Simulations of SIN mutations and histone variants in human nucleosomes reveal altered protein-DNA and core histone interactions

Alterations in the stability of a nucleosome exert predominant influence on chromatin structure and eukaryotic gene expression. In an attempt to investigate the mononucleosome stability using computational approaches, we have simulated the structure of a human mononucleosome and have compared their energies under the influence of core mutations, tail substitutions, variant histones, and orthologs. We observe that mutant nucleosomes carrying SIN (SWI Independent) mutations do not alter the overall nucleosomal structure but cause local structural changes leading to significant changes in energy and hence the stability. We observe that the nucleosome stability is altered by the substitution of only certain critical lysine residues on the H3 tails. Interestingly, the incorporation of variants H2A.Z and H3.3 lower nucleosome stability as evidenced by small energy changes. However, the substitution of histone orthologs did not alter structural stability. Our simulations to determine the nucleosome stability using energy trends emphasize the role of mutations, variants, and orthologs as determinants of chromatin structure at the nucleosome core particle level. The destabilization we observe on the human nucleosome with core mutations show similar trends of instability as validated experimentally in yeast.     

Availability of hyperacetylated H4 histone in intact nucleosomes to specific antibodies

Specific antibodies against the tetra-acetylated form of H4 histone have been elicited in the rabbit. They do not cross-react with the non-, mono-, and di-acetylated forms of the histone molecule but a slight cross-reactivity with the tri-acetylated form of H4 histone is observed. Our studies also show that hyperacetylated H4 histones are recognized by the antibodies in intact nucleosomes.     

Reassociation of histone H1 with nucleosomes.

The role of histone H1 in nucleosome heterogeneity and structure has been studied using a reconstitution procedure. Histone H1 and non-histone proteins are removed selectively from enzymatically fragmented chromatin by Dowex 50W-X2 treatment. The resulting "stripped" chromatin then is reassociated with purified histone H1 using step gradient dialysis. Material reconstituted in this manner was examined by gel electrophoresis, protein cross-linking, and chromatin fingerprinting. The results demonstrate that the histone H1 molecule efficiently binds to nucleosomes with fidelity in an apparent noncooperative manner. Polynucleosomes possess two specific binding sites for histone H1 per histone octamer; the first binding site is of higher affinity than the second. The 160-base pair nuclease digestion barrier and nucleosome electrophoretic class (MIII)n are established upon binding the 1st histone H1 molecule. Upon binding the 2nd histone H1 molecule, polynucleosomes assume a highly compact conformation. The experimental approach introduced here should permit determining whether nucleosomes possess independent specific binding sites for other chromosomal proteins, and should allow reconstitution of the other electrophoretic forms of nucleosomes which we have described previously.     

Absence of histone H2B in nucleosomes containing histone TH2B and interaction of immunoglobulin with nucleosomes

Nucleosomes containing histone TH2B were isolated from chromatin subunits of rat testis nuclei (MNT) by incubating with anti-TH2B immunoglobulin (IgTH2B) which was covalently attached to agarose gels. Electrophoretic separation of histones of these isolated nucleosomes revealed that histone H2B was completely absent, suggesting that histone TH2B, the variant of H2B, existed in nucleosomes only as TH2B X TH2B and that TH2B X H2B was not likely to exist in chromatin. Sucrose gradient ultracentrifugation of mixtures of MNT and IgTH2B revealed that when excess amounts of immunologically active IgTH2B were present, complexes of higher sedimentation coefficients than MNT X IgTH2B were formed, but with limited amounts of active IgTH2B, only MNT X IgTH2B was formed. When purified IgTH2B was coated on polystyrene tubes and incubated with MNT, those MNT immobilized by the tube-coated IgTH2B adsorbed IgTH2B from diluted antiserum during subsequent incubation. Those results suggested the absence of steric hindrance in the binding of IgTH2B to MNT X IgTH2B. When MNT was coated on polystyrene tubes and incubated with DNase and then with dilute anti-TH2B antiserum, it was found that DNase digestion increased the binding of immunoglobulin to the tubes approximately 76%. Interaction of chromatin subunits of rat liver nuclei (MNL) with anti-TH2B antiserum was negligible, but DNase digestion of MNL coated on tubes was followed by considerable interaction with anti-TH2B antiserum. Those results indicated DNase unmasked at least part of the determinants encased by DNA. Anti-H2B immunoglobulin (IgH2B) interacted with histone H2B and TH2B to the same extent, and interacted significantly to a lesser extent with either MNT or MNL. DNase digestion of MNT and MNL increased binding of IgH2B approximately 170 and 117%, respectively.     

H3.H4 tetramer directs DNA and core histone octamer assembly in the nucleosome core particle.

The way in which histones interact with DNA during in vitro assembly of nucleohistone has been examined. Chicken erythrocyte core histones H2A, H2B, H3, and H4 and lambdaDNA in 2 M NaCl were allowed to interact by stepwise decrease in the salt concentration. Binding, although weak, was first observed at 1.4 M NaCl and was essentially completed at 0.6 M NaCl. Analysis of the DNA-bound histones revealed that each of the histones in the pairs H2A,H2B and H3,H4 was always present in equimolar amounts and that the relative proportion of each pair was constant between 1.4 and 0.8 M NaCl. Evidence is presented suggesting that binding occurred via complexes of the four histones, the nature of which is likely to reflect the equilibrium among the octamer and its products of dissociation (Ruiz-Carrillo, A., & Jorcano, J.L. (1979) Biochemistry (preceding paper in this issue)). The presence of complexes of the four core histones is, however not required for the correct assembly of the nucleosome core particle. Nucleohistones obtained by adding at progressively lower ionic strengths the dimer H2A.H2B to the H3.H4-DNA complex (split reconstitutions) had the same characteristics as those assembled with the core histone complexes.     

The role of histone H1 and non-structured domains of core histones in maintaining the orientation of nucleosomes within the chromatin fiber

Calf thymus chromatin was digested with trypsin and the structural alterations which occurred were followed by flow linear dichroism. After a sharp initial increase, the amplitude of the positive signal gradually decreased followed by a change of the sign of the dichroism and further increase of the negative signal up to a plateau. These changes of the dichroism were compared to the respective changes in the histone pattern. It was shown that the positive dichroism of chromatin did not depend on the condensation state of chromatin, and that the orientation of the nucleosomes along the chromatin fiber was maintained by the globular domain of H1 and the non-structured parts of core histones.     

Influence of histone tails and H4 tail acetylations on nucleosome-nucleosome interactions

Nucleosome–nucleosome interaction plays a fundamental role in chromatin folding and self-association. The cation-induced condensation of nucleosome core particles (NCPs) displays properties similar to those of chromatin fibers, with important contributions from the N-terminal histone tails. We study the self-association induced by addition of cations [Mg²⁺, Ca²⁺, cobalt(III)hexammine³⁺, spermidine³⁺ and spermine⁴⁺] for NCPs reconstituted with wild-type unmodified histones and with globular tailless histones and for NCPs with the H4 histone tail having lysine (K) acetylations or lysine-to-glutamine mutations at positions K5, K8, K12 and K16. In addition, the histone construct with the single H4K16 acetylation was investigated. Acetylated histones were prepared by a semisynthetic native chemical ligation method. The aggregation behavior of NCPs shows a general cation-dependent behavior similar to that of the self-association of nucleosome arrays. Unlike nucleosome array self-association, NCP aggregation is sensitive to position and nature of the H4 tail modification. The tetra-acetylation in the H4 tail significantly weakens the nucleosome–nucleosome interaction, while the H4 K → Q tetra-mutation displays a more modest effect. The single H4K16 acetylation also weakens the self-association of NCPs, which reflects the specific role of H4K16 in the nucleosome–nucleosome stacking. Tailless NCPs can aggregate in the presence of oligocations, which indicates that attraction also occurs by tail-independent nucleosome–nucleosome stacking and DNA–DNA attraction in the presence of cations. The experimental data were compared with the results of coarse-grained computer modeling for NCP solutions with explicit presence of mobile ions.     

Selective removal of histone H1 from nucleosomes at low ionic strength

The method for removal of histone H 1 from chromatin by treatment with ion-exchange resin AG 50 WX 2 in the presence of 100 mM NaCl and 50 mM phosphate buffer (Thoma and Koller, 1977, Cell, 12, 101–107) results in production not only of H1-depleted chromatin but also free DNA. We have now modified this procedure so that the nucleosome is treated with the cation exchange resin in two steps, first in 50 mM sodium phosphate buffer and then in 50 mM sodium phosphate and 50 mM NaCl whereby histone H 1 is selectively removed without a release of free DNA at low resin concentrations.Abbreviations NaP Sodium phosphate buffer of molarities and pH as stated in the text - SDS Sodium dodecyl sulfate     

H3 and H4 histone tails play a central role in the interactions of recombinant NCPs

Using small-angle x-ray scattering, we probe the effect of histone tails on both internucleosomal interactions and nucleosome conformation. To get insight into the specific role of H3 and H4 histone tails, perfectly monodisperse recombinant nucleosome core particles were reconstituted, either intact or deprived of both H3 and H4 histone tails (gH3gH4). The main result is that H3 and H4 histone tails are necessary to induce attractive interactions between NCPs. A pair potential model was used to describe interactions between NCPs. At all salt concentrations, interactions between gH3gH4 NCPs are best described by repulsive interactions exclusively. For intact NCPs, an additional attractive term, with a 5–10 kT magnitude and 20 Å range, is required to account for interparticle interactions above 50 mM monovalent salt. Regarding conformation, intact NCPs in solution are similar to NCPs in 3D crystals. gH3gH4 NCPs instead give rise to slightly different small-angle x-ray scattering curves that can be understood as a more opened conformation of the particle, where DNA ends are slightly detached from the core.     

Phosphoacetylation of histone H3 on c-fos- and c-jun-associated nucleosomes upon gene activation

The induction of immediate-early (IE) genes, including proto-oncogenes c-fos and c-jun, correlates well with a nucleosomal response, the phosphorylation of histone H3 and HMG-14 mediated via extracellular signal regulated kinase or p38 MAP kinase cascades. Phosphorylation is targeted to a minute fraction of histone H3, which is also especially susceptible to hyperacetylation. Here, we provide direct evidence that phosphorylation and acetylation of histone H3 occur on the same histone H3 tail on nucleosomes associated with active IE gene chromatin. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies that specifically recognize the doubly-modified phosphoacetylated form of histone H3. Analysis of the associated DNA shows that histone H3 on c-fos- and c-jun-associated nucleosomes becomes doubly-modified, the same H3 tails becoming both phosphorylated and acetylated, only upon gene activation. This study reveals potential complications of occlusion when using site-specific antibodies against modified histones, and shows also that phosphorylated H3 is more sensitive to trichostatin A (TSA)-induced hyperacetylation than non-phosphorylated H3. Because MAP kinase-mediated gene induction is implicated in controlling diverse biological processes, histone H3 phosphoacetylation is likely to be of widespread significance.     

Effects of histone acetylation by Piccolo NuA4 on the structure of a nucleosome and the interactions between two nucleosomes

We characterized the effect of histone acetylation on the structure of a nucleosome and the interactions between two nucleosomes. In this study, nucleosomes reconstituted with the Selex "Widom 601" sequence were acetylated with the Piccolo NuA4 complex, which acetylates mainly H4 N-terminal tail lysine residues and some H2A/H3 N-terminal tail lysine residues. Upon the acetylation, we observed directional unwrapping of nucleosomal DNA that accompanies topology change of the DNA. Interactions between two nucleosomes in solution were also monitored to discover multiple transient dinucleosomal states that can be categorized to short-lived and long-lived (～1 s) states. The formation of dinucleosomes is strongly Mg(2+)-dependent, and unacetylated nucleosomes favor the formation of long-lived dinucleosomes 4-fold as much as the acetylated ones. These results suggest that the acetylation of histones by Piccolo NuA4 disturbs not only the structure of a nucleosome but also the interactions between two nucleosomes. Lastly, we suggest a structural model for a stable dinucleosomal state where the two nucleosomes are separated by ～2 nm face-to-face and rotated by 34° with respect to each other.     

Human histone acetyltransferase 1 protein preferentially acetylates H4 histone molecules in H3.1-H4 over H3.3-H4

In mammalian cells, canonical histone H3 (H3.1) and H3 variant (H3.3) differ by five amino acids and are assembled, along with histone H4, into nucleosomes via distinct nucleosome assembly pathways. H3.1-H4 molecules are assembled by histone chaperone CAF-1 in a replication-coupled process, whereas H3.3-H4 are assembled via HIRA in a replication-independent pathway. Newly synthesized histone H4 is acetylated at lysine 5 and 12 (H4K5,12) by histone acetyltransferase 1 (HAT1). However, it remains unclear whether HAT1 and H4K5,12ac differentially regulate these two nucleosome assembly processes. Here, we show that HAT1 binds and acetylates H4 in H3.1-H4 molecules preferentially over H4 in H3.3-H4. Depletion of Hat1, the catalytic subunit of HAT1 complex, results in reduced H3.1 occupancy at H3.1-enriched genes and reduced association of Importin 4 with H3.1, but not H3.3. Finally, depletion of Hat1 or CAF-1p150 leads to changes in expression of a H3.1-enriched gene. These results indicate that HAT1 differentially impacts nucleosome assembly of H3.1-H4 and H3.3-H4.