Chromatin superstructure-dependent crosslinking with DNA of the histone H5 residues Thr1, His25 and His62.

The points of histone H5 interactions with DNA within nucleosomes and chromatin at different levels of compaction are delineated by identification of H5 amino acid residues that can be covalently bound to DNA. Three major crosslinkable points of H5 are His25, His62 (both within the globular part of the molecule), and N-terminal Thr1. His25 interacts with the terminal regions of nucleosomal DNA; His62 appears to bind more distal segments of the linker DNA. The His25-DNA crosslink predominates in the isolated mononucleosomes and persists throughout the chromatin condensation states studied, from extended oligonucleosomal chains to nuclei. His62 is the strongest crosslinking site in nuclei; in oligonucleosomes, the predominance of the His62-DNA crosslink requires the number of nucleosomes in the chain to be above some critical value. The Thr1-DNA crosslink is generated only in decondensed poly- or oligonucleosomes, but not in mononucleosomes. Thus, underlying the higher-order folding transitions of the nucleosomal chain is the restructuring of H5-DNA interactions.

One-domain interaction of histone H4 with nucleosomal core DNA is restricted to a narrow DNA segment.

The interaction of histone H4 with DNA in the nucleosomal core particle has been studied by crosslinking DNA to proteins through their lysine residues. We have compared the crosslinked peptides of H4 at the detected DNA-binding sites: H4(55), H4(65), H4(88), located, respectively, at about 55, 65 and 88 nucleotides from the core DNA termini. For all these binding sites, the patterns of crosslinked peptides were shown to be very similar. This suggests the presence of a single DNA-binding domain in the H4 molecule. The H4-binding sites are located within a narrow DNA segment close to one another on the complementary strands across the DNA grooves, overlap with sites +/- 1 of the DNA sharp bending [(1984) Nature 311, 532-537] and with the strong binding sites for histone H3: H3(75) and H3(85).

Mechanism of Lac repressor switch-off: orientation of the Lac repressor DNA-binding domain is reversed upon inducer binding.

Lac repressor's DNA-binding domains contain helix-turn-helix motif which, though similar to those of phage lambda Cro protein, are oriented differently with respect to DNA: in the specific complexes with Lac operator, N termini of the repressor's subunits are facing inwards. We demonstrate that, in the presence of an inducer, the repressor's N termini cross-link to the operator's outermost nucleotides. We suggest that the inducer fixes the repressor's DNA-binding domains in the Cro-type configuration and thus garbles its recognition surface. Since the Cro-type configuration is perfectly suitable for binding the DNA, this also explains how the switched-off repressor retains its non-specific DNA-binding.

An estimate of the extent of folding of nucleosomal DNA by laterally asymmetric neutralization of phosphate groups.

We attempt quantitative implementation of a previous suggestion that asymmetric charge neutralization of DNA phosphate groups may provide part of the driving force for nucleosome folding. Polyelectrolyte theory can be used to estimate the effective compressive force acting along the length of one side of the DNA surface when a fraction of the phosphate groups are neutralized by histones bound to that side. A standard engineering formula then relates the force to the bending amplitude caused by it. Calculated bending amplitudes are consistent with the curvature of nucleosomal DNA and the overall extent of charge neutralization by the histones. The relation of the model to various aspects of nucleosome folding, including the detailed path of core-particle DNA, is discussed. Several other DNA-protein complexes are listed as examples of possible asymmetric charge-induced bending.

A highly basic histone H4 domain bound to the sharply bent region of nucleosomal DNA.

A nucleosomal core particle is composed of two each of histones H2A, H2B, H3 and H4 located inside the particle with approximately 47 base pairs (bp) of DNA wrapped around the octamer in about 1.8 turns of a left-handed superhelix. The path of the superhelix is not smooth; the DNA is sharply bent, or kinked, at positions symmetrically disposed at a distance of about one and four double-helical turns in both directions from the nucleosomal dyad axis (designated as sites +/- 1 and +/- 4 respectively). This non-uniform bending is considered archetypal to other DNA-protein complexes, but its mechanism is not clear (reviewed in ref. 4). DNA-histone chemical cross-linking within the core particle has revealed strong binding of each of the two histone H4 molecules to DNA at a distance of 1.5 helical turns either side of the nucleosomal dyad axis (sites +/- 1.5). In each of these sites, a single flexible domain of H4 was previously shown to contact three points, at about nucleotides 55 and 65 on one strand and nucleotide 88 on the complementary strand, numbering from the 5' terminus of each 147-base strand; these three locations are closely juxtaposed across the highly compressed minor and major grooves (Fig. 1). Here we report that the amino-acid residue of histone H4 cross-linked at the 1.5 site is histidine-18, embedded in a highly basic cluster Lys-Arg-His-Arg-Lys-Val-Leu-Arg which is probably involved in the sharp bending of the DNA double helix at the +/- 1 sites.

Nucleosomal structure at hyperacetylated loci probed in nuclei by DNA-histone crosslinking.

Chemically induced histone-DNA crosslinking in nuclei is used to monitor structural changes in chromosomal domains containing hyperacetylated histones. Core particles harbouring the crosslinks are immunofractionated with antibodies specific for acetylated histones. Crosslinking is revealed by gel separation of tryptic peptides from core histones that carry 32P-labelled residual nucleotide. The large number of DNA-histone crosslinks retained indicates that acetylated core histone tails are not totally displaced from the DNA. Changes in the patterns of crosslinked peptides imply a restructuring of hyperacetylated histone-DNA interactions at several points within the nucleosome. This demonstrates that a distinct conformational state is adopted in acetylated nucleosomes, known to be concentrated at transcriptionally active loci.

Change in the pattern of histone binding to DNA upon transcriptional activation.

Patterns of histone binding to DNA of transcriptionally active D. melanogaster hsp70 genes within the nuclei have been analyzed by two methods of histone-DNA chemical cross-linking. When cross-linking is restricted to the central, "globular" regions of histones, it drops most for H1, to an intermediate extent for H2A and H2B, and least for H3 and H4 in transcriptionally active versus transcriptionally silent chromatin. When it occurs via histone terminal regions as well, cross-linking is quantitatively similar for active and inactive chromatin. Neither cross-linking method detects histones on the hsp70 promoter region. It appears that chromatin activation decreases histone binding to DNA via the "globular" regions, known to be essential for the folding of nucleosomes and the 30 nm chromatin fibril, but does not significantly affect the interaction of flexible and loosely bound histone "tails" with DNA. The role of these histone-DNA interaction changes in the unfolding of active chromatin and RNA polymerase reading through histone-bound DNA is discussed.