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.     

Nucleosome assembly on telomeric sequences

The organization of telomeric chromatin differs from that of bulk chromatin in some peculiar features, such as the unusually short nucleosomal spacing found in vertebrates. Telomeric DNAs are straight, since they consist mostly of 6-8-bp repeated sequences, therefore out of phase with the B DNA period. This feature should be of relevance in nucleosome formation, suggesting the usefulness of studying simple model systems of nucleosome assembly. We reconstituted nucleosomes in vitro, by using purified histone octamers and/or by octamer transfer from chicken erythrocyte nucleosomes, onto telomeric sequences from human, Arabidopsis thaliana, and Saccharomyces cerevisiae. All of these telomeres contain GGG and GGT triplets but are characterized by different repeat lengths (6, 7, and 8 bp). The free energies involved in the association process are the highest among the biological sequences so far assayed, suggesting a main role of DNA flexibility in the assembly of telomeric chromatin. Digestion studies with DNase I, hydroxyl radicals, exonuclease III, and lambda exonuclease indicate that telomeric nucleosomes are characterized by multiple translational positioning without rotational phasing, whereas the telomeric DNA folding around the histone octamer shows the canonical periodicity of about 10.2 bp. The experimental results and a theoretical simulation of DNase I digestion indicate a multiple nucleosome positioning with the periodicity of telomeric DNA. This suggests a main role of local chemical recognition between telomeric sequences and the histone octamer in nucleosome assembly.     

In vitro reconstitution of Artemia satellite chromatin

We report the characterization of an in vitro chromatin assembly system derived from Artemia embryos and its application to the study of AluI-113 satellite DNA organization in nucleosomes. The system efficiently reconstitutes chromatin templates by associating DNA, core histones, and H1. The polynucleosomal complexes show physiological spacing of repeat length 190 +/- 5 base pairs, and the internucleosomal distances are modulated by energy-using activities that contribute to the dynamics of chromatin conformation. The assembly extract was used to reconstitute tandemly repeated AluI-113 sequences. The establishment of preferred histone octamer/satellite DNA interactions was observed. In vitro, AluI-113 elements dictated the same nucleosome translational localizations as found in vivo. Specific rotational constraints seem to be the central structural requirement for nucleosome association. Satellite dinucleosomes showed decreased translational mobility compared with mononucleosomes. This could be the consequence of interactions between rotationally positioned nucleosomes separated by linker DNA of uniform length. AluI-113 DNA led to weak cooperativity of nucleosome association in the proximal flanking regions, which decreased with distance. Moreover, the structural properties of satellite chromatin can spread, thus leading to a specific organization of adjacent nucleosomes.     

In vitro binding of H1 histone subtypes to nucleosomal organized mouse mammary tumor virus long terminal repeat promotor

The binding of all known linker histones, named H1a through H1e, including H1(0) and H1t, to a model chromatin complex based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, we found a dissociation constant of 8-16 nM to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes varied between 2 and 4 nM. Most of the H1 histones, namely H1a, H1c, H1d/e, and H1(0), completely aggregate polynucleosomes (1.3 kilobase pairs, 6 nucleosomes) at 270-360 nM, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome. To form aggregates with the histones H1t and H1b, however, greater amounts of protein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes. S phase-specific phosphorylation with one to three phosphate groups at specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes. In contrast, highly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucleosome but a low chromatin aggregation capability. These findings suggest that specific S phase or mitotic phosphorylation sites act independently and have distinct functional roles.     

The organization of histones and DNA in chromatin: evidence for an arginine-rich histone kernel

We have examined the role played by various histones in the organization of the DNA of the nucleosome, using staphylococcal nuclease as a probe of DNA conformation. When this enzyme attacks chromatin, a series of fragments evenly spaced at 10 base pair intervals is generated, reflecting the histone-DNA interactions within the nucleosome structure. To determine what contribution the various histones make to DNA organization, we have studied the staphylococcal nuclease digestion patterns of complexes of DNA with purified histones. Virtually all possible combinations of homogeneous histones were reconstituted onto DNA. Exhaustive digestion of a complex containing the four histones H2A, H2B,H3, and H4 yields a DNA fragment pattern very similar to that of whole chromatin. The only other combinations of histones capable of inducing chromatin-like DNA organization are H2A/H2B/H4 and those mixtures containing both H3 and H4. From an examination of the kinetics of digestion of H3/H4 reconstitutes, we conclude that although the other histones have a role in DNA organization within the nucleosome, the arginine-rich histone pair, H3/H4, can organize DNA segments the length of the nucleosome core in the absence of all other histones.     

Nucleosome assembly on CTG triplet repeats

Expansion of CTG repeat sequences is associated with several human genetic diseases. We have examined the consequences of CTG repeat expansion for nucleosome assembly and positioning. Short CTG repeats are found within the most favored DNA sequences yet defined for nucleosome assembly. We find that as few as six CTG repeats will facilitate nucleosome assembly to a similar extent as the 50 or more repeats found in disease genes. Thus an increase in nucleosome stability on expansion of existing triplet repeats is unlikely to explain the acquisition of the disease phenotype. However, the CTG repeat sequence is efficiently wrapped around the histone octamer, preferring to associate with histones at the nucleosomal dyad. Thus short segments CTG repeat sequence will facilitate the assembly of a stable positioned nucleosome which might contribute to the expansion phenomenon and the functional organization of chromatin.