Histone-DNA contacts in structure/function relationships of nucleosomes as revealed by crosslinking.

We describe studies of histone-DNA contacts in the nucleosome using the method of covalent zero length protein-DNA crosslinking. These studies show that in intact nuclei isolated from different sources the linear sequential arrangement of histone-DNA contacts in the nucleosomal core is essentially the same. However, the relative strength of certain contacts varies and correlates with the level of chromatin activity and condensation. These altered contacts are located in the sharply bent regions of the nucleosomal DNA and are supposed to be sensitive to the structural changes that may occur during nucleosome functions. Studies of the mechanism of these alterations revealed that the difference in strength of these contacts is attributed to the different conformational state of the nucleosomal core and is caused by stretching of the nucleosomal DNA upon chromatin decondensation during its activation. Histone-terminal domains may be involved in this process through posttranslational modifications affecting chromatin condensation. The described localization of the histone H2A C-terminal domain in the nucleosome by crosslinking demonstrates the ability of this methodology to determine the location of histone-terminal domains and thereby elucidate their role in nucleosome function. Results of the described experiments suggest that chromatin decondensation may alter the nucleosomal DNA conformation and affect the histone-DNA contacts resulting in a structural transition that may play a role in rendering the nucleosome competent for transcription and/or replication.

Nucleosome structural transition during chromatin unfolding is caused by conformational changes in nucleosomal DNA.

We have recently reported that certain core histone-DNA contacts are altered in nucleosomes during chromatin unfolding (Usachenko, S. I., Gavin I. M., and Bavykin, S. G. (1996) J. Biol. Chem. 271, 3831-3836). In this work, we demonstrate that these alterations are caused by a conformational change in the nucleosomal DNA. Using zero-length protein-DNA cross-linking, we have mapped histone-DNA contacts in isolated core particles at ionic conditions affecting DNA stiffness, which may change the nucleosomal DNA conformation. We found that the alterations in histone-DNA contacts induced by an increase in DNA stiffness in isolated core particles are identical to those observed in nucleosomes during chromatin unfolding. The change in the pattern of micrococcal nuclease digestion of linker histone-depleted chromatin at ionic conditions affecting chromatin compaction also suggests that the stretching of the linker DNA may alter the nucleosomal DNA conformation, resulting in a structural transition in the nucleosome which may play a role in rendering the nucleosome competent for transcription and/or replication.

Alterations in nucleosome core structure in linker histone-depleted chromatin.

We have previously shown that the sequential arrangement of histone-DNA contacts is essentially the same in the nucleosomal core of sea urchin sperm nuclei, where chromatin is highly condensed and repressed, and in nuclei from lily bud sepals or yeast, where chromatin is highly active in transcription and replication and is significantly or completely depleted of histone H1. However, the difference in the strength of some histone-DNA contacts has not been understood or discussed. In this work, we demonstrate that some of these differences are due to a conformational change in the nucleosomal core. We show that the nucleosomal core in linker histone-depleted chromatin is in a different conformational state compared with the nucleosomal core in folded chromatin or in isolated core nucleosomes. This conformational state is characterized by altered strengths in the histone H4 and H2A/H2B contacts with the regions of sharply bent nucleosomal DNA around sites +/-1 and +/-4 and site +/-5, respectively. We demonstrate that this conformation, which we call the "stretched nucleosome," is a general feature of unfolded linker histone-depleted chromatin and may occur during chromatin activation. Our results suggest that this nucleosome structural alteration does not depend on chromatin sources and histone variants studied in this work. In addition, we show that this alteration is reversible and is caused by the stretching of linker DNA during chromatin unfolding.

Rearrangement of the histone H2A C-terminal domain in the nucleosome.

Using zero-length covalent protein-DNA crosslinking, we have mapped the histone-DNA contacts in nucleosome core particles from which the C- and N-terminal domains of histone H2A were selectively trimmed by trypsin or clostripain. We found that the flexible trypsin-sensitive C-terminal domain of histone H2A contacts the dyad axis, whereas its globular domain contacts the end of DNA in the nucleosome core particle. The appearance of the histone H2A contact at the dyad axis occurs only in the absence of linker DNA and does not depend on the absence of linker histones. Our results show the ability of the histone H2A C-terminal domain to rearrange. This rearrangement might play a biological role in nucleosome disassembly and reassembly and the retention of the H2A-H2B dimer (or the whole octamer) during the passing of polymerases through the nucleosome.

Structure of nucleosomes and organization of internucleosomal DNA in chromatin.

We have compared the mononucleosomal pattern produced by micrococcal nuclease digestion of condensed and unfolded chromatin and chromatin in nuclei from various sources with the repeat length varying from 165 to 240 base-pairs (bp). Upon digestion of isolated H1-containing chromatin of every tested type in a low ionic strength solution (unfolded chromatin), a standard series of mononucleosomes (MN) was formed: the core particle, MN145, and H1-containing, MN165, MN175, MN185, MN195, MN205 and MN215 (the indexes give an approximate length of the nucleosomal DNA that differs in these particles by an integral number of 10 bp). In addition to the pattern of unfolded chromatin, digestion of whole nuclei or condensed chromatin (high ionic strength of Ca2+) gave rise to nuclei-specific, H1-lacking MN155. Digestion of H1-lacking chromatin produced only MN145, MN155 and MN165 particles, indicating that the histone octamer can organize up to 165 bp of nucleosomal DNA. Although digestion of isolated sea urchin sperm chromatin (repeat length of about 240 bp) at a low ionic strength gave a typical "unfolded chromatin pattern", digests of spermal nuclei contained primarily MN145, MN155, MN235 and MN245 particles. A linear arrangement of histones along DNA (primary organization) of the core particle was found to be preserved in the mononucleosomes, with the spacer DNA length from 10 to 90 bp on one (in MN155) or both sides of core DNA being a multiple of about 10 bp. In MN235, the core particle occupies preferentially a central position with the length of the spacer DNA on both sides of the core DNA being usually about 30 + 60 or 40 + 50 bp. Histone H1 is localized at the ends of these particles, i.e. close to the centre of the spacer DNA. The finding that globular part of histones H3 and sea urchin sperm H2B can covalently bind to spacer DNA suggests their involvement in the organization of chromatin superstructure. Our data indicate that decondensation of chromatin is accompanied by rearrangement of histone H1 on the spacer DNA sites adjacent to the core particle and thus support a solenoid model for the chromatin superstructure in nuclei in which the core DNA together with the spacer DNA form a continuous superhelix.

[Structure of nucleosomes and organization of inter-nucleosomal DNA in chromatin]. / Struktura nukleosom i organizatsiia mezhnukleosomnoi DNK v khromatine.

We have compared mononucleosomes that were obtained by hydrolysis of chromatin micrococcal nuclease from a number of sources with the length of a nucleosomal repeat 185--245 b. p. long. For hydrolysis of chromatin isolated from nuclei, a series of nucleosomes was formed: MN145 (core particle), MN165, MN175...MN205, MN215, the lengths of their DNAs differing (by approximately 10.n b.p. where n = 1, 2, 3...) by a factor of 10. A feature of hydrolysis of chromatin in nuclei was the appearance of an additional H1-depleted MN155 particle. It is suggested that upon isolation of chromatin from nuclei, its partial decompactization takes place. This decompactization changes the character of nuclease splitting and seems to be connected with rearrangement of histone H1. These observations demonstrate that besides core particles MN145 and chromatosomes MN165, the major particles of digest of nuclei appear to be MN155, and for isolated chromatin--MN175. Unlike this standard picture, mainly MN145, MN155, MN235 and MN245 are formed upon hydrolysis of sea urchin sperm nuclei.

[Comparison of the primary structure of reconstructed minimal nucleosomes and nucleosomes isolated from the chromatin]. / Sravnenie pervichnoi organizatsii rekonstruirovannykh i vydelennykh iz khromatina minimal'nykh nukleosom.

We have reconstructed nucleosomes from a histone octamer (H2A, H2B, H3, H4)2 and DNA 146 b.p. or 2-3 thousands b.p. in length. Comparison by means of DNA-histone cross-links of the primary organization of minimal nucleosomes obtained by reconstruction or isolated from chromatin of chicken erythrocyte nuclei has demonstrated a high similarity in histone location on their DNAs. Simultaneously, there have been observed some variations in the character of interaction for all core histones with DNA on nucleosomes. Thus, the cross-link of histone H4 with DNA of a core particle at H4 sites (65), unlike H4(55) and H4(88) sites, significantly depends on the superstructure of chromatin, ionic strength of solution and the presence of denaturating agents. All these differences are expected to probe the existence of conformational isomers for core particles. (Bracketed is the distance from the histone interaction site with the DNA of the core particle to the DNA 5'-terminus.)

Primary organization of nucleosomal core particles is invariable in repressed and active nuclei from animal, plant and yeast cells.

A refined map for the linear arrangement of histones along DNA in nucleosomal core particles has been determined by DNA-protein crosslinking. On one strand of 145-bp core DNA, histones are aligned in the following order: (5') H2B25,35-H455,65-H375,85,95/H488-H2B105,11 5-H2A118-H3135,145/H2A145 (3') (the subscripts give approximate distance in nucleotides of the main histone contacts from the 5'-end). Hence, the histone tetramer (H3,H4)2 and two dimers (H2A-H2B) are arranged on double-stranded core DNA in a symmetrical and rather autonomous way: H2A/H3-(H2A-H2B)-(H3,H4)2-(H2B-H2A)-H3/H2A. The primary organization was found to be very similar in core particles isolated from repressed nuclei of sea urchin sperm and chicken erythrocytes, from active in replication and transcription nuclei of Drosophila embryos and yeast and from somatic cells of lily. These data show that (i) the core structure is highly conserved in evolution and (ii) the overall inactivation of chromatin does not affect the arrangement of histones along DNA and thus does not seem to be regulated on this level of the core structure.

[Primary organization of nucleosome core particles in active and repressed nuclei]. / Pervichnaia organizatsiia minimal'nykh nukleosom aktivnykh i repressirovannykh iader.

A refined high-resolution map for the linear arrangement of histones along DNA in the nucleosomal core particles has been determined by DNA-protein crosslinking. Histones are aligned on one strand of the 145 bp core DNA in the following order: (5') H2B25,35--H455,65--H375,85,, 95/H488--H2B105, 115--H2A118--H3135, 145/H2A145 (3') (the subscripts indicate the approximate distance in nucleotides of the main histone binding sites from the 5'-end of the core DNA). This suggests a symmetrical and rather autonomous arrangement of the histone tetramer (H3, H4)2 and two dimers (H2A, H2B) on the double-stranded core DNA: H2A/H3--(H2A, H2B)--(H3, H4)2--(H2B, H2A)--H3/H2A. The arrangement of histones on DNA was found to be very similar for the cores isolated from the repressed nuclei of sea urchin sperm and chicken erythrocytes and from the active in transcription and replication Drosophila embryo and yeast nuclei. This indicates that the core nucleosome structure is highly conserved through evolution and that the overall inactivation of chromatin does not affect the primary organization of the cores. A new binding site H2B58 was found for a sea urchin spermal variant of H2B which contains an additional basic segment within the N-terminal part of the molecule. The core isolation procedure was shown to introduce changes into the core structure which are reflected in the appearance of a new binding site H2A75.