of β-turns, but differ in the proportions of α-helix and non-hydrogen bonded conformations. In spite of the large differences in the proportions of secondary structure motifs between salmon and squid protamines, they appear to be equally efficient in promoting tight hexagonal packing of the DNA molecules in sperm nuclei.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases of the central nervous system. The aggregation of the amyloid-β peptide, Aβ(1-42), is believed to play an important role in the pathogenesis of AD. Histone H1 is found in the cytoplasm of neurons in AD, and it has been shown to interact with aggregated amyloid-β peptides and with amyloid fibrils. We have used Thioflavin T (ThT) fluorescence enhancement, circular dichroism spectroscopy (CD), coprecipitation, and transmission electron microscopy (TEM) to study the interaction of histone H1 with Aβ(1-42). Both freshly prepared (monomeric) Aβ(1-42) and histone H1 solutions showed negative CD bands typical of the random coil. Mixing Aβ(1-42) and histone H1 led to the loss of the random coil, which was replaced mostly by β-structure. Therefore, both Aβ(1-42) and histone H1 behave as intrinsically disordered proteins with coupled binding and folding. Mutual structure induction demonstrates the interaction of Aβ(1-42) and histone H1. The interaction was confirmed by coprecipitation followed by SDS-PAGE. Mutual structure induction was also observed with the H1 terminal domains. Incubation of Aβ(1-42) for 1 week in the presence of histone H1 led to the formation of laminar aggregates and thick bundles, characterized by the parallel association of large numbers of fibrils. The aggregates were particularly large and ordered with the H1 subtype H1.2. Further aging of the complexes led to tight compaction of fibril bundles and to fiber growth. Stabilization of fibril-fibril interactions appeared to be determined by the C-terminal domain of histone H1. In summary, these observations indicate that histone H1 has at least two effects: it helps the folding of Aβ monomers and stabilizes the parallel association of fibrils.

Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation.

Chicken erythrocyte nuclei were digested with micrococcal nuclease and fractionated by centrifugation in low-salt buffer into soluble and insoluble fractions. Post-translational modifications of the purified linker histones of both fractions were analyzed by LC–ESI–MS/MS. All six histone H1 subtypes (H1.01, H1.02, H1.03, H1.10, H1.1L and H1.1R) and histone H5 were identified. Mass spectrometry analysis enabled the identification of a wide range of PTMs, including Nα-terminal acetylation, acetylation, formylation, phosphorylation and oxidation. A total of nine new modifications in chicken linker histones were mapped, most of them located in the N-terminal and globular domains. Relative quantification of the modified peptides showed that linker histone PTMs were differentially distributed among both chromatin fractions, suggesting their relevance in the regulation of chromatin structure. The analysis of our results combined with previously reported data for chicken and some mammalian species showed that most of the modified positions were conserved throughout evolution, highlighting their importance in specific linker histone functions and epigenetics. Biological significance Post-translational modifications of linker histones could have a role in the regulation of gene expression through the modulation of chromatin higher-order structure and chromatin remodeling. Finding new PTMs in linker histones is the first step to elucidate their role in the histone code. In this manuscript we report nine new post-translational modifications of the linker histones from chicken erythrocytes, one in H5 and eight in the H1 subtypes. Chromatin fractionated by centrifugation in low-salt buffer resulted in two fractions with different contents and compositions of linker histones and enriched in specific core histone PTMs. Of particular interest is the fact that linker histone PTMs were differentially distributed in both chromatin fractions, suggesting specific functions. Future studies are needed to establish the interplay between PTMs of linker and core histones in order to fully understand chromatin regulation. A protein sequence alignment summarizing the PTMs found to date in chicken, mouse, rat and humans showed that, while many of the modified positions were conserved between these species, the type of modification often varied depending on the species or the cellular type. This finding suggests an important role for the PTMs in the regulation of linker histone functions.

H1 linker histones are involved both in the maintenance of chromatin higher-order structure and in gene regulation. H1 binds to linker DNA regions on the surface of the nucleosome. In higher eukaryotes, H1 contains three distinct domains: a short N-terminal domain (NTD), a central globular domain, and a long C-terminal domain (CTD). Terminal domains determine subtype specificity and to a large extent the linker DNA binding and chromatin condensing properties of histone H1. This review is focused on the recent numerous studies that have provided insights in the role of H1 terminal domains in chromatin dynamics. The N- and C-terminal domains behave as intrinsically disordered proteins with coupled binding and folding. We examine the potential kinetic advantages of intrinsic disorder in the recognition of the specific H1 binding sites in chromatin. As typical intrinsically disordered regions, H1 terminal domains are post-translationally modified. Post-translational modifications in the NTD determine the interaction of histone H1 with other proteins involved in heterochromatin formation and transcriptional regulation, while phosphorylation by cyclin-dependent kinases modulates the secondary structure of the CTD and chromatin condensation. We review the arguments in favor of the involvement of H1 hyperphosphorylation in metaphase chromatin condensation and of partial phosphorylation in interphase chromatin relaxation. In addition, the interplay of histone H1 and other chromatin architectural proteins, such as proteins of the high-mobility group, protamines, and MeCP2, is associated with changes in chromatin structure.

Histone H1 is involved in chromatin structure and gene regulation. H1 also performs functions outside cell nuclei, which may depend on its properties as a lipid-binding protein. The H1 CTD behaves as an intrinsically disordered protein (IDP) with coupled binding and folding. Here, we used neutral detergents and anionic SDS to study the contribution of hydrophobic interactions to the folding of the CTD. In the presence of neutral detergents, the CTD folded with proportions of secondary structure motifs similar to those observed in the DNA complexes. These results identify a folding pathway for the CTD based on hydrophobic interactions, and independent of charge compensation. The CTD is phosphorylated to different extents by cyclin-dependent kinases. The general effect of phosphorylation in the presence of detergents was a decrease in the α-helix content and an increase in that of the β-structure. The greatest effect was observed in the fully phosphorylated CTD (three phosphate groups) in the presence of anionic SDS (7:1, detergent/CTD molar ratio); in these conditions, the CTD became an all-β protein, with 83% β-structure and no α-helix. The CTD in all-β conformation readily formed ribbon-like fibers. The entire H1 also formed fibers when fully phosphorylated in the CTD. Fibers were of the amyloid type, as judged by strong birefringence in the presence of Congo red and thioflavin fluorescence enhancement. Amyloid fiber formation was only observed in SDS, suggesting that it requires the joint effects of partial charge neutralization and hydrophobic interactions, together with the all-β potential provided by full phosphorylation.

Knowledge of the structural properties of linker histones is important to the understanding of their role in higher-order chromatin structure and gene regulation. Here we study the conformational properties of the peptide Ac-EKTPVKKKARKAAGGAKRKTSG-NH(2) (NE-1) by circular dichroism and (1)H-NMR. This peptide corresponds to the positively charged region of the N-terminal domain, adjacent to the globular domain, of mouse histone H1e (residues 15-36). This is the most abundant H1 subtype in many kinds of mammalian somatic cells. NE-1 is mainly unstructured in aqueous solution, but in the presence of the secondary-structure stabilizer trifluoroethanol (TFE) it acquires an alpha-helical structure. In 90% TFE solution the alpha-helical population is approximately 40%. In these conditions, NE-1 is structured in two alpha-helices that comprise almost all the peptide, namely, from Thr17 to Ala27 and from Gly29 to Thr34. Both helical regions are highly amphipathic, with the basic residues on one face of the helix and the apolar ones on the other. The two helical elements are separated by a Gly-Gly motif. Gly-Gly motifs at equivalent positions are found in many vertebrate H1 subtypes. Structure calculations show that the Gly-Gly motif behaves as a flexible linker between the helical regions. The wide range of relative orientations of the helical axes allowed by the Gly-Gly motif may facilitate the tracking of the phosphate backbone by the helical elements or the simultaneous binding of two nonconsecutive DNA segments in chromatin.

H1 linker histones are involved both in the maintenance of higher-order chromatin structure and in gene regulation. Histone H1 exists in multiple isoforms, is evolutionarily variable and undergoes a large variety of post-translational modifications. We review recent progress in the understanding of the folding and structure of histone H1 domains with an emphasis on the interactions with DNA. The importance of intrinsic disorder and hydrophobic interactions in the folding and function of the carboxy-terminal domain (CTD) is discussed. The induction of a molten globule-state in the CTD by macromolecular crowding is also considered. The effects of phosphorylation by cyclin-dependent kinases on the structure of the CTD, as well as on chromatin condensation and oligomerization, are described. We also address the extranuclear functions of histone H1, including the interaction with the β-amyloid peptide.

Histone H1 preferentially binds and aggregates scaffold-associated regions (SARs) via the numerous homopolymeric oligo(dA).oligo(dT) tracts present within these sequences. Here we show that the mammalian somatic subtypes H1a,b,c,d,e and H1 degrees and the male germline-specific subtype H1t, all preferentially bind to the Drosophila histone SAR. Experiments with the isolated domains show that whilst the C-terminal domain maintains strong and preferential binding, the N-terminal and globular domains show weak binding and poor specificity for the SAR. The preferential binding of SAR by the H1 molecule thus appears to be determined by its highly basic C-terminal domain. Salmine, a typical fish protamine, which could have its evolutionary origin in histone H1, also shows preferential binding to the SAR. The interaction of distamycin, a minor groove binder with high affinity for homopolymeric oligo(dA).oligo(dT) tracts, abolishes preferential binding of the C-terminal domain of histone H1 and protamine to the SAR, suggesting the involvement of the DNA minor groove in the interaction.

H1 subtypes are involved in chromatin higher-order structure and gene regulation. H1 has a characteristic three-domain structure. We studied the length variation of the available H1 subtypes and showed that the length of the N-terminal and C-terminal domains was more variable than that of the central domain. The N-terminal and C-terminal domains were of low sequence complexity both at the nucleotide and at the amino acid level, whereas the globular domain was of high complexity. In most subtypes, low complexity was due only to cryptic simplicity, which reflects the clustering of a number of short and often imperfect sequence motifs. However, a subset of subtypes from eubacteria, plants, and invertebrates contained tandem repeats of short amino acid motifs (four to 12 residues), which could amount to a large proportion of the terminal domains. In addition, some other subtypes, such as those of Drosophila and mammalian H1t, were only marginally simple. The coexistence of these three kinds of subtypes suggests that the terminal domains could have originated in the amplification of short sequence motifs, which would then have evolved by point mutation and further slippage.

Histone H1 subtypes are involved in chromatin higher-order structure. The representation of the subtypes varies greatly depending on the cellular and developmental context. We have estimated the rates of nucleotide substitution for several H1 subtypes, including mammalian and amphibian H1 degree, avian H5, and mammalian H1a-e and H1t, with the aim of finding evidence for their functional differentiation. The rates of nonsynonymous substitution differ among the subtypes by almost one order of magnitude. Such a wide variation in the degree of tolerance of amino acid substitutions is consistent with the functional differentiation of the subtypes. H1 has a characteristic three-domain structure. The rate ratios among the domains of the molecule are not systematically maintained in the different subtypes. This suggests the assumption of differentiated functions by the individual domains in chromatin structure. We have estimated the average time of divergence of H1a-e and H1t paralogs as 406 +/- 80 Myr. The lack of evidence for concerted evolution of H1a-e and H1t since long before the mammalian radiation further supports the functional differentiation of the subtypes.

We have examined the central nervous system (CNS) of developing and adult transgenic mice carrying sequences upstream of the histone H1 zero gene fused to the E. coli beta-galactosidase gene (lac Z). The transgene is induced in a subset of the neuronal population during postnatal development, coinciding with neuronal terminal differentiation. At postnatal day 9, the earliest time at which the transgene product can be detected, positive neurons are observed in the granular layer of the cerebellar cortex and in the pyramidal fields of the hippocampus. The transgene is then induced in other areas of the CNS, such as the neocortex, thalamus, hypothalamus, olfactory bulb, globus pallidus superior and inferior colliculus, substantia nigra, pontine nuclei and brain stem. Induction is unrelated with determination and quiescence, which are essentially prenatal. The overlapping of the temporal and regional patterns of transgene activity with those of the endogenous protein shows that the accumulation of H1 zero in differentiating neurons is at least in part under transcriptional control. In the light of these results, the H1 zero gene appears as the only mammalian histone gene that specifically responds to terminal differentiation. However, not all terminally differentiated neurons express H1 zero at detectable levels. For instance, Purkinje cells are negative. In neurons, terminal differentiation appears thus as a necessary, but not a sufficient condition for increased H1 zero expression.

We have studied the interaction of the isolated C-terminal domain of histone H1 with linear DNA using precipitation curves and electron microscopy. The C-terminal domain shows a salt-dependent transition towards cooperative binding, which reaches completion at 60 mM NaCl. At this salt concentration, the C-terminal domain binds to some of the DNA molecules, leaving the rest free. A binding site of 22 base-pairs can be calculated from the stoichiometry of the precipitated fractions. The C-terminal domain condenses the DNA in toroidal particles. The average inner radius of the particles is of the order of 195 A. Consideration of the value of the inner radius of the toroids in the light of counterion condensation theory suggests that in these complexes the isolated C-terminal domain is capable of nearly full electrostatic neutralization of the DNA phosphate charge.

Background Histone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1° for long chromatin fragments (30–35 nucleosomes) in physiological salt (0.14 M NaCl) at constant stoichiometry. Results The H1 complement of native chromatin was perturbed by adding an additional amount of one of the subtypes. A certain amount of SAR (scaffold-associated region) DNA was present in the mixture to avoid precipitation of chromatin by excess H1. SAR DNA also provided a set of reference relative affinities, which were needed to estimate the relative affinities of the subtypes for chromatin from the distribution of the subtypes between the SAR and the chromatin. The amounts of chromatin, SAR and additional H1 were adjusted so as to keep the stoichiometry of perturbed chromatin similar to that of native chromatin. H1 molecules freely exchanged between the chromatin and SAR binding sites. In conditions of free exchange, H1a was the subtype of lowest affinity, H1b and H1c had intermediate affinities and H1d, H1e and H1° the highest affinities. Subtype affinities for chromatin differed by up to 19-fold. The relative affinities of the subtypes for chromatin were equivalent to those estimated for a SAR DNA fragment and a pUC19 fragment of similar length. Avian H5 had an affinity ~12-fold higher than H1e for both DNA and chromatin. Conclusion H1 subtypes freely exchange in vitro between chromatin binding sites in physiological salt (0.14 M NaCl). The large differences in relative affinity of the H1 subtypes for chromatin suggest that differential affinity could be functionally relevant and thus contribute to the functional differentiation of the subtypes. The conservation of the relative affinities for SAR and non-SAR DNA, in spite of a strong preference for SAR sequences, indicates that differential affinity alone cannot be responsible for the heterogeneous distribution of some subtypes in cell nuclei.

We have studied the secondary structure of the carboxyl-terminal domains of linker histone H1 subtypes H1(0) (C-H1(0)) and H1t (C-H1t), free in solution and bound to DNA, by IR spectroscopy. The carboxyl-terminal domain has little structure in aqueous solution but becomes extensively folded upon interaction with DNA. The secondary structure elements present in the bound carboxyl-terminal domain include the alpha-helix, beta-structure, turns, and open loops. The structure of the bound domain shows a significant dependence on salt concentration. In low salt (10 mm NaCl), there is a residual amount of random coil, 7% in C-H1(0) and 12% in C-H1t. In physiological salt concentrations (140 mm NaCl), the carboxyl termini become fully structured. Under these conditions, C-H1(0) contained 24% alpha-helix, 25% beta-structure, 17% open loops, and 33% turns. The latter component could include a substantial proportion of the 3(10) helix. Despite their low sequence identity (approximately 30%), the representation of the different structural motifs in C-H1t was similar to that in C-H1(0). Examination of the changes in the amide I components in the 20-80 degrees C temperature interval showed that the secondary structure of the DNA-bound C-H1t is for the most part extremely stable. The H1 carboxyl-terminal domain appears to belong to the so-called disordered proteins, undergoing coupled binding and folding.

The 5' flanking region and the 5'untranslated region (5' UTR) of the rat N-methyl-D-aspartate receptor subunit 2A were cloned and sequenced using polymerase chain reaction-mediated chromosome walking. The complementary DNA (cDNA) was obtained by rapid amplification of 5'cDNA ends (5'RACE). The comparison of the cDNA and the genomic sequences showed that the 5'UTR contained two introns and three exons, the third exon overlapping the beginning of the coding region. Transcriptional initiation sites were identified by 5'RACE and RNA-protection assays, using total rat brain RNA. The main start sites were found at -591, -577, -560 and -541 nucleotides 5' of the AUG. The promoter region lacked TATA and CAAT positioning elements. A CpG island of about 700 bp overlapped the 5' flanking sequences and the 5' UTR. The CpG island was inside a wider GC-rich region (66% GC) spanning the entire 5' UTR. Comparison of the rat sequences with the human sequences from the Human Genome Data Bank revealed that the 5' UTR exon 2 was extremely conserved with 95.8% sequence identity, as were the initial 640 bp of 5' flanking sequences, with 78% sequence identity. Beyond this point, sequence identity dropped abruptly to 44%. Putative recognition sequences for the transcription factors S8, Sp1, GATA, AML1 and NF-kappaB were identified in both the rat and human promoter sequences.

The H1° gene has a long 3′ untranslated region (3′UTR) of 1,125 nucleotides in the rat and 1,310 in humans. Analysis of the sequences shows that they have features of simple DNA that suggest involvement of replication slippage in their evolution. These features include the length imbalance between the rat and human sequences; the abundance of single-base repeats, two-base runs and other simple motifs clustered along the sequence; and the presence of single-base repeat length polymorphisms in the rat and mouse sequences. Pairwise comparisons show numerous short insertions/deletions, often flanked by direct repeats. In addition, a proportion of short insertions/deletions results from length differences in conserved single-base repeats. Quantification of the sequence simplicity shows that simple sequences have been more actively incorporated in the human lineage than in the rodent lineage. The combination of insertions/deletions and nucleotide substitutions along the sequence gives rise to three main regions of homology: a highly variable central region flanked by more conserved regions nearest the coding region and the polyA addition site.

The binding of the intermediate proteins phi 1 and phi 3 from the mussel Mytilus edulis to DNA was studied in comparison with the typical protamine from the squid Loligo vulgaris using precipitation curves, thermal denaturation and X-ray diffraction techniques. The properties of protein phi 1 appear to be very close to those of typical protamines while the properties of protein phi 3 are notably different. The method of reconstitution influences the structural properties of the complexes. This effect is most pronounced in the case of protein phi 3. The structural heterogeneity of the protein component in the complexes is discussed in the light of these observations.

Neuronal nuclei have a low H1 content. A stoichiometry of 0.47 molecule/nucleosome, on average, is calculated for rat brain cortical neurons by comparing its H1 content with that of liver nuclei. The H1 fraction of rat cerebral cortex neurons has been resolved into five subtypes, H1a--e, that have the same mobility as the unphosphorylated H1 forms of other rat tissues. The subtypes H1a--d decay exponentially during postnatal development and are substituted to different extents by H1e. The higher replacement rate is shown by H1a with an apparent half-lifetime of about 5 days. The corresponding values for H1b, H1c and H1d are 11, 21 and 15 days. Several conclusions can be drawn from the observation of postnatal changes in H1 subtype proportions. The low H1 content of neuronal nuclei does not imply the presence of notable peculiarities in subtype composition or in subtype substitution pattern. There is turnover of H1 in differentiating neurons once cell proliferation and DNA replication have ceased. The relative rates of synthesis and/or degradation of the subtypes differ in germinal cells and in neurons. Comparison with previous results on H1 degrees accumulation also shows that in cortical neurons the regulation of the subtypes H1a--e differs from that of H1 degrees.

Neutron scatter studies have been made on gently prepared chicken erythrocyte chromatin over a range of ionic strength. At low ionic strength the mass per unit length of the '10 nm nucleofilament corresponds to one nucleosome per 8--12 nm and a DNA packing ratio of between 6 and 9. From the contrast dependence of the cross-section radius of gyration of the nucleofilament the following parameters have been obtained; RgDNA' the cross-section radius of gyration (Rg) when DNA dominates the scatter; RgP, the cross-section Rg when protein dominates the scatter; Rc, the cross-section Rg at infinite contrast and alpha, the constant which describes the dependence of the cross-section Rg on contrast variation. From our understanding of the structure of the core particle, various arrangement of core particles in the nucleofilament have been tested. In models consistent with the above parameters the core particles are arranged edge-to-edge or with the faces of the core particles inclined to within 20 degrees to the axis of the nucleofilament. With increase of ionic strength the transition to the second-order chromatin structure has been followed. This gave the interesting result that above 20 microM NaCL or 0.4 mM MgCL2 the cross-section Rg increases abruptly to about 9 nm with a packing ratio of 0.2 nucleosome/mn and with further increase of ionic strength the Rg increases to 9.5 nm while the packing ratio increases threefold to 0.6 nucleosome/nm. This suggests a family of supercoils of nucleosomes which contract with increasing ionic strength. In its most contracted form the diameter of the hydrated supercoil has been found from the radial distribution function to be 34 nm. Models for the arrangements of core particles in the 34-nm supercoil are discussed.

Rat brain cortical neurons originate from germinal cells during a period of 6 days immediately before birth. Upon leaving the proliferative layer neurons become irreversibly quiescent. We have previously reported the presence of core histone nonallelic variants in terminally differentiated rat brain cortical neurons. Although the functional significance of core histone variants is unknown, several lines of evidence suggest that the processes of variant replacement could be involved in the structural and functional differentiation of chromatin. Here we describe the changes in core histone composition that occur during postnatal development. The changes in chromatin composition are already apparent at birth, suggesting that the change in synthesis patterns is related to the arrest of cell proliferation and neuron commitment. During postnatal development H2A.2, H2A.x, and H3.3 accumulate, whereas H2A.1, H3.1, and H3.2 decrease. H2A.z is the only variant that remains constant. The time courses of replacement and the observed variant proportions when the variant composition approaches the equilibrium suggest that all H2A variants are synthesized either in germinal cells or in neurons, whereas H3.1 and H3.2 seem to be synthesized only in germinal cells. The extent of the replacement of H3.1 and H3.2 by H3.3 shows that the exchange process affects most of the chromatin. The half-life times of H2A.1 and H3.2 were calculated from their respective exponential decays. Values of 65 days or less and 142 days were found for H2A.1 and H3.2, respectively. The preferential replacement of H2A.1 over H3.2 reinforces the view that the histone core does not degrade as a single unit.

The condensation of DNA by the C-terminal domain of histone H1 has been studied by circular dichroism in physiological salt concentration (0.14 M NaF). As the intact H1 molecule, its C-terminal domain induces the so-called psi state of DNA that is characterized by a nonconservative circular dichroism spectrum which is currently attributed to ordered aggregation of the DNA molecules. On a molar basis, intact H1 and its C-terminal domain give spectra of similar intensity. Neither the globular domain of H1 nor an N-terminal fragment, that includes both the globular and N-terminal domains, has any effect on the conservative circular dichroism of DNA. From these results it is concluded that the condensation of DNA mediated by histone H1 is mainly due to its C-terminal domain. The effect of the salt concentration and the size of DNA molecules on the circular dichroism of the complexes are also examined.

Neutron scattering studies have been applied to chromatin core particles in solution, using the contrast variation technique. On the basis of the contrast dependance of the radius of gyration and the radial distribution function it is shown that the core particle consists of a core containing most of the histone around which is wound the DNA helix,following a path with a mean radius of 4.5 nm,in association with a small proportion of the histones. Separation of the shape from the internal structure, followed by model calculations shows that the overall shape of the particle is that of a flat cylinder with dimensions ca. 11x11x6 nm. Further details of the precise folding of the DNA cannot be deduced from the data, but detailed model calculations support concurrent results from crystallographic studies(25).Images

In the present report it is shown that histone bands in urea-acetic acid or Triton-urea-acetic acid-polyacrylamide gels can be stained with the fluorescent dye 1-anilinonaphthalene 8-sulfonate and visualized by transillumination of the gel with an uv-light source. The 1-anilinonaphthalene 8-sulfonate staining method described here for urea and Triton-urea gels is rapid (it can be completed in 90 min) and allows the detection of less than 1 μg of histone per band.

The complexes formed by DNA and different types of protamines are studied by X-ray diffraction. The protamines used have considerable differences in their molecular weight and in the content of various amino acids. However, in all cases it is found that protamines stabilize DNA in the B form. The molecules pack in the hexagonal system with a single DNA molecule per unit cell. It is concluded that bridges of protamine between DNA molecules are an essential feature in the formation of nucleoprotamine complexes. The salts of DNA with arginine and L-arginyl-L-arginine are also studied. Their X-ray diffraction patterns show features intermediate between those of nucleoprotamine and the alkaline metal salts of DNA.

The pattern of non-allelic variants of core histones was investigated in terminally differentiated rat cerebral cortex neurons. At 30 days two major H2A variants are present, H2A.1 and .2, together with two minor components, .x and .z. H2B has two variants, H2B.1 and .2, and H3 presents three variants, H3.1, .2 and .3. The ubiquitinated adducts of all H2A and H2B variants can be recognised on two-dimensional electrophoresis as forming a pattern similar to that of the unmodified species. uH2A amounts to 12–14% of total H2A. A11 H2A variants appear to be equally modified. uH2B amounts to 1–2% of total H2B.

Rat cerebral cortex neurons contain the five histone HI subtypes Hla-e and the subtype HIO present in other mammalian somatic tissues. The four subtypes Hla-d decay exponentially during postnatal development and are partially or totally replaced by Hle that becomes the major Bl subtype in adults. Hl0 accumulates in a period restricted to neuronal terminal differentiation. Here we study the synthesis of the Bl subtypes in cortical neurons and their neuroblasts by in vivo labeling with [14C]lysine. The subtype synthesis pattern of neuroblasts has been determined by labeling gravid rats during the period of proliferation of cortical neurons and synthesis in neurons has been studied by postnatal labeling. The subtype Hla is synthesized in neuroblasts but not in neurons and is therefore rapidly removed from neuronal chromatin. The synthesis of Hlb and Hld is much lower in neurons than in neuroblasts so that these subtypes are replaced to a large extent during postnatal development. Ole is synthesized at levels much higher than the other subtypes both in neurons and neuroblasts, but its very high turnover, about one order of magnitude faster than that of Hle in neurons, favors its partial replacement during postnatal development. Comparison of the synthesis rates of Hl0 in newborn and 30-day-old rats shows that the accumulation of HIO in differentiating neurons is due to an increased level of synthesis.

Core histones can be modified by aceylation and this modification has been correlated with the modulation of chromatin condensation and histone deposition. We have now studied the levels of acetylation of the core histones in rat brain cortical neurons from the middle of the period of neuronal proliferation through postnatal development and aging. The results show that the level of acetylation of H4 decreases with age. The kinetics of H4 deacetylation show a perinatal fast phase followed by a much slower phase that spans the rest of the period examined. H4 deacetylation is accounted for by the decrease of the monoacetylated species, the proportions of the more highly acetylated species remaining essentially constant. By contrast to histone H4, the overall levels of acetylation and the proportions of the different acetylated species of H2A, H2B and H3 remain unchanged throughout the period examined. Furthermore, the variants belonging to a given histone class always show the same level of acetylation. The fact that in neurons the level of monoacetylated H4 decreases during development and aging, in sharp contrast with the constancy of the levels of all other acetylated histone species, raises the possibility that in interphase chromatin monoacetylated H4 may have a central role in the modulation of chromatin structure. The results also suggest that the slow decrease of the proportion of monoacetylated H4 may imply a gradual loss of chromatin structural plasticity and thus lead to aging.