DNA Folding in Chromatin at the Supra-nucleosome Level

Division Of Basic Sciences - Nci

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Abstract

Eukaryotic DNA is extremely tightly packed in the nucleus, nevertheless its sequence is effectively recognized by numerous protein factors. To elucidate structural mechanisms of this recognition one needs to have a detailed information about the second level of DNA organization, or 30-nm fibers. To tackle this problem, we computed all possible configurations of the two-start chromatin fibers with DNA linkers L = 10 - 70 bp (nucleosome repeat length, NRL = 157 - 217 bp). As a result, we observed two different families of conformations (i.e., topoisomers) characterized by different DNA topologies. The optimal geometry of a fiber depends on the linker length: the fibers with linkers L = 10n and 10n+5 bp have DNA linking numbers per nucleosome delta(Lk) = -1.5 and -1.0, respectively. In other words, the level of DNA supercoiling is directly related to the nucleosome spacing in chromatin. Thus, we made an important step toward resolving the long-standing discrepancy known as the linking-number paradox. We hypothesize that topological polymorphism of chromatin fibers described above may play a role in the process of transcription, which is known to generate different levels of DNA supercoiling upstream and downstream from RNA polymerase. A genome-wide analysis of the NRL distribution in yeast genes confirmed this assumption. We also found that the two fiber topoisomers (with L = 10n and 10n+5 bp) differ not only in their equilibrium configuration and average DNA linking number, but also in their dynamics. In particular, the novel 10n+5 topoisomer is characterized by an increased plasticity, which makes chromatin more accessible to transcription factors (TFs). In addition, genomic DNA is made accessible by partial unwrapping of nucleosomes. As follows from the force spectroscopy measurements, the chromatin fiber is so mobile that nucleosomes lose their stacking under small external forces F = 2-3 pN, whereas at F = 4-5 pN, the nucleosomes are significantly unwrapped. (These forces are well below the tension produced by RNA polymerase; therefore, the observed unwrapping corresponds to 'native' conditions.) Importantly, the nucleosome breathing occurs asymmetrically, with one end opened much stronger than the other one. We were able to explain this non-trivial effect theoretically, considering a non-linear adhesion energy function describing interactions between DNA and histone core. This observation may have profound implications for transcription and other DNA-related cellular functions. According to our data, asymmetric unwrapping of nucleosomal DNA exposes 50-60 bp at one end (compared to 20-30 bp at both ends in the case of symmetric unwrapping). Therefore, asymmetric breathing of nucleosomes increases accessibility of DNA to TFs. The nucleosome positioning is not entirely determined by underlying DNA sequence, but rather is modulated by a plethora of various factors. We provide the bioinformatic data indicating that the sequence-specific interaction between DNA and histone tails may be one of such factors. We have initiated experiments to test this assumption, analyzing positioning of nucleosomes reconstituted with histones H2A and H4 lacking the N-tails, on human BRCA1 gene in vitro. This may be of general biological interest because, if our hypothesis is confirmed, it would be for the first time (to the best of our knowledge) when the epigenetic regulation of nucleosome positioning is demonstrated. This, in turn, would open the prospects for revealing structural mechanisms of DNA recognition by TFs in the context of the three-dimensional organization of chromatin. In particular, we are interested in the nucleosome rearrangement in the vicinity of CTCF binding sites (many of which are involved in stabilization of chromatin loops), in Alu repeats that are abundant around the CTCF sites, and the p53 REs frequently occurring in these repeats. Remodeling the DNA wrapping around the histone core can either facilitate the TF binding by exposing the cognate DNA site, or, by contrast, hinder the binding by placing the TF site inside the DNA loop. This consideration is directly related to the DNA recognition by p53 (see the second project).