NucleoMap: A computational tool for identifying nucleosomes in ultra-high resolution contact maps

Although poorly positioned nucleosomes are ubiquitous in the eukaryotic genome, they are difficult to identify with existing nucleosome identification methods. Recently available enhanced high-throughput chromatin conformation capture techniques such as Micro-C, DNase Hi-C, and Hi-CO characterize nucleosome-level chromatin proximity, probing the positions of mono-nucleosomes and the spacing between nucleosome pairs at the same time, enabling nucleosome profiling in poorly positioned regions. Here we develop a novel computational approach, NucleoMap, to identify nucleosome positioning from ultra-high resolution chromatin contact maps. By integrating nucleosome read density, contact distances, and binding preferences, NucleoMap precisely locates nucleosomes in both prokaryotic and eukaryotic genomes and outperforms existing nucleosome identification methods in both precision and recall. We rigorously characterize genome-wide association in eukaryotes between the spatial organization of mono-nucleosomes and their corresponding histone modifications, protein binding activities, and higher-order chromatin functions. We also find evidence of two tetra-nucleosome folding structures in human embryonic stem cells and analyze their association with multiple structural and functional regions. Based on the identified nucleosomes, nucleosome contact maps are constructed, reflecting the inter-nucleosome distances and preserving the contact distance profiles in original contact maps.     

Probing SWI/SNF remodeling of the nucleosome by unzipping single DNA molecules

Chromatin-remodeling enzymes can overcome strong histone-DNA interactions within the nucleosome to regulate access of DNA-binding factors to the genetic code. By unzipping individual DNA duplexes, each containing a uniquely positioned nucleosome flanked by long segments of DNA, we directly probed histone-DNA interactions. The resulting disruption-force signatures were characteristic of the types and locations of interactions and allowed measurement of the positions of nucleosomes with 2.6-base-pair (bp) precision. Nucleosomes remodeled by yeast SWI/SNF were moved bidirectionally along the DNA, resulting in a continuous position distribution. The characteristic distance of motion was approximately 28 bp per remodeling event, and each event occurred with a catalytic efficiency of 0.4 min(-1) per nM SWI/SNF. Remodeled nucleosomes had essentially identical disruption signatures to those of unremodeled nucleosomes, indicating that their overall structure remained canonical. These results impose substantial constraints on the mechanism of SWI/SNF remodeling.     

Prediction of nucleosome occupancy in Saccharomyces cerevisiae using position-correlation scoring function

Knowledge of the detailed organization of nucleosomes across genomes and the mechanisms of nucleosome positioning is critical for the understanding of gene regulation and expression. In the present work, the bias of 4-mer frequency in nucleosome and linker sequences of the S. cerevisiae genome was analyzed statistically. A novel position-correlation scoring function algorithm based on the bias of 4-mer frequency in linker sequences was presented to distinguish nucleosome vs linker sequences. Five-fold cross-validation demonstrated that the algorithm achieved a good performance with mean area under the receiver operator characteristics curve of 0.981. Next, the algorithm was used to predict nucleosome occupancy throughout the S. cerevisiae genome and relatively high correlation coefficients with experiment maps of nucleosome positioning were obtained. Besides, the distinct nucleosome depleted regions in the vicinity of regulatory sites were confirmed. The results suggest that intrinsic DNA sequence preferences in linker regions have a significant impact on the nucleosome occupancy.     

DNase I digestion reveals alternating asymmetrical protection of the nucleosome by the higher order chromatin structure

DNase I was used to probe the higher order chromatin structure in whole nuclei. The digestion profiles obtained were the result of single-stranded cuts and were independent of pH, type of divalent ion and chromatin repeat length. Furthermore, the protection from digestion of the DNA at the entry/exit points on the nucleosome was found to be caused not by the H1/H5 histone tails, but by the compact structure that these proteins support. In order to resolve symmetry ambiguities, DNase I digestion fragments over several nucleosome repeat lengths were analysed quantitatively and compared with computer simulations using combinations of the experimentally obtained rate constants (some of which were converted to 0 to simulate steric protection from DNase I digestion). A clear picture of precisely defined, alternating, asymmetrically protected nucleosomes emerged. The linker DNA is inside the fibre, while the nucleosomes are positioned above and below a helical path and/or with alternating orientation towards the dyad axis. The dinucleosomal modulation of the digestion patterns comes from alternate protection of cutting sites inside the nucleosome and not from alternating exposure to the enzyme of the linker DNA.     

Site-specific repair of cyclobutane pyrimidine dimers in a positioned nucleosome by photolyase and T4 endonuclease V in vitro.

Since genomic DNA is folded into nucleosomes, and DNA damage is generated all over the genome, a central question is how DNA repair enzymes access DNA lesions and how they cope with nucleosomes. To investigate this topic, we used a reconstituted nucleosome (HISAT nucleosome) as a substrate to generate DNA lesions by UV light (cyclobutane pyrimidine dimers, CPDs), and DNA photolyase and T4 endonuclease V (T4-endoV) as repair enzymes. The HISAT nucleosome is positioned precisely and contains a long polypyrimidine region which allows one to monitor formation and repair of CPDs over three helical turns. Repair by photolyase and T4-endoV was inefficient in nucleosomes compared with repair in naked DNA. However, both enzymes showed a pronounced site-specific modulation of repair on the nucleosome surface. Removal of the histone tails did not substantially enhance repair efficiency nor alter the site specificity of repair. Although photolyase and T4-endoV are different enzymes with different mechanisms, they exhibited a similar site specificity in nucleosomes. This implies that the nucleosome structure has a decisive role in DNA repair by exerting a strong constraint on damage accessibility. These findings may serve as a model for damage recognition and repair by more complex repair mechanisms in chromatin.     

Are nucleosome positions in vivo primarily determined by histone–DNA sequence preferences?

Large-scale and genome-wide studies have concluded that ∼80% of the yeast (Saccharomyces cerevisiae) genome is occupied by positioned nucleosomes. In vivo this nucleosome organization can result from a variety of mechanisms, including the intrinsic DNA sequence preferences for wrapping the DNA around the histone core. Recently, a genome-wide study was reported using massively parallel sequencing to directly compare in vivo and in vitro nucleosome positions. It was concluded that intrinsic DNA sequence preferences indeed have a dominant role in determining the in vivo nucleosome organization of the genome, consistent with a genomic code for nucleosome positioning. Some other studies disagree with this view. Using the large amount of data now available from several sources, we have attempted to clarify a fundamental question concerning the packaging of genomic DNA: to what extent are nucleosome positions in vivo determined by histone-DNA sequence preferences? We have analyzed data obtained from different laboratories in the same way, and have directly compared these data. We also identify possible problems with some of the experimental designs used and with the data analysis. Our findings suggest that DNA sequence preferences have only small effects on the positioning of individual nucleosomes throughout the genome in vivo.     

The divalent cations Ca2+ and Mg2+ play specific roles in stabilizing histone-DNA interactions within nucleosomes that are partially redundant with the core histone tail domains

We previously reported that reconstituted nucleosomes undergo sequence-dependent translational repositioning upon removal of the core histone tail domains under physiological conditions, indicating that the tails influence the choice of position. We report here that removal of the core histone tail domains increases the exposure of the DNA backbone in nucleosomes to hydroxyl radicals, a nonbiased chemical cleavage reagent, indicative of an increase in the motility of the DNA on the histone surface. Moreover, we demonstrate that the divalent cations Mg(2+) and Ca(2+) can replace the role of the tail domains with regard to stabilization of histone-DNA interactions within the nucleosome core and restrict repositioning of nucleosomes upon tail removal. However, when nucleosomes were incubated with Mg(2+) after tail removal, the original distribution of translational positions was not re-established, indicating that divalent cations increase the energy barrier between translational positions rather than altering the free energy differences between positions. Interestingly, other divalent cations such as Zn(2+), Fe(2+), Co(2+), and Mn(2+) had little or no effect on the stability of histone-DNA interactions within tailless nucleosomes. These results support the idea that specific binding sites for Mg(2+) and Ca(2+) ions exist within the nucleosome and play a critical role in nucleosome stability that is partially redundant with the core histone tail domains.