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.     

Nucleosomes protect DNA from DNA methylation in vivo and in vitro

Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo.     

Dynamics of nucleosome invasion by DNA binding proteins

Nucleosomes sterically occlude their wrapped DNA from interacting with many large protein complexes. How proteins gain access to nucleosomal DNA target sites in vivo is not known. Outer stretches of nucleosomal DNA spontaneously unwrap and rewrap with high frequency, providing rapid and efficient access to regulatory DNA target sites located there; however, rates for access to the nucleosome interior have not been measured. Here we show that for a selected high-affinity nucleosome positioning sequence, the spontaneous DNA unwrapping rate decreases dramatically with distance inside the nucleosome. The rewrapping rate also decreases, but only slightly. Our results explain the previously known strong position dependence on the equilibrium accessibility of nucleosomal DNA, which is characteristic of both selected and natural sequences. Our results point to slow nucleosome conformational fluctuations as a potential source of cell-cell variability in gene activation dynamics, and they reveal the dominant kinetic path by which multiple DNA binding proteins cooperatively invade a nucleosome.     

Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites

We have previously shown that nucleosomes are conformationally dynamic: DNA sequences that in the time-average are buried inside nucleosomes are nevertheless transiently accessible, even to large proteins (or any other macromolecule). We refer to this dynamic behavior as "site exposure". Here we show that: (i) the equilibrium constants describing this dynamic site exposure decrease progressively from either end of the nucleosomal DNA in toward the middle; and (ii) these position-dependent equilibrium constants are strongly dependent on the nucleosomal DNA sequence. The progressive decrease in equilibrium constant with distance inside the nucleosome supports the hypothesis that access to sites internal to a nucleosome is provided by progressive (transient) release of DNA from the octamer surface, starting from one end of the nucleosomal DNA. The dependence on genomic DNA sequence implies that a specific genomic DNA sequence could be a major determinant of target site occupancies achieved by regulatory proteins in vivo, by either governing the time-averaged accessibility for a given nucleosome position, or biasing the time-averaged positioning (of mobile nucleosomes), which in turn is a major determinant of site accessibility.     

Collaborative competition mechanism for gene activation in vivo

The mechanism by which gene regulatory proteins gain access to their DNA target sites is not known. In vitro, binding is inherently cooperative between arbitrary DNA binding proteins whose target sites are located within the same nucleosome. We refer to such competition-based cooperativity as collaborative competition. Here we show that arbitrarily chosen foreign DNA binding proteins, LexA and Tet repressor, cooperate with an adjacently binding endogenous activator protein, Gcn4, to coactivate expression of chromosomal reporter genes in Saccharomyces cerevisiae. Coactivation requires that the cooperating target sites be within a nucleosome-length distance; it leads to increased occupancy by Gcn4 at its binding site; and it requires both Gcn5 and Swi/Snf which, at an endogenous Gcn4-dependent promoter, act subsequent to Gcn4 binding. These results imply that collaborative competition contributes to gene regulation in vivo. They further imply that, even in the presence of the cell's full wild-type complement of chromatin remodeling factors, competition of regulatory proteins with histone octamer for access to regulatory target sites remains a quantitative determinant of gene expression levels. We speculate that initial target site recognition and binding may occur via spontaneous nucleosomal site exposure, with remodeling factor action required downstream to lock in higher levels of regulatory protein occupancy.     

Multiple Nhp6 molecules are required to recruit Spt16-Pob3 to form yFACT complexes and to reorganize nucleosomes

The Saccharomyces cerevisiae Nhp6 protein contains a DNA-binding motif that is similar to those found in the high mobility group B family of chromatin proteins. Nhp6 bound to nucleosomes and made at least two changes in them: the nucleosomal DNA became more sensitive to DNase I at specific sites, and the nucleosomes became competent to bind Spt16-Pob3 to form yFACT.nucleosome complexes. Both changes occurred at similar concentrations of Nhp6, suggesting that they reflect the same structural reorganization of the nucleosome. Nucleosomes have multiple binding sites for Nhp6, and structural reorganization was associated with a concentration of Nhp6 about 10-fold higher than that needed for simple binding. We propose that the coordinated action of multiple Nhp6 molecules is required to convert nucleosomes to an alternative form as the first step in a two-step reorganization of nucleosomes with the second step being dependent on Spt16-Pob3. The presence of linker DNA had only subtle effects on these processes, indicating that both Nhp6 and yFACT act on core nucleosome structure rather than on the interaction between nucleosomes and adjacent DNA. These results suggest that Nhp6 and the related high mobility group B proteins may have a general role in promoting rearrangements of chromatin by initiating the destabilization of core nucleosomal structure.     

Superhelicity of nucleosomal DNA changes its double-helical repeat

Winding DNA in a superhelix can be considered a process consisting of two smooth deformations: bending and twisting. The extra twist angle introduced by winding DNA into the nucleosomal superhelix is calculated by means of the Crick formula to be -0.5 degrees per base pair (bp). This is equivalent to a change of -0.15 +/- 0.015 bp in the DNA double-helical repeat. Free DNA in solution is known to have a helical repeat of 10.55 +/- 0.1 bp. On the other hand, a weighted average of various estimates of the DNA repeat in the nucleosome is 10.38 +/- 0.02. The difference happens to be perfectly accounted for by the superhelicity of the nucleosomal DNA. This implies that the latter is essentially nonconstrained .