RESUMEN

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.

Asunto(s)

RESUMEN

It is important to establish the structural properties of linker histones to understand the role they play in chromatin higher order structure and gene regulation. Here, we use CD, NMR, and IR spectroscopy to study the conformation of the amino-terminal domain of histone H1 degrees, free in solution and bound to the DNA. The NH(2)-terminal domain has little structure in aqueous solution, but it acquires a substantial amount of alpha-helical structure in the presence of trifluoroethanol (TFE). As in other H1 subtypes, the basic residues of the NH(2)-terminal domain of histone H1 degrees are clustered in its COOH-terminal half. According to the NMR results, the helical region comprises the basic cluster (Lys(11)-Lys(20)) and extends until Asp(23). The fractional helicity of this region in 90% TFE is about 50%. His(24) together with Pro(25) constitute the joint between the NH(2)-terminal helix and helix I of the globular domain. Infrared spectroscopy shows that interaction with the DNA induces an amount of alpha-helical structure equivalent to that observed in TFE. As coulombic interactions are involved in complex formation, it is highly likely in the complexes with DNA that the minimal region with alpha-helical structure is that containing the basic cluster. In chromatin, the high positive charge density of the inducible NH(2)-terminal helical element may contribute to the binding stability of the globular domain.

Asunto(s)

RESUMEN

We have studied the conformation of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), free in solution and bound to the DNA, by Fourier-transform infrared spectroscopy. The peptide belongs to the COOH-terminal domain of histone H1(0) (residues 99-121) and is adjacent to the central globular domain of the protein. In aqueous (D(2)O) solution the amide I' is dominated by component bands at 1643 cm(-1) and 1662 cm(-1), which have been assigned to random coil conformations and turns, respectively. In accordance with previous NMR results, the latter component has been interpreted as arising in turn-like conformations in rapid equilibrium with unfolded states. The peptide becomes fully structured either in 90% trifluoroethanol (TFE) solution or upon interaction with the DNA. In these conditions, the contributions of turn (1662 cm(-1)) and random coil components virtually disappear. In TFE, the spectrum is dominated by the alpha-helical component (1654 cm(-1)). The band at 1662 cm(-1) shifts to 1670 cm(-1), and has been assigned to the COOH-terminal TPKK motif in a more stable turn conformation. A band at 1637 cm(-1), also present in TFE, has been assigned to 3(10) helical structure. The amide I' band of the complexes with the DNA retains the components that were attributed to 3(10) helix and the TPKK turn. In the complexes with the DNA, the alpha-helical component observed in TFE splits into two components at 1657 cm(-1) and 1647 cm(-1). Both components are inside the spectral region of alpha-helical structures. Our results support the presence of inducible helical and turn elements, both sharing the character of DNA-binding motifs.

Asunto(s)

RESUMEN

The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone H1(o) (residues 99-121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i,i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C(alpha) protons, span from Pro100 to Val116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the alpha-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) beta-turn conformation, a sigma-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

The H10 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.

Asunto(s)

RESUMEN

We have determined the complete coding sequence of the histone-encoding H1(0) gene from rat PC12 cells. Southern and Northern analyses suggest that rat H1(0) is encoded by a single-copy gene which generates an mRNA of about 2.2 kb. Comparison of the rat, mouse and human amino-acid sequences shows that the C-terminal domain of the protein is much more variable than the N-terminal and central domains. Rates of non-synonymous and synonymous nucleotide substitution have been calculated. The rate of non-synonymous substitution is about 2.5-times higher in the rodent lineage than in the human lineage.

Asunto(s)

RESUMEN

The core histone classes H2A, H2B, H3, and H4 are the main group of proteins responsible for the folding of DNA in nucleosomes. Each of the core histone classes except H4 is composed of nonallelic variants. The core histone variant composition changes during postnatal development in rat cerebral cortex neurons; H2A.1, H2B.1, H3.1 and H3.2 decay exponentially, whereas H2A.2, H2A.x, H2B.2, and H3.3 accumulate. H2A.z is the only variant that remains constant. We have studied the synthesis of core histone variants in cortical neurons and their neuroblasts by in vivo labeling with [14C]lysine. The variant synthesis pattern of neuroblasts has been determined by labeling gravid rats during the period of proliferation of the brain cortical neurons of the fetuses, and synthesis in neurons has been studied by postnatal labeling. The incorporation of H2A.1 is about twice that of H2A.2, both in neurons and neuroblasts. Despite its higher synthesis rate, the proportion of H2A.1 decreases during postnatal development indicating that the turnover of H2A.1 is faster than that of H2A.2. Differential turnover and a change in synthesis rate are both involved in determining the relative concentrations of H2B.1 and H2B.2 in neurons. H3.1 and H3.2 are synthesized in neuroblasts, but not in neurons, and are thus replaced by H3.3 in neuronal chromatin. The fact that the synthesis pattern of immature neurons from newborns does not differ from that of mature neurons indicates that the changes in the synthesis pattern of core histones occur at the arrest of cell proliferation and are unrelated to the state of differentiation of the cells.

Asunto(s)

RESUMEN

This study has analysed by immunocytochemistry the expression pattern of histone H1 zero after the osmotically induced activation of transcription in supraoptic nucleus neurons of the rat. In control rats, histone H1 zero was constitutively expressed in neuronal and glial cell nuclei of supraoptic nucleus. After chronic neuronal stimulation by intermittent salt-loading, the majority of neuronal cell nuclei exhibited a marked reduction of immunostaining, which was confirmed by densitometric analysis of immunoreactivity. This effect was reversible, since optical density values returned to control levels when the stimulation of supraoptic neurons was suppressed by rehydration. Ultrastructural immunocytochemistry of histone H1 zero showed that immunogold particles specifically decorated chromatin fibers, with the highest accumulation of particles being on the condensed inactive chromatin. These results indicate that transcriptional activation in supraoptic neurons is accompanied by a depletion of the chromatin-associated histone H1 zero, and also suggest that this transcription-dependent expression of histone H1 zero may be involved in regulating chromatin condensation and gene expression in mature neurons that constitutively express this protein.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

The cellular distribution of histone H1(0) has been examined immunohistochemically in the rat brain. H1(0) accumulates in neurons and glial cells during postnatal development. In neurons, immunoreactivity increases progressively from about postnatal day 10, and reaches a distribution pattern similar to that of adult rats by postnatal day 20. Immunoreactivity in glial cells shows a prominent increase from postnatal day 20 to adult age. The accumulation of H1(0) during postnatal development appears to be correlated with terminal differentiation and maturation. Although immunoreactive neurons are widely distributed in all areas of the central nervous system, many neurons do not express immunoreactivity. For instance in the cerebellum, Purkinje neurons are negative. In females, the number of immunoreactive neurons in the arcuate area of the hypothalamus increases during postnatal development. In contrast, the percentage of immunoreactive neurons in males is low at all ages studied. The expression of H1(0) in the ventromedial part of the arcuate is reversibly and negatively regulated during the estrous cycle by the level of plasma estradiol. Ovariectomy increases the number of immunoreactive neurons while the restoration of the physiological levels of estradiol results in the opposite effect. Early postnatal androgenization of females suppresses the increment in the number of immunoreactive neurons in both the dorsolateral and the ventromedial parts of the arcuate during postnatal development, thus leading to permanently decreased levels of H1(0) immunoreactivity in postpuberal females.

Asunto(s)

RESUMEN

Rat cerebral cortex neurons contain the five histone H1 subtypes H1a-e and the subtype H1 zero present in other mammalian somatic tissues. The four subtypes H1a-d decay exponentially during postnatal development and are partially or totally replaced by H1e that becomes the major H1 subtype in adults. H1 zero accumulates in a period restricted to neuronal terminal differentiation. Here we study the synthesis of the H1 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 H1a is synthesized in neuroblasts but not in neurons and is therefore rapidly removed from neuronal chromatin. The synthesis of H1b and H1d is much lower in neurons than in neuroblasts so that these subtypes are replaced to a large extent during postnatal development. H1c 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 H1e in neurons, favors its partial replacement during postnatal development. Comparison of the synthesis rates of H1 zero in newborn and 30-day-old rats shows that the accumulation of H1 zero in differentiating neurons is due to an increased level of synthesis.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

In this paper we have studied the kinetics of psi-DNA structure formation induced by H1 and H1 peptides containing the C-terminal domain, namely the CTB peptide, obtained by thrombin digestion, and the CNBS peptide, derived from N-bromosuccinimide treatment of H1. The time course for the formation of the psi structure has been followed by measuring the changes in ellipticity at 270 nm as a function of time under different experimental conditions. In all cases studied here, we have observed the existence of two elementary processes: one fast, the other slow. Kinetic experiments performed with high molecular weight DNA showed that the greater the salt concentration, the higher was the apparent rate of psi structure formation. In complexes formed with sonicated DNA and H1, CNBS and CTB, we observed that the greater the content of the C-terminal domain, the higher was the apparent rate at which the final psi structure was reached. Thus, the presence of increasing amounts of either salt or C-terminal domain facilitates the formation of the psi structure. The molecular basis for these phenomena is discussed. The influence of the order of addition of the different components of the complex on the kinetics of psi structure induction is also studied.

Asunto(s)

RESUMEN

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.

Asunto(s)

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Histone H1(0) is found in tissues with little or no cellular proliferation and has been shown to accumulate during cellular terminal differentiation. Two subtypes of H1(0), H1 a and H1 b, are present in any tissue where the protein has been detected. We report here the first evidence of an age-dependent change in the proportions of H1 subtypes. In rat cerebral cortex neurons the proportion of H1 a rises from 44% of total H1 at birth to about 80% at day 300. These results show that terminally differentiated neurons synthesize and exchange H1 at a significant rate.

Asunto(s)

RESUMEN

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. All H2A variants appear to be equally modified. uH2B amounts to 1-2% of total H2B.

Asunto(s)