A critical epitope for substrate recognition by the nucleosome remodeling ATPase ISWI

The ATPase ISWI is the catalytic core of several nucleosome remodeling complexes, which are able to alter histone–DNA interactions within nucleosomes such that the sliding of histone octamers on DNA is facilitated. Dynamic nucleosome repositioning may be involved in the assembly of chromatin with regularly spaced nucleosomes and accessible regulatory sequence elements. The mechanism that underlies nucleosome sliding is largely unresolved. We recently discovered that the N-terminal ‘tail’ of histone H4 is critical for nucleosome remodeling by ISWI. If deleted, nucleosomes are no longer recognized as substrates and do not stimulate the ATPase activity of ISWI. We show here that the H4 tail is part of a more complex recognition epitope which is destroyed by grafting the H4 N-terminus onto other histones. We mapped the H4 tail requirement to a hydrophilic patch consisting of the amino acids R₁₇H₁₈R₁₉ localized at the base of the tail. These residues have been shown earlier to contact nucleosomal DNA, suggesting that ISWI recognizes an ‘epitope’ consisting of the DNA-bound H4 tail. Consistent with this hypothesis, the ISWI ATPase is stimulated by isolated H4 tail peptides ISWI only in the presence of DNA. Acetylation of the adjacent K₁₂ and K₁₆ residues impairs substrate recognition by ISWI.     

Stability of nucleosome placement in newly repaired regions of DNA

Rearrangements of chromatin structure during excision repair of UV-damaged DNA appear to involve unfolding of nucleosomal DNA while repair is taking place, followed by refolding of this DNA into a native nucleosome structure. Recently, we found that repair patches are not distributed uniformly along the DNA in nucleosome core particles immediately following their refolding into nucleosomes (Lan, S. Y., and Smerdon, M. J. (1985) Biochemistry, 24,7771). Therefore, the distribution of repair patches in nucleosome core DNA was used to monitor the stability of nucleosome placement in these regions. Our results indicate that in nondividing human cells undergoing excision repair there is a slow change in the positioning of nucleosomes in newly repaired regions of chromatin, resulting in the eventual randomization of repair patches in nucleosome core DNA. Furthermore, the nonrandom placement of nucleosomes observed just after the refolding event is not re-established during DNA replication. Possible mechanisms for this change in nucleosome placement along the DNA are discussed.     

Regulation of histone synthesis and nucleosome assembly

Histone deposition onto nascent DNA is the first step in the process of chromatin assembly during DNA replication. The process of nucleosome assembly represents a daunting task for S-phase cells, partly because cells need to rapidly package nascent DNA into nucleosomes while avoiding the generation of excess histones. Consequently, cells have evolved a number of nucleosome assembly factors and regulatory mechanisms that collectively function to coordinate the rates of histone and DNA synthesis during both normal cell cycle progression and in response to conditions that interfere with DNA replication.     

DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila

Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC.     

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.     

The incorporation of the novel histone variant H2AL2 confers unusual structural and functional properties of the nucleosome

In this work we have studied the properties of the novel mouse histone variant H2AL2. H2AL2 was used to reconstitute nucleosomes and the structural and functional properties of these particles were studied by a combination of biochemical approaches, atomic force microscopy (AFM) and electron cryo-microscopy. DNase I and hydroxyl radical footprinting as well as micrococcal and exonuclease III digestion demonstrated an altered structure of the H2AL2 nucleosomes all over the nucleosomal DNA length. Restriction nuclease accessibility experiments revealed that the interactions of the H2AL2 histone octamer with the ends of the nucleosomal DNA are highly perturbed. AFM imaging showed that the H2AL2 histone octamer was complexed with only ~130 bp of DNA. H2AL2 reconstituted trinucleosomes exhibited a type of a ‘beads on a string’ structure, which was quite different from the equilateral triangle 3D organization of conventional H2A trinucleosomes. The presence of H2AL2 affected both the RSC and SWI/SNF remodeling and mobilization of the variant particles. These unusual properties of the H2AL2 nucleosomes suggest a specific role of H2AL2 during mouse spermiogenesis.     

Histone protein surface accessibility dictates direction of RSC-dependent nucleosome mobilization

Chromatin remodeling enzymes use energy derived from ATP hydrolysis to mobilize nucleosomes and alter their structure to facilitate DNA access. The Remodels the Structure of Chromatin (RSC) complex has been extensively studied, yet aspects of how this complex functionally interacts with nucleosomes remain unclear. We introduce a steric mapping approach to determine how RSC activity depends on interaction with specific surfaces within the nucleosome. We find that blocking SHL + 4.5/–4.5 via streptavidin binding to the H2A N-terminal tail domains results in inhibition of RSC nucleosome mobilization. However, restriction enzyme assays indicate that remodeling-dependent exposure of an internal DNA site near the nucleosome dyad is not affected. In contrast, occlusion of both protein faces of the nucleosome by streptavidin attachment near the acidic patch completely blocks both remodeling-dependent nucleosome mobilization and internal DNA site exposure. However, we observed partial inhibition when only one protein surface is occluded, consistent with abrogation of one of two productive RSC binding orientations. Our results indicate that nucleosome mobilization requires RSC access to the trailing but not the leading protein surface, and reveals a mechanism by which RSC and related complexes may drive unidirectional movement of nucleosomes to regulate cis-acting DNA sequences in vivo.     

Chromatin assembled in the presence of cytosine arabinoside has a short nucleosome repeat.

Incubation of MSB cells with cytosine arabinoside (1-beta-D-arabinofuranosylcytosine, ara-C) inhibits 3H-thymidine incorporation into nascent DNA while nucleosome core histone synthesis proceeds in molar stoichiometry at about 20% of control rates. The excess nascent histone is incorporated into chromatin and nucleosome cores are assembled normally on the small amount of DNA which is synthesized at submaximal levels of ara-C. This DNA becomes packaged into a shortened nucleosome repeat, however. These results indicate that the nucleosome core is a strongly conserved unit of chromatin replication and suggest that the stoichiometry of nascent histone to DNA may be one factor influencing the establishment of the nucleosome repeat length. It cannot be the only factor, however, since the closely packed nucleosomes made in the presence of ara-C begin to return to their normal spacing within six hours after reversal.     

Theoretical analysis of epigenetic cell memory by nucleosome modification

Chromosomal regions can adopt stable and heritable alternative states resulting in bistable gene expression without changes to the DNA sequence. Such epigenetic control is often associated with alternative covalent modifications of histones. The stability and heritability of the states are thought to involve positive feedback where modified nucleosomes recruit enzymes that similarly modify nearby nucleosomes. We developed a simplified stochastic model for dynamic nucleosome modification based on the silent mating-type region of the yeast Schizosaccharomyces pombe. We show that the mechanism can give strong bistability that is resistant both to high noise due to random gain or loss of nucleosome modifications and to random partitioning upon DNA replication. However, robust bistability required: (1) cooperativity, the activity of more than one modified nucleosome, in the modification reactions and (2) that nucleosomes occasionally stimulate modification beyond their neighbor nucleosomes, arguing against a simple continuous spreading of nucleosome modification.     

The coexistence of the nucleosome positioning code with the genetic code on eukaryotic genomes

It is known that there are several codes residing simultaneously on the DNA double helix. The two best-characterized codes are the genetic code—the code for protein production, and the code for DNA packaging into nucleosomes. Since these codes have to coexist simultaneously on the same DNA region, both must be degenerate to allow this coexistence. A-tracts are homopolymeric stretches of several adjacent deoxyadenosines on one strand of the double helix, having unusual structural properties, which were shown to exclude nucleosomes and as such are instrumental in setting the translational positioning of DNA within nucleosomes. We observe, cross-kingdoms, a strong codon bias toward the avoidance of long A-tracts in exon regions, which enables the formation of high density of nucleosomes in these regions. Moreover, long A-tract avoidance is restricted exclusively to nucleosome-occupied exon regions. We show that this bias in codon usage is sufficient for enabling DNA organization within nucleosomes without constraints on the actual code for proteins. Thus, there is inter-dependency of the two major codes within DNA to allow their coexistence. Furthermore, we show that modulation of A-tract occurrences in exon versus non-exon regions may result in a unique alternation of the diameter of the ‘30-nm’ fiber model.