Chromatin condensing functions of the linker histone C-terminal domain are mediated by specific amino acid composition and intrinsic protein disorder.

Linker histones bind to the nucleosomes and linker DNA of chromatin fibers, causing changes in linker DNA structure and stabilization of higher order folded and oligomeric chromatin structures. Linker histones affect chromatin structure acting primarily through their approximately 100-residue C-terminal domain (CTD). We have previously shown that the ability of the linker histone H1 degrees to alter chromatin structure was localized to two discontinuous 24-/25-residue CTD regions (Lu, X., and Hansen, J. C. (2004) J. Biol. Chem. 279, 8701-8707). To determine the biochemical basis for these results, we have characterized chromatin model systems assembled with endogenous mouse somatic H1 isoforms or recombinant H1 degrees CTD mutants in which the primary sequence has been scrambled, the amino acid composition mutated, or the location of various CTD regions swapped. Our results indicate that specific amino acid composition plays a fundamental role in molecular recognition and function by the H1 CTD. Additionally, these experiments support a new molecular model for CTD function and provide a biochemical basis for the redundancy observed in H1 isoform knockout experiments in vivo.

Linker histones stabilize the intrinsic salt-dependent folding of nucleosomal arrays: mechanistic ramifications for higher-order chromatin folding.

Defined nucleosomal arrays reconstituted from core histone octamers and twelve 208 bp tandem repeats of Lytechinus 5S rDNA (208-12 nucleosomal arrays) possess the ability to form an unstable folded species in MgCl2 whose extent of compaction equals that of canonical higher-order 30 nm diameter chromatin structures [Schwarz, P. M., and Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. To address the mechanistic functions of linker histones in chromatin condensation, purified histone H5 has been assembled with 208-12 nucleosomal arrays in 50 mM NaCl. Novel purification procedures subsequently were developed that yielded preparations of 208-12 chromatin model systems in which a majority of the sample contained both one histone octamer per 5S rDNA repeat and one molecule of histone H5 per histone octamer. The integrity of the purified 208-12 chromatin has been extensively characterized under low-salt conditions using analytical ultracentrifugation, quantitative agarose gel electrophoresis, electron cryomicroscopy, and nuclease digestion. Results indicate that histone H5 binding to 208-12 nucleosomal arrays constrains the entering and exiting linker DNA in a way that produces structures that are indistinguishable from native chicken erythrocyte chromatin. Folding experiments performed in NaC1 and MgC12 have shown that H5 binding markedly stabilizes both the intermediate and extensively folded states of nucleosomal arrays without fundamentally altering the intrinsic nucleosomal array folding pathway. These results provide new insight into the mechanism of chromatin folding by demonstrating for the first time that distinctly different macromolecular determinants are required for formation and stabilization of higher-order chromatin structures.

Interleukin-1 alpha- and beta-, interleukin-6- and tumour necrosis factor-alpha-like immunoreactivities in chronic granulomatous skin conditions.

Paraformaldehyde-fixed tissue of chronic granulomatous skin conditions, such as cutaneous leishmaniasis, granuloma annulare, leprosy and hidroadenitis, was investigated for the presence of interleukin-1 alpha-, interleukin-1 beta-, interleukin-6- and tumour necrosis factor-alpha-like immunoreactivities among the cellular infiltrates. There was a weak to strong cytoplasmic labelling of plasma cells for interleukin-6 and tumour necrosis factor-alpha at the periphery of the granulomatous mass and around the skin appendages. The interleukin-6-like immunoreactivity seemed to be correlated with the coarseness of the chromatin material of the cells, being more intense with coarse chromatin. The cytoplasmic labelling for interleukin-1 alpha and interleukin-1 beta in the plasma cells was less intense. Epitheloid, Langhans' giant cells and small round cells exhibited a weak to moderate cytoplasmic labelling for interleukin-1 alpha and interleukin-1 beta, whereas the staining intensity for interleukin-6 and tumour necrosis factor-alpha was weak to strong. In addition, there was staining of the stroma in the centre of granuloma with antisera against interleukin-1 alpha, interleukin-1 beta, interleukin-6 and tumour necrosis factor-alpha. This area contained few cells, suggesting that the granuloma was in a resolution process. A contribution of interleukin-6 and tumour necrosis factor-alpha to the granulomatous reaction, at least during the maintenance period, is suggested by the occurrence of these cytokines in the skin conditions studied. The findings are also consistent with a suggested role of B cells in the late stages of the granulomatous reaction. In addition, they are in line with the reported declining role of interleukin-1 in the maintenance of granuloma.

Formation and stability of higher order chromatin structures. Contributions of the histone octamer.

Unique roles have been identified for the histone octamer in the formation and stabilization of higher order chromatin structures. Histone octamers were assembled onto 12 tandem repeats of Lytechinus 5 S rDNA, at either saturating or subsaturating ratios. The extent of oligonucleosome folding and intermolecular association in divalent salts was monitored using analytical and differential sedimentation techniques. Saturated oligonucleosomes (12 nucleosomes/DNA) sedimented at 29 S in very low salt buffer. In 1.0-2.0 mM MgCl2, saturated oligonucleosomes formed a maximally folded 55 S structure whose extent of compaction was equivalent to that of classical higher order 30-nm diameter chromatin structures. These results are in marked contrast to those obtained previously in NaCl, where the maximally folded oligonucleosome species sedimented at only approximately 40 S (Hansen, J. C., Ausio, J., Stanik, V. H., and van Holde, K. E. (1989) Biochemistry 28, 9129-9136). Mg(2+)-dependent formation of the 55 S conformation was inhibited by histone octamer depletion; the maximum sedimentation coefficient observed for rDNA molecules containing 10-11 nucleosomes in 2.0 mM MgCl2 was only 40 S. Above 2.0 mM MgCl2, the equilibrium was progressively shifted toward formation of large associated oligonucleosome species. The implications of these results to the mechanism of chromatin folding and its relationship to the biological activity of the chromatin fiber are discussed.