Dissection of the unusual structural and functional properties of the variant H2A.Bbd nucleosome

The histone variant H2A.Bbd appeared to be associated with active chromatin, but how it functions is unknown. We have dissected the properties of nucleosome containing H2A.Bbd. Atomic force microscopy (AFM) and electron cryo-microscopy (cryo-EM) showed that the H2A.Bbd histone octamer organizes only approximately 130 bp of DNA, suggesting that 10 bp of each end of nucleosomal DNA are released from the octamer. In agreement with this, the entry/exit angle of the nucleosomal DNA ends formed an angle close to 180 degrees and the physico-chemical analysis pointed to a lower stability of the variant particle. Reconstitution of nucleosomes with swapped-tail mutants demonstrated that the N-terminus of H2A.Bbd has no impact on the nucleosome properties. AFM, cryo-EM and chromatin remodeling experiments showed that the overall structure and stability of the particle, but not its property to interfere with the SWI/SNF induced remodeling, were determined to a considerable extent by the H2A.Bbd docking domain. These data show that the whole H2A.Bbd histone fold domain is responsible for the unusual properties of the H2A.Bbd nucleosome.     

Sequence-specific recognition of DNA in the nucleosome by pyrrole-imidazole polyamides

The ability of DNA-binding proteins to recognize their cognate sites in chromatin is restricted by the structure and dynamics of nucleosomal DNA, and by the translational and rotational positioning of the histone octamer. Here, we use six different pyrrole-imidazole polyamides as sequence-specific molecular probes for DNA accessibility in nucleosomes. We show that sites on nucleosomal DNA facing away from the histone octamer, or even partially facing the histone octamer, are fully accessible and that nucleosomes remain fully folded upon ligand binding. Polyamides only failed to bind where sites are completely blocked by interactions with the histone octamer. Removal of the amino-terminal tails of either histone H3 or histone H4 allowed these polyamides to bind. These results demonstrate that much of the DNA in the nucleosome is freely accessible for molecular recognition in the minor groove, and also support a role for the amino-terminal tails of H3 and H4 in modulating accessibility of nucleosomal DNA.     

Contribution of histones H2A and H2B to the folding of nucleosomal DNA

We have studied the structural properties of nucleosomal particles deficient in histones H2A and H2B produced by modification of histone amino groups with dimethylmaleic anhydride [Jordano, J., Montero, F., & Palacián, E. (1984) Biochemistry (preceding paper in this issue)]. Digestion with DNase I of residual particles containing only 15% of the original H2A . H2B complement produces only discrete DNA fragments no longer than 70 nucleotides. As compared with the original nucleosomes, thermal denaturation of the residual particles shows a decrease from 140 to about 90 in the number of nucleotide base pairs per particle that melt at the highest temperature transition as well as a drop in the temperature of this transition. Circular dichroism spectra of the residual particles give ellipticity values around 275 nm, much higher than those corresponding to the control nucleosomes, which appears to indicate a loss in the compact DNA tertiary structure. When regeneration of the modified amino groups of the residual particles takes place in the presence of the complementary fraction containing histones H2A and H2B, but not in its absence, nucleosomal particles with the structural properties of the original nucleosomes are reconstituted. Therefore, the structural change observed in the residual particles can be assigned to the lack of histones H2A and H2B and not to the modified amino groups of the histones present in the residual particles. The results are consistent with the stabilization by histones H2A and H2B of a DNA length of 50-70 base pairs per nucleosome.     

Disparity in the DNA translocase domains of SWI/SNF and ISW2

An ATP-dependent DNA translocase domain consisting of seven conserved motifs is a general feature of all ATP-dependent chromatin remodelers. While motifs on the ATPase domains of the yeast SWI/SNF and ISWI families of remodelers are highly conserved, the ATPase domains of these complexes appear not to be functionally interchangeable. We found one reason that may account for this is the ATPase domains interact differently with nucleosomes even though both associate with nucleosomal DNA 17–18 bp from the dyad axis. The cleft formed between the two lobes of the ISW2 ATPase domain is bound to nucleosomal DNA and Isw2 associates with the side of nucleosomal DNA away from the histone octamer. The ATPase domain of SWI/SNF binds to the same region of nucleosomal DNA, but is bound outside of the cleft region. The catalytic subunit of SWI/SNF also appears to intercalate between the DNA gyre and histone octamer. The altered interactions of SWI/SNF with DNA are specific to nucleosomes and do not occur with free DNA. These differences are likely mediated through interactions with the histone surface. The placement of SWI/SNF between the octamer and DNA could make it easier to disrupt histone–DNA interactions.     

Tetrameric structure of centromeric nucleosomes in interphase Drosophila cells

Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3). However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical “beads-on-a-string” appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity.     

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.     

Structural changes of nucleosomal particles and isolated core-histone octamers induced by chemical modification of lysine residues

Treatment of nucleosomal particles and isolated core-histone octamers with dimethylmaleic anhydride, but not with acetic anhydride, is accompanied by a biphasic release of the two H2A.H2B dimers, the first dimer being more easily released than the second. With both kinds of particles, 50% of histones H2A and H2B are released for modification of approximately 35% of the histone amino groups. The similar behavior of nucleosomal particles and isolated core-histone octamers is consistent with the same structure of the histone octamer in the nucleosomal particle and in the free octamer in 2 M NaCl. The described release of H2A.H2B dimers allows the preparation of nucleosomal particles deficient in one H2A.H2B dimer and of the histone hexamers H2A.H2B.(H3.H4)2. For more extensive modifications, both reagents, acetic and dimethylmaleic anhydrides, cause the dissociation of nucleosomal particles with liberation of double-stranded DNA, which suggests that lysine amino groups are involved in the binding of histones to DNA. The modified nucleosomal particles are more sensitive to ionic strength than those untreated, and the presence of salt (NaCl) increases the extent of DNA release. The histones corresponding to the liberated DNA, except H2A and H2B released with dimethylmaleic anhydride, are apparently bound to the DNA-containing particles as extra histones.     

CENP-A Nucleosome is a Sensitive Allosteric Scaffold for DNA and Chromatin Factors

Centromeric loci of chromosomes are defined by nucleosomes containing the histone H3 variant CENP-A, which bind their DNA termini more permissively than their canonical counterpart, a feature that is critical for the mitotic fidelity. A recent cryo-EM study demonstrated that the DNA termini of CENP-A nucleosomes, reconstituted with the Widom 601 DNA sequence, are asymmetrically flexible, meaning one terminus is more clearly resolved than the other. However, an earlier work claimed that both ends could be resolved in the presence of two stabilizing single chain variable fragment (scFv) antibodies per nucleosome, and thus are likely permanently bound to the histone octamer. This suggests that the binding of scFv antibodies to the histone octamer surface would be associated with CENP-A nucleosome conformational changes, including stable binding of the DNA termini. Here, we present computational evidence that allows to explain at atomistic level the structural rearrangements of CENP-A nucleosomes resulting from the antibody binding. The antibodies, while they only bind the octamer façades, are capable of altering the dynamics of the nucleosomal core, and indirectly also the surrounding DNA. This effect has more drastic implications for the structure and the dynamics of the CENP-A nucleosome in comparison to its canonical counterpart. Furthermore, we find evidence that the antibodies bind the left and the right octamer façades at different affinities, another manifestation of the DNA sequence. We speculate that the cells could use induction of similar allosteric effects to control centromere function.     

Nucleosomes undergo slow spontaneous gaping

In eukaryotes, DNA is packaged into a basic unit, the nucleosome which consists of 147 bp of DNA wrapped around a histone octamer composed of two copies each of the histones H2A, H2B, H3 and H4. Nucleosome structures are diverse not only by histone variants, histone modifications, histone composition but also through accommodating different conformational states such as DNA breathing and dimer splitting. Variation in nucleosome structures allows it to perform a variety of cellular functions. Here, we identified a novel spontaneous conformational switching of nucleosomes under physiological conditions using single-molecule FRET. Using FRET probes placed at various positions on the nucleosomal DNA to monitor conformation of the nucleosome over a long period of time (30–60 min) at various ionic conditions, we identified conformational changes we refer to as nucleosome gaping. Gaping transitions are distinct from nucleosome breathing, sliding or tightening. Gaping modes switch along the direction normal to the DNA plane through about 5–10 angstroms and at minutes (1–10 min) time scale. This conformational transition, which has not been observed previously, may be potentially important for enzymatic reactions/transactions on nucleosomal substrate and the formation of multiple compression forms of chromatin fibers.     

Separation of nucleosomes containing histones H1 and H5

Hen erythrocyte chromatin was digested with staphylococcal nuclease and fractionated by electrophoresis in polyacrylamide gels. Instead of the three bands described for mouse carcinoma chromatin, four main discrete components (MN1, MN2, MN2E and MN3) were resolved in the mononucleosome fraction of erythrocyte chromatin. MN2 contained all five histones and a DNA fragment of 165–180 base pairs. MN2E comprised four nucleosomal histones plus histone H5 (but not H1) and a DNA fragment of 170–190 base pairs. The relatively nuclease resistant MN3 fraction of erythrocyte nucleosomes contained H1 but no H5 histone. A more accurate analysis of the MN2 fraction in mouse carcinoma nucleosomes revealed some additional microheterogeneity depending on the presence of two different subfractions of H1.     

Tetrameric Structure of Centromeric Nucleosomes in Interphase Drosophila Cells

Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3). However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical “beads-on-a-string” appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity.     

Nucleosomes structure and dynamics: effect of CHAPS

Dynamics of nucleosomes and spontaneous unwrapping of DNA are fundamental property of the chromatin enabling access to nucleosomal DNA for regulatory proteins. Probing of such dynamics of nucleosomes performed by single molecule techniques revealed a large scale dynamics of nucleosomes including their spontaneous unwrapping. Dissociation of nucleosomes at low concentrations is a complicating issue for studies with single molecule techniques. In this paper, we tested the ability of 3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) to prevent dissociation of nucleosomes. The study was performed with mononucleosome system assembled with human histones H2A, H2B, H3 and H4 on the DNA substrate containing sequence 601 that provides the sequencespecific assembly of nucleosomes. We used Atomic Force Microscopy (AFM) to directly identify nucleosomes and analyze their structure at the nanometer level. These studies showed that in the presence of CHAPS at millimolar concentrations, nucleosomes, even at sub-nanomolar concentrations, remain intact over days compared to a complete dissociation of the same nucleosome sample over 10 min in the absence of CHAPS. Importantly, CHAPS does not change the conformation of nucleosomes as confirmed by the AFM analysis. Moreover, 16 µM CHAPS stabilizes nucleosomes in over one hour incubation in the solution containing as low as 0.4 nM in nucleosomes. The stability of nucleosomes is slightly reduced at physiological conditions (150 mM NaCl), although the nucleosomes dissociate rapidly at 300 mM NaCl. The sequence specificity of the nucleosome in the presence of CHAPS decreased suggesting that the histone core translocates along the DNA substrate utilizing sliding mechanism.     

The histone variant macro-H2A preferentially forms "hybrid nucleosomes"

The histone domain of macro-H2A, which constitutes the N-terminal one third of this histone variant, is only 64% identical to major H2A. We have shown previously that the main structural differences in a nucleosome in which both H2A moieties have been replaced by macro-H2A reside in the only point of contact between the two histone dimers, the L1-L1 interface of macro-H2A. Here we show that the L1 loop of macro-H2A is responsible for the increased salt-dependent stability of the histone octamer, with implications for the nucleosome assembly pathway. It is unknown whether only one or both of the H2A-H2B dimers within a nucleosome are replaced with H2A variant containing nucleosomes in vivo. We demonstrate that macro-H2A preferentially forms hybrid nucleosomes containing one chain each of major H2A and macro-HA in vitro. The 2.9-A crystal structure of such a hybrid nucleosome shows significant structural differences in the L1-L1 interface when comparing with homotypic major H2A- and macro-H2A-containing nucleosomes. Both homotypic and hybrid macro-nucleosome core particles (NCPs) are resistant to chaperone-assisted H2A-H2B dimer exchange. Together, our findings suggest that the histone domain of macro-H2A modifies the dynamic properties of the nucleosome. We propose that the possibility of forming hybrid macro-NCP adds yet another level of complexity to variant nucleosome structure and function.     

Unfolding of nucleosome cores induced by chemical acetylation of histones

Relative accessibility of nucleosomal histones to acetic anhydride during acetylation has been studied as a function of concentration, pH and ionic strength of the solution using high-resolution gel-electrophoresis. It was shown that about 80% of lysine residues in nucleosomal histones and 100% of the same residues in histone complexes without DNA in 2 M NaCl are accessible to the modification, which is proved by the localization of the majority of lysine residues in nucleosomes near the surface of the histone octamer, by their participation in ionic interactions with DNA and, probably, in histone-histone contacts. Gel-electrophoretic experiments with nucleosomes and studies of the histone resistance to mild trypsinolysis indicated that neither nucleosomes themselves nor histone octamers are affected even though 50% of lysine residues in histones have been acetylated. The process of acetylation is accompanied by the growing tendency of histones to participate in mild trypsinolysis and by a gradual decline in electrophoretic mobility and in the value of the sedimentation constant. The circular dichroism spectra and the microscopic appearance of nucleosomes are also markedly changed. These results suggest that a gradual unfolding of nucleosomes occurs when 5 or more lysine residues in the nucleosomal histones have been acetylated.     

The mechanics behind DNA sequence-dependent properties of the nucleosome

Chromatin organization and composition impart sophisticated regulatory features critical to eukaryotic genomic function. Although DNA sequence-dependent histone octamer binding is important for nucleosome activity, many aspects of this phenomenon have remained elusive. We studied nucleosome structure and stability with diverse DNA sequences, including Widom 601 derivatives with the highest known octamer affinities, to establish a simple model behind the mechanics of sequence dependency. This uncovers the unique but unexpected role of TA dinucleotides and a propensity for G|C-rich sequence elements to conform energetically favourably at most locations around the histone octamer, which rationalizes G|C% as the most predictive factor for nucleosome occupancy in vivo. In addition, our findings reveal dominant constraints on double helix conformation by H3-H4 relative to H2A-H2B binding and DNA sequence context-dependency underlying nucleosome structure, positioning and stability. This provides a basis for improved prediction of nucleosomal properties and the design of tailored DNA constructs for chromatin investigations.