Nucleosome Positioning,Nucleosome Spacing and the Nucleosome Code

Abstract

Nucleosome positioning has been the subject of intense study for many years. The properties of micrococcal nuclease, the enzyme central to these studies, are discussed. The various methods used to determine nucleosome positions in vitro and in vivo are reviewed critically. These include the traditional low resolution method of indirect end-labelling, high resolution methods such as primer extension, monomer extension and nucleosome sequencing, and the high throughput methods for genome-wide analysis (microarray hybridisation and parallel sequencing). It is established that low resolution mapping yields an averaged chromatin structure, whereas high resolution mapping reveals the weighted superposition of all the chromatin states in a cell population. Mapping studies suggest that yeast DNA contains information specifying the positions of nucleosomes and that this code is made use of by the cell. It is proposed that the positioning code facilitates nucleosome spacing by encoding information for multiple alternative overlapping nucleosomal arrays. Such a code might facilitate the shunting of nucleosomes from one array to another by ATP-dependent chromatin remodelling machines.     

Nucleosome recognition and spacing by chromatin remodelling factor ISW1a

Nucleosomes are actively positioned along DNA by ATP-dependent, chromatin remodelling factors. A structural model for the ISW1a chromatin remodelling factor from Saccharomyces cerevisiae in complex with a dinucleosome substrate was constructed from the X-ray structures of ISW1a (ΔATPase) with and without DNA bound, two different cryo-EM (cryo-electron microscopy) structures of ISW1a (ΔATPase) bound to a nucleosome, and site-directed photo-cross-linking analyses in solution. The X-ray structure of ISW1a (ΔATPase) with DNA bound suggests that DNA sequence may be involved in nucleosome recognition and thereby specificity of promoter interaction. The model suggests how the highly ordered nucleosome arrays observed by mapping nucleosomes in genes and their promoter regions could be generated by a chromatin remodelling factor.     

Reading sequence-directed computational nucleosome maps

Recently developed latest version of the sequence-directed single-base resolution nucleosome mapping reveals existence of strong nucleosomes and chromatin columnar structures (columns). Broad application of this simple technique for further studies of chromatin and chromosome structure requires some basic understanding as to how it works and what information it affords. The paper provides such an introduction to the method. The oscillating maps of singular nucleosomes, of short and long oligonucleosome columns, are explained, as well as maps of chromatin on satellite DNA and occurrences of counter-phase (antiparallel) nucleosome neighbors.     

Analysis of Chromatin Structure in the Control Regions of the <Emphasis Type="Italic">Chlamydomonas HSP70A</Emphasis> and <Emphasis Type="Italic">RBCS2</Emphasis> Genes

We have used DNaseI and micrococcal nuclease sensitivity assays to determine the chromatin structures in the control regions of the Chlamydomonas reinhardtii HSP70A and RBCS2 genes. Both genes appear to be organized into nucleosome arrays, which exhibit shorter nucleosome repeat lengths than bulk chromatin. In HSP70A we have identified up to four confined DNaseI hypersensitive sites, three of them localize to the promoter region, a fourth one to the fourth intron. Three hypersensitive sites map close to putative heat shock elements, one close to a CCAAT-box. All hypersensitive sites are located to internucleosomal linkers. Alternative nucleosome positions at half-nucleosomal phasing were constitutively detected in the HSP70A promoter region, indicating local chromatin remodelling. Upon heat shock, dramatic changes in the nucleosome structure of HSP70A were detected that particularly affected the promoter, but also a region within the fourth intron. In contrast, light induction entailed no change in HSP70A chromatin. In the RBCS2 control region we identified a strong DNaseI hypersensitive site that maps close to a CCAAT-box. This site forms the boundary of a nucleosome array with a region of ~700 bp apparently devoid of nucleosomes. This study demonstrates that chromatin structure may be determined readily at fairly high resolution in Chlamydomonas, suggesting this organism as a well-suited model for studying the role of chromatin structure on gene expression in photosynthetic eukaryotes.     

Sin mutations alter inherent nucleosome mobility

Previous studies have identified sin mutations that alleviate the requirement for the yeast SWI/SNF chromatin remodelling complex, which include point changes in the yeast genes encoding core histones. Here we characterise the biochemical properties of nucleosomes bearing these mutations. We find that sin mutant nucleosomes have a high inherent thermal mobility. As the SWI/SNF complex can alter nucleosome positioning, the higher mobility of sin mutant nucleosomes provides a means by which sin mutations may substitute for SWI/SNF function. The location of sin mutations also provides a new opportunity for insights into the mechanism for nucleosome mobilisation. We find that both mutations altering histone DNA contacts at the nucleosome dyad and mutations in the dimer-tetramer interface influence nucleosome mobility. Furthermore, incorporation of H2A.Z into nucleosomes, which also alters dimer-tetramer interactions, affects nucleosome mobility. Thus, variation of histone sequence or subtype provides a means by which eukaryotes may regulate access to chromatin through alterations to nucleosome mobility.     

DNA sequence plays a major role in determining nucleosome positions in yeast CUP1 chromatin.

The role of DNA sequence in determining nucleosome positions in vivo was investigated by comparing the positions adopted by nucleosomes reconstituted on a yeast plasmid in vitro using purified core histones with those in native chromatin containing the same DNA, described previously. Nucleosomes were reconstituted on a 2.5 kilobase pair DNA sequence containing the yeast TRP1ARS1 plasmid with CUP1 as an insert (TAC-DNA). Multiple, alternative, overlapping nucleosome positions were mapped on TAC-DNA. For the 58 positioned nucleosomes identified, the relative positioning strengths and the stabilities to salt and temperature were determined. These positions were, with a few exceptions, identical to those observed in native, remodeled TAC chromatin containing an activated CUP1 gene. Only some of these positions are utilized in native, unremodeled chromatin. These observations suggest that DNA sequence is likely to play a very important role in positioning nucleosomes in vivo. We suggest that events occurring in yeast CUP1 chromatin determine which positions are occupied in vivo and when they are occupied.     

Predicting nucleosome positions on the DNA: combining intrinsic sequence preferences and remodeler activities

Nucleosome positions on the DNA are determined by the intrinsic affinities of histone proteins to a given DNA sequence and by the ATP-dependent activities of chromatin remodeling complexes that can translocate nucleosomes with respect to the DNA. Here, we report a theoretical approach that takes into account both contributions. In the theoretical analysis two types of experiments have been considered: in vitro experiments with a single reconstituted nucleosome and in vivo genome-scale mapping of nucleosome positions. The effect of chromatin remodelers was described by iteratively redistributing the nucleosomes according to certain rules until a new steady state was reached. Three major classes of remodeler activities were identified: (i) the establishment of a regular nucleosome spacing in the vicinity of a strong positioning signal acting as a boundary, (ii) the enrichment/depletion of nucleosomes through amplification of intrinsic DNA-sequence-encoded signals and (iii) the removal of nucleosomes from high-affinity binding sites. From an analysis of data for nucleosome positions in resting and activated human CD4⁺ T cells [Schones et al., Cell 132, p. 887] it was concluded that the redistribution of a nucleosome map to a new state is greatly facilitated if the remodeler complex translocates the nucleosome with a preferred directionality.     

Nucleotide sequence-directed mapping of the nucleosomes

The concept of sequence-dependent deformational anisotropy of DNA proposed earlier is further elaborated and a computational procedure is developed for the sequence-directed mapping of the nucleosomes along chromatin DNA nucleotide sequences. The deformational anisotropy is found to be nonuniform along the molecule of the nucleosomal DNA, suggesting that the DNA superhelix in the nucleosome is slightly oval rather than circular in projection. The number of superhelical turns in the nucleosome core particle is estimated to be 2.0 +/- 0.2. Preliminary mapping of the nucleosomes in various chromatin DNA sequences yields the distribution of linker lengths which shows several minima separated by about 10 base-pairs. This is explained by sterical exclusion effects due to overlapping of the nucleosomes in space when some specific linker lengths are chosen. The mapping procedure described is tested by comparing its results with all the most accurate experimental mapping data reported so far. The comparison demonstrates that the exact positions of all the nucleosomes appear to be determined exclusively by the nucleotide sequences.     

Chromatin remodelling in mammalian cells by ISWI-type complexes--where, when and why?

The specific location of nucleosomes on DNA has important inhibitory or activating roles in the regulation of DNA-dependent processes as it affects the DNA accessibility. Nucleosome positions depend on the ATP-coupled activity of chromatin-remodelling complexes that translocate nucleosomes or evict them from the DNA. The mammalian cell harbors numerous different remodelling complexes that possess distinct activities. These can translate a variety of signals into certain patterns of nucleosome positions with specific functions. Although chromatin remodellers have been extensively studied in vitro, much less is known about how they operate in their cellular environment. Here, we review the cellular activities of the mammalian imitation switch proteins and discuss mechanisms by which they are targeted to sites where their activity is needed.