Pedro Suau

Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin

BackgroundHistone 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.ResultsThe 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.ConclusionH1 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.

Phosphorylation of the carboxy-terminal domain of histone H1: effects on secondary structure and DNA condensation

Linker histone H1 plays an important role in chromatin folding. Phosphorylation by cyclin-dependent kinases is the main post-translational modification of histone H1. We studied the effects of phosphorylation on the secondary structure of the DNA-bound H1 carboxy-terminal domain (CTD), which contains most of the phosphorylation sites of the molecule. The effects of phosphorylation on the secondary structure of the DNA-bound CTD were site-specific and depended on the number of phosphate groups. Full phosphorylation significantly increased the proportion of β-structure and decreased that of α-helix. Partial phosphorylation increased the amount of undefined structure and decreased that of α-helix without a significant increase in β-structure. Phosphorylation had a moderate effect on the affinity of the CTD for the DNA, which was proportional to the number of phosphate groups. Partial phosphorylation drastically reduced the aggregation of DNA fragments by the CTD, but full phosphorylation restored to a large extent the aggregation capacity of the unphosphorylated domain. These results support the involvement of H1 hyperphosphorylation in metaphase chromatin condensation and of H1 partial phosphorylation in interphase chromatin relaxation. More generally, our results suggest that the effects of phosphorylation are mediated by specific structural changes and are not simply a consequence of the net charge.

Differential kinetics of histone H1o accumulation in neuronal and glial cells from rat cerebral cortex during postnatal development

The accumulation of histone H1(0) as been studied in neuronal and glial nuclei from rat cerebral cortex during postnatal development. In neurons H1 degrees represents approximately 2% of the H1 content at birth and remains unchanged until day 8. Beyond this point H1 accumulates rapidly until day 18, where it levels off at 16% of H1. The midpoint of the transition is at day 14. In glial cells H1 represents approximately 2.5% of the H1 at birth. It starts to accumulate between days 18 and 21; its concentration raises rapidly up to day 30 slowing down from then on. At day 300 (the farthest point examined) it represents 21% of H1. These results are discussed in relation to the events of the postnatal development of the cerebral cortex in the rat. It is concluded that H1 probably does not suppress cell proliferation.

Condensation of DNA by the C-terminal domain of histone H1 A circular dichroism study

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.

An inducible helix-Gly-Gly-helix motif in the N-terminal domain of histone H1e: a CD and NMR study.

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‐NH2 (NE‐1) by circular dichroism and 1H‐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 α‐helical structure. In 90% TFE solution the α‐helical population is ∼40%. In these conditions, NE‐1 is structured in two α‐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.

Role of Charge Neutralization in the Folding of the Carboxy-Terminal Domain of Histone H1

H1 linker histones are involved in chromatin structure and gene regulation. The carboxy-terminal domain (CTD) of histone H1 is very basic with approximately 40% Lys residues, approximately 75% of which are present as doublets. The CTD has little structure in diluted solution but becomes cooperatively folded upon interaction with DNA. The DNA-bound CTD contains alpha-helix, beta-structure, turns, and flexible regions. We studied the effects of charge neutralization on the secondary structure of the CTD independently of DNA interaction through deprotonation of the epsilon-amino groups of the Lys side chains at alkaline pH. Alkaline pH induces extensive folding of the CTD with proportions of secondary structure similar to those observed in the complexes with DNA. The CTD is phosphorylated by cyclin-dependent kinases. In the fully phosphorylated CTD, alkaline pH induces a higher amount of beta-sheet and a lower amount of alpha-helix, as observed in the complexes with DNA. These results, together with structure predictions, suggest that the increased hydrophobicity of Lys side chains accompanying charge neutralization is responsible for the folding of the CTD upon interaction with DNA.

Differential expression and gonadal hormone regulation of histone H1° in the developing and adult rat brain

Abstract The cellular distribution of histone H1° has been examined immunohistochemically in the rat brain. H1° 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° 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° in the ventromedial part of the arcuate is reversiby 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° immunoreactivity in postpuberal females.

Core histone variants and ubiquitinated histones 2A and 2B of rat cerebral cortex neurons

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.

Secondary structure of protamine in sperm nuclei: an infrared spectroscopy study

BackgroundProtamines 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.ResultsWe 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.ConclusionDNA-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.

Interplay between histone H1 structure and function.

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.