Histone acetylation reduces nucleosome core particle linking number change

Nucleosome core particles differing in their levels of histone acetylation have been formed on a closed circular DNA that contains a tandemly repeated 207 bp nucleosome positioning sequence. The effect of acetylation on the linking number per nucleosome particle has been determined. With increasing levels of acetylation, the negative linking number change per nucleosome decreases from -1.04 +/- 0.08 for control to -0.82 +/- 0.05 for highly acetylated nucleosomes. These results indicate that histone acetylation has the ability to release negative supercoils previously constrained by nucleosomes into a closed chromatin loop and in effect function as a eukaryotic gyrase.     

Characterization of histone H2A and H2B variants and their post-translational modifications by mass spectrometry

The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2A and H2B variants derived from Jurkat cells. A combination of mass spectrometric techniques, HPLC separations, and enzymatic digestions using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin were used to identify histone H2A and H2B subtypes and their modifications. We identified nine histone H2A and 11 histone H2B subtypes, among them proteins that only had been postulated at the gene level. The two main H2A variants, H2AO and H2AC, as well as H2AL were either acetylated at Lys-5 or phosphorylated at Ser-1. For the replacement histone H2AZ, acetylation at Lys-4 and Lys-7 was found. The main histone H2B variant, H2BA, was acetylated at Lys-12, -15, and -20. The analysis of core histone subtypes with their modifications provides a first step toward an understanding of the functional significance of the diversity of histone structures.     

Model of the packing of DNA and histone octamer in the structure of the nucleosome

A model is proposed which describes the packing of polypeptide chains of histone molecules in the octamer (H3--H4--H2A--H2B)2, and interlocation of DNA and octamer in the nucleosome. DNA packing in the nucleosome is provided for by electrostatic interactions between DNA phosphates and cationic groups located on the globular part surface of histones octamer. The cationic groups of N- and C-end regions of the histone molecules (histones H3 and H4 in particular) additionally stabilize the nucleosome structure.     

Chromatin compaction at the mononucleosome level

Using a previously described FRET technique, we measured the distance between the ends of DNA fragments on which nucleosomes were reconstituted from recombinant and native histones. This distance was analyzed in its dependence on the DNA fragment length, concentration of mono- and divalent counterions, presence of linker histone H1, and histone modifications. We found that the linker DNA arms do not cross under all conditions studied but diverge slightly as they leave the histone core surface. Histone H1 leads to a global approach of the linker DNA arms, confirming the notion of a "stem structure". Increasing salt concentration also leads to an approach of the linker DNAs. To study the effect of acetylation, we compared chemically acetylated recombinant histones with histones prepared from HeLa cells, characterizing the sites of acetylation by mass spectroscopy. Nucleosomes from chemically acetylated histones have few modifications in the core domain and form nucleosomes normally. Acetylating all histones or selectively only H3 causes an opening of the nucleosome structure, indicated by the larger distances between the linker DNA ends. Selective acetylation of H4 distances the linker ends for short fragments but causes them to approach each other for fragments longer than 180 bp.     

Specific contributions of histone tails and their acetylation to the mechanical stability of nucleosomes

The distinct contributions of histone tails and their acetylation to nucleosomal stability were examined by mechanical disruption of individual nucleosomes in a single chromatin fiber using an optical trap. Enzymatic removal of H2A/H2B tails primarily decreased the strength of histone-DNA interactions located approximately +/-36bp from the dyad axis of symmetry (off-dyad strong interactions), whereas removal of the H3/H4 tails played a greater role in regulating the total amount of DNA bound. Similarly, nucleosomes composed of histones acetylated to different degrees by the histone acetyltransferase p300 exhibited significant decreases in the off-dyad strong interactions and the total amount of DNA bound. Acetylation of H2A/H2B appears to play a particularly critical role in weakening the off-dyad strong interactions. Collectively, our results suggest that the destabilizing effects of tail acetylation may be due to elimination of specific key interactions in the nucleosome.     

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.     

Opposing roles of H3- and H4-acetylation in the regulation of nucleosome structure—a FRET study

Using FRET in bulk and on single molecules, we assessed the structural role of histone acetylation in nucleosomes reconstituted on the 170 bp long Widom 601 sequence. We followed salt-induced nucleosome disassembly, using donor–acceptor pairs on the ends or in the internal part of the nucleosomal DNA, and on H2B histone for measuring H2A/H2B dimer exchange. This allowed us to distinguish the influence of acetylation on salt-induced DNA unwrapping at the entry–exit site from its effect on nucleosome core dissociation. The effect of lysine acetylation is not simply cumulative, but showed distinct histone-specificity. Both H3- and H4-acetylation enhance DNA unwrapping above physiological ionic strength; however, while H3-acetylation renders the nucleosome core more sensitive to salt-induced dissociation and to dimer exchange, H4-acetylation counteracts these effects. Thus, our data suggest, that H3- and H4-acetylation have partially opposing roles in regulating nucleosome architecture and that distinct aspects of nucleosome dynamics might be independently controlled by individual histones.     

Acetylation of histone H3 lysine 56 regulates replication-coupled nucleosome assembly

Chromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. Here we show that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106.     

A statistical thermodynamic model applied to experimental AFM population and location data is able to quantify DNA-histone binding strength and internucleosomal interaction differences between acetylated and unacetylated nucleosomal arrays

Imaging of nucleosomal arrays by atomic force microscopy allows a determination of the exact statistical distributions for the numbers of nucleosomes per array and the locations of nucleosomes on the arrays. This precision makes such data an excellent reference for testing models of nucleosome occupation on multisite DNA templates. The approach presented here uses a simple statistical thermodynamic model to calculate theoretical population and positional distributions and compares them to experimental distributions previously determined for 5S rDNA nucleosomal arrays (208-12,172-12). The model considers the possible locations of nucleosomes on the template, and takes as principal parameters an average free energy of interaction between histone octamers and DNA, and an average wrapping length of DNA around the octamers. Analysis of positional statistics shows that it is possible to consider interactions between nucleosomes and positioning effects as perturbations on a random positioning noninteracting model. Analysis of the population statistics is used to determine histone-DNA association constants and to test for differences in the free energies of nucleosome formation with different types of histone octamers, namely acetylated or unacetylated, and different DNA templates, namely 172-12 or 208-12 5S rDNA multisite templates. The results show that the two template DNAs bind histones with similar affinities but histone acetylation weakens the association of histones with both templates. Analysis of locational statistics is used to determine the strength of specific nucleosome positioning tendencies by the DNA templates, and the strength of the interactions between neighboring nucleosomes. The results show only weak positioning tendencies and that unacetylated nucleosomes interact much more strongly with one another than acetylated nucleosomes; in fact acetylation appears to induce a small anticooperative occupation effect between neighboring nucleosomes.     

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.     

Nucleosome linking number change controlled by acetylation of histones H3 and H4

High levels of acetylation of lysines in the amino-terminal domains of all four core histones, H2A, H2B, H3, and H4, have been shown to reduce the linking number change per nucleosome core particle in reconstituted minichromosomes (Norton, V. G., Imai, B. S., Yau, P., and Bradbury, E. M. (1989) Cell 57, 449-457). Because there is evidence to suggest that the acetylations of H3 and H4 have functions that are distinct from those of H2A and H2B, we have determined the nucleosome core particle linking number change in minichromosomes containing fully acetylated H3 and H4 and very low levels of acetylation in H2A and H2B. This linking number change was -0.81 +/- 0.05, in close agreement with the linking number change for hyperacetylated nucleosome core particles which contain high levels of acetylation in all four core histones (approximately 70% of full acetylation in H3 and H4). Therefore, high levels of acetylation of H3 and H4 alone are responsible for the reduction in the linking number change per nucleosome core particle.     

Nucleosome recognition by the Piccolo NuA4 histone acetyltransferase complex

The mechanisms by which multisubunit histone acetyltransferase (HAT) complexes recognize and perform efficient acetylation on nucleosome substrates are largely unknown. Here, we use a variety of biochemical approaches and compare histone-based substrates of increasing complexity to determine the critical components of nucleosome recognition by the MOZ, Ybf2/Sas3, Sas2, Tip60 family HAT complex, Piccolo NuA4 (picNuA4). We find the histone tails to be dispensable for binding to both nucleosomes and free histones and that the H2A, H3, and H2B tails do not influence the ability of picNuA4 to tetra-acetylate the H4 tail within the nucleosome. Most notably, we discovered that the histone-fold domain (HFD) regions of histones, particularly residues 21-52 of H4, are critical for tight binding and efficient tail acetylation. Presented evidence suggests that picNuA4 recognizes the open surface of the nucleosome on which the HFD of H4 is located. This binding mechanism serves to direct substrate access to the tails of H4 and H2A and allows the enzyme to be "tethered", thereby increasing the effective concentration of the histone tail and permitting successive cycles of H4 tail acetylation.     

Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains

Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 ? resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q. These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.