Abstract
The differently acetylated subfractions of histone H4 isolated from cuttlefish testis and from calf thymus were separated by ion exchange chromatography on sulfopropyl-Sephadex, using a shallow linear gradient of guanidine hydrochloride in the presence of 6 M urea at pH 3.0. The tetra-, tri-, di-, mono-, and nonacetylated forms of cuttlefish H4 represent 2, 6.4, 18, 32.2, and 41.4% of the whole histone, respectively. The di-, mono-, and nonacetylated forms of calf H4 represent 11.7, 41.3, and 44% of the whole histone, respectively. The acetylation sites were determined in each subfraction by identification of the acetylated peptides. In each acetylated H4 subfraction, the acetylated tryptic peptides were identified by peptide mapping and amino acid analysis with reference to the peptide map of nonacetylated H4. In cuttlefish testis H4, lysine 12 is the main site of acetylation in the monoacetylated subfraction; lysines 5 and 12 are found acetylated in diacetylated H4; lysines 5, 12, and 16 are found acetylated in triacetylated H4. From these results and the stoichiometry of the different H4 subfractions, it can be concluded that lysine 5 is acetylated after lysine 12. In calf thymus, lysine 16 is the only site of acetylation in the monoacetylated subfraction. All the diacetylated forms are acetylated in lysine 16, the second site of acetylation being, in decreasing order, lysine 12, lysine 5, or lysine 8. These observations suggest that acetylation occurs in a sequential manner. Moreover, the sites of acetylation depend upon the biological event in which acetylation is involved.
Highlights
From the Unite 409 Centre National de la Recherche Scientifique, Znstitut de Recherchesur le Cancer, Place de Verdun, 59045 Lille Cedex, France
It has been suggested that these isolated from cuttlefish testis and from calf thymus amino-terminal regions could, in interacting with the DNA, were separated by ion exchange chromatography on play a role either in the nucleosomal organization and/or in sulfopropyl-Sephadex, using a shallow linear gradient the formation and the keeping of higher order structures of of guanidine hydrochloride in thepresence of 6M urea the chromatin [3,4,5].By contrast with the amino-terminal at p H 3.0
Lysines 5, 12,and 16 are found acetylated in triacety- In this paper, we present a study of histone H4 4 subfraclated H4.From these results and the stoichiometry of tions of known degreeof acetylation, isolated from twodifferthe different H4 subfractions, it can be concludedthat ent tissues, cuttlefish testis and calf thymus, where histone lysine 5 is acetylated after lysine 12
Summary
Isolation of Histone H4"Histone H4 was directlyobtained in pure form, from cuttlefish testis or calf thymus chromatin by a slight modification of the method of Johns [12,13,14]. Lane 1 , calf thymus histone H4; lane 2, total acetic acid hydrolysate; lane 3, mixture of peptides 1-23 and 69-102; lanes 4,5, 6, fractions 1, 3, and 4 from Fig. 2A. The mixture of peptides obtained was first fractionated on a Sephadex G-50 fine column (5 X 150 cm) equilibrated and eluted with 10 mM HCl saturated with chloroform. Separation of the Different Acetylated Forms of Histone H4 The small peaks which appear before fractions 3 and 4 on Sulfopropyl-Sephadex-The chromatographic separations contain fragment 1-24 resulting from an incomplete enucleaon sulfopropyl-Sephadex were performed at pH 3.0 At this tion of aspartyl residue at position 24 in H4 histone (data not pH, histone aggregation is minimized, and carbamylation by shown). Graphic fractions 1-5 corresponding t o H4 tetra-, H4 tri-, H4 Peptide Mappingof H4 Subfractiorz-The degree of acetydi-, H4 mono-, and H4 nonacetylated, respectively, and rep- lation, as well as the site(s) of acetylation of the different resenting 2, 6.4, 18,32.2, and 41.4% of the whole H4, respec- subspecies of histone H4, were checked by mapping the tryptic tively
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