Imma Ponte

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.

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.

Transcriptional activation of Histone H1° during neuronal terminal differentiation

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.

SEQUENCE SIMPLICITY AND EVOLUTION OF THE 3' UNTRANSLATED REGION OF THE HISTONE H1 GENE

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.

Cloning and analysis of the coding region of the histone H1° -encoding gene from rat PC12 cells

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.