P. Michalon et al., STRUCTURAL-ANALYSIS OF TRYPANOSOMA-BRUCEI-BRUCEI CHROMATIN BY LIMITEDPROTEOLYSIS - AN ELECTRICAL-BIREFRINGENCE STUDY, European journal of biochemistry, 216(2), 1993, pp. 387-394
The sensitive electric-birefringence method was used to reveal structu
ral differences between the soluble chromatin of procyclic Trypanosoma
brucei brucei and the chromatin of the higher eukaryotes. The orienta
tion of the nucleosomal chains and the presence of extended DNA were a
nalysed from the sign and amplitude of the steady-state birefringence,
and the conformational properties (overall dimensions and flexibility
) were studied in relation to the orientational relaxation times. In c
ontrast to the higher eukaryotes, the birefringence of T. brucei bruce
i is negative and of low amplitude, corresponding to that of H1-deplet
ed rat liver nucleosomes. Furthermore, the relaxation times are very s
mall, about 10 mus. If salt is added, the birefringence as well as the
relaxation time decreases dramatically, indicating that condensation
affects T. brucei brucei chromatin although it behaves like nucleosome
filaments, with less stable DNA-protein interaction than for the high
er eukaryotes. However, this condensation does not induce the formatio
n of regular higher-order structure. This complies with the hypothesis
that typical histone H1 is absent from T. brucei brucei chromatin and
that a protein or protein domain fulfils the role of histone H1. The
accessibility and structural role of histone-like proteins in T. bruce
i brucei chromatin were also investigated using limited proteolysis wi
th enzymes covalently bound to nylon spheres. The analysis of protein
products obtained after digestion with immobilized trypsin and subtili
sin shows that proteins a and d, which are classified as H3 and H4 his
tones, respectively, are the first to be attacked. The changes in chro
matin conformation indicate that chromatin undergoes a structural tran
sition, leading to decondensation, as indicated by increases in negati
ve birefringence and relaxation time, and to a change in its orientati
on mechanism, indicated by the appearance of a permanent moment. This
result is very interesting since, in rat liver, H4 was very resistant
and was the last histone to be attacked, suggesting internal location
and its involvement in nucleosome stabilization rather than higher-ord
er condensation. Therefore, in T. brucei brucei chromatin, the charact
eristic properties of proteins a and d (their composition and interact
ion with DNA), as well as their external location on the nucleosome su
rface, suggest that if these proteins play a role similar to that play
ed by H3 and H4 in higher eukaryotes, probably through their N-termina
l regions and interaction either with DNA or protein domains, the mech
anisms involved in chromatin compaction are quite different. Since the
re is no binding with H1, it may be hypothesized that a (non-histone)
protein, or core protein domains, may play a part in chromatin compact
ion and gene expression regulation comparable to that of H1 in higher
eukaryotes.