Spontaneous access to DNA target sites in folded chromatin fibers

DNA wrapped in nucleosomes is sterically occluded from many protein complexes that must act on it; how such complexes gain access to nucleosomal DNA is not known. In vitro studies on isolated nucleosomes show that they undergo spontaneous partial unwrapping conformational transitions, which make the wrapped nucleosomal DNA transiently accessible. Thus, site exposure might provide a general mechanism allowing access of protein complexes to nucleosomal DNA. However, existing quantitative analyses of site exposure focused on single nucleosomes, while the presence of neighbor nucleosomes and concomitant chromatin folding might significantly influence site exposure. In this work, we carried out quantitative studies on the accessibility of nucleosomal DNA in homogeneous nucleosome arrays. Two striking findings emerged. Organization into chromatin fibers changes the accessibility of nucleosomal DNA only modestly, from ∼ 3-fold decreases to ∼ 8-fold increases in accessibility. This means that nucleosome arrays are intrinsically dynamic and accessible even when they are visibly condensed. In contrast, chromatin folding decreases the accessibility of linker DNA by as much as ∼ 50-fold. Thus, nucleosome positioning dramatically influences the accessibility of target sites located inside nucleosomes, while chromatin folding dramatically regulates access to target sites in linker DNA.     

基于DNA变形能的核小体定位预测方法研究进展

真核细胞中,作为染色质基本结构单元的核小体参与调控基因的转录、DNA复制、重组以及RNA剪接等诸多生物学过程。阐明核小体定位机制并准确预测核小体在染色体上的位置对解读染色质结构与功能有重要生物学意义。在过去30多年时间里,研究人员发展了多种预测核小体位置的方法。最理想的方法应考虑DNA序列、组蛋白修饰和染色质重塑等影响核小体定位的诸多因素,然而现实中,捕捉主要因素的模型也往往具有很高的鲁棒性和实用价值。DNA序列偏好性是在全基因组尺度上影响核小体定位的最重要因素之一,因此基于DNA序列的核小体定位预测方法也最常见。这种方法可大致分为两类,即基于DNA序列信息的生物信息学模型和基于DNA变形能的生物物理学模型。本文重点介绍生物物理学模型近些年取得的主要进展。     

A critical role for linker DNA in higher-order folding of chromatin fibers

Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.     

Nucleosome positioning in relation to nucleosome spacing and DNA sequence-specific binding of a protein

Nucleosome positioning is an important mechanism for the regulation of eukaryotic gene expression. Folding of the chromatin fiber can influence nucleosome positioning, whereas similar electrostatic mechanisms govern the nucleosome repeat length and chromatin fiber folding in vitro. The position of the nucleosomes is directed either by the DNA sequence or by the boundaries created due to the binding of certain trans-acting factors to their target sites in the DNA. Increasing ionic strength results in an increase in nucleosome spacing on the chromatin assembled by the S-190 extract of Drosophila embryos. In this study, a mutant lac repressor protein R3 was used to find the mechanisms of nucleosome positioning on a plasmid with three R3-binding sites. With increasing ionic strength in the presence of R3, the number of positioned nucleosomes in the chromatin decreased, whereas the internucleosomal spacings of the positioned nucleosomes in a single register did not change. The number of the positioned nucleosomes in the chromatin assembled in vitro over different plasmid DNAs with 1-3 lac operators changed with the relative position and number of the R3-binding sites. We found that in the presence of R3, nucleosomes were positioned in the salt gradient method of the chromatin assembly, even in the absence of a nucleosome-positioning sequence. Our results show that nucleosome-positioning mechanisms are dominant, as the nucleosomes can be positioned even in the absence of regular spacing mechanisms. The protein-generated boundaries are more effective when more than one binding site is present with a minimum distance of approximately 165 bp, greater than the nucleosome core DNA length, between them.     

High-Resolution Mapping of Sequence-Directed Nucleosome Positioning on Genomic DNA

We have mapped in vitro nucleosome positioning on the sheep β-lactoglobulin gene using high-throughput sequencing to characterise the DNA sequences recovered from reconstituted nucleosomes. This methodology surpasses previous approaches for coverage, accuracy and resolution and, most importantly, offers a simple yet rapid and relatively inexpensive method to characterise genomic DNA sequences in terms of nucleosome positioning capacity. We demonstrate an unambiguous correspondence between in vitro and in vivo nucleosome positioning around the promoter of the gene; identify discrete, sequence-specific nucleosomal structures above the level of the canonical core particle—a feature that has implications for regulatory protein access and higher-order chromatin packing; and reveal new insights into the involvement of periodically organised dinucleotide sequence motifs of the type GG and CC and not AA and TT, as determinants of nucleosome positioning—an observation that supports the idea that the core histone octamer can exploit different patterns of sequence organisation, or structural potential, in the DNA to bring about nucleosome positioning.     

In silico approaches reveal the potential for DNA sequence-dependent histone octamer affinity to influence chromatin structure in vivo

Nucleosome positioning signals embedded within the DNA sequence have the potential to influence the detailed structure of the higher-order chromatin fibre. In two previous studies of long stretches of DNA, encompassing the chicken beta-globin and ovine beta-lactoglobulin genes, respectively, we mapped the relative affinity of every site for the core histone octamer. In both cases a periodic arrangement of the in vitro positioning sites suggests that they might influence the folding of a nucleosome chain into higher-order structure; this hypothesis was borne out in the case of the beta-lactoglobulin gene, where the distribution of the in vitro positioning sites is related to the positions nucleosomes actually occupy in sheep liver cells. Here, we have exploited the in vitro nucleosome positioning datasets to simulate nucleosomal organisation using in silico approaches. We use the high-resolution, quantitative positioning maps to define a one-dimensional positioning energy lattice, which can be populated with a defined number of nucleosomes. Monte Carlo techniques are employed to simulate the behaviour of the model at equilibrium to produce a set of configurations, which provide a probability-based occupancy map. Employing a variety of techniques we show that the occupancy maps are a sensitive function of the histone octamer density (nucleosome repeat length) and find that a minimal change in this property can produce dramatic localised changes in structure. Although simulations generally give rise to regular periodic nucleosomal arrangements, they often show octamer density-dependent discontinuities, which tend to co-localise with sequences that adopt distinctive chromatin structure in vivo. Furthermore, the overall organisation of simulated chromatin structures are more closely related to the situation in vivo than is the original in vitro positioning data, particularly at a nucleosome density corresponding to the in vivo state. Although our model is simplified, we argue that it provides a unique insight into the influence that DNA sequence can have in determining chromatin structure and could serve as a useful basis for the incorporation of other parameters.     

Nucleosome positioning signals in the DNA sequence of the human and mouse H19 imprinting control regions

We have investigated the sequences of the mouse and human H19 imprinting control regions (ICRs) to see whether they contain nucleosome positioning information pertinent to their function as a methylation-regulated chromatin boundary. Positioning signals were identified by an in vitro approach that employs reconstituted chromatin to comprehensively describe the contribution of the DNA to the most basic, underlying level of chromatin structure. Signals in the DNA sequence of both ICRs directed nucleosomes to flank and encompass the short conserved sequences that constitute the binding sites for the zinc finger protein CTCF, an essential mediator of insulator activity. The repeat structure of the human ICR presented a conserved array of strong positioning signals that would preferentially flank these CTCF binding sites with positioned nucleosomes, a chromatin structure that would tend to maintain their accessibility. Conversely, all four CTCF binding sites in the mouse sequence were located close to the centre of positioning signals that were stronger than those in their flanks; these binding sites might therefore be expected to be more readily incorporated into positioned nucleosomes. We found that CpG methylation did not effect widespread repositioning of nucleosomes on either ICR, indicating that allelic methylation patterns were unlikely to establish allele-specific chromatin structures for H19 by operating directly upon the underlying DNA-histone interactions; instead, epigenetic modulation of ICR chromatin structure is likely to be mediated principally at higher levels of control. DNA methylation did, however, both promote and inhibit nucleosome positioning at several sites in both ICRs and substantially negated one of the strongest nucleosome positioning signals in the human sequence, observations that underline the fact that this epigenetic modification can, nevertheless, directly and decisively modulate core histone-DNA interactions within the nucleosome.     

DNA engineering shows that nucleosome phasing on the African green monkey alpha-satellite is the result of multiple additive histone-DNA interactions

The mechanism underlying sequence-specific positioning of nucleosomes on DNA was investigated. African green monkey alpha-satellite DNA was reconstituted in vitro with histones. Histone octamers were found to adopt one major and several minor positions on the satellite repeat unit, very similar to those positions found previously in vitro, demonstrating that sequence-specific histone-DNA interactions are responsible for nucleosome positioning on this DNA. In order to understand the nature of these interactions in more detail, we have constructed a variant satellite fragment containing an insertion of half a helical DNA turn. The parent fragment directs histones to one major and two overlapping minor positions that are all affected by the insertion. All three frames respond in a unique fashion to the additional five base-pairs. From a quantitative analysis of the nucleosome positions on the engineered fragment, consensus "phasing boxes" as the basis for nucleosome positioning can be ruled out. Instead, our results argue very strongly that nucleosome positioning is due to the independent contribution of many different DNA-histone contacts along the entire core particle, in an apparently additive fashion.     

Reconstitution experiments show that sequence-specific histone-DNA interactions are the basis for nucleosome phasing on mouse satellite DNA

The molecular basis underlying the sequence-specific positioning of nucleosomes on DNA was investigated. We previously showed that histone octamers occupy multiple specific positions on mouse satellite DNA in vivo and have now reconstituted the 234 bp mouse satellite repeat unit with pure core histones into mononucleosomes. Histones from mouse liver or chicken erythrocytes bind to the DNA in multiple precisely defined frames in perfect phase with a diverged 9 bp subrepeat of the satellite DNA. This is the first time that nucleosome positions on a DNA in vivo have been compared to those found on the same DNA by in vitro reconstitution. Most of the nucleosomes occupy identical positions in vivo and in vitro. There are, however, some characteristic differences. We conclude that sequence-dependent histone-DNA interactions play a decisive role in the positioning of nucleosomes in vivo, but that the nucleosome locations in native chromatin are subject to additional constraints.