Evidence indicating proximity in the nucleosome between the histone H4 N termini and the globular domain of histone H1.
Proteolysis of rat liver chromatin by the Arg-C peptidase, clostripain, is characterized by a progressive fragmentation of the N-terminal segments of the four core histones H2A, H2B, H3 and H4, until a well-defined limit digest is reached. This work addresses the case of histone H4. Two intermediate proteolytic sites are identified for this histone, i.e. Arg3 and Arg17, before the limit digest is achieved through cleavage of the polypeptide chain after Arg19. The accessibility of these intermediate sites depends strongly on the presence or absence of histone H1. When H1 is absent, both intermediate sites of histone H4 are similarly accessible, whereas one of them, Arg3, becomes totally inaccessible in the presence of histone H1. Di- and trinucleosomes were used with the aim of avoiding any interference with superstructural effects which can occur with longer polynucleosomes in the presence of H1. We also investigated the accessibility of the Arg sites of H1 that are located primarily in the central globular domain of this histone. In free histone H1, all the centrally located Arg sites are accessible to clostripain. In contrast, in the chromatin-bound state none of these sites is accessible. Besides the arginyl sites in the central globular domain of H1, two Arg residues are observed with the most abundant H1d variant in rat chromatin, one in the N-terminal region and the other in the C-terminal region. The restricted number of proteolytic fragments observed with chromatin-bound H1 is accounted for by the cleavage of H1 after these Arg residues located on the outside of the globular domain. Our results suggest that mutual steric effects are at play between histones H1 and H4 and indicate that the N termini of both histones H4 in the nucleosome lie in close proximity to the globular domain of H1. Based on these observations and taking into account the known structural features of the nucleosome, we propose a model for positioning the N-terminal segments of both histones H4 at the periphery of the nucleoprotein structure. In this model both H4 segments are located within the expanded DNA minor grooves, at periods +/- 1, symmetrically disposed relatively to the nucleosome dyad axis. This arrangement brings the amino ends of both H4 molecules in close contact with the H1 globular domain thus accounting for the observed inaccessibility of the Arg3 site of H4 in the presence of H1.
Banères
J L
JL
URA-CNRS 1111 Chimie des Médiateurs et Physico-Chimie des Interactions Biologiques, Faculté de Pharmacie, Montpellier, France.
Essalouh
L
L
Jariel-Encontre
I
I
Mesnier
D
D
Garrod
S
S
Parello
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D003546:Cysteine Endopeptidases; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D051381:Rats
D006657:Histones; D009707:Nucleosomes; D001120:Arginine; D003546:Cysteine Endopeptidases; C019913:clostripain