Nucleosome positioning as a determinant of chromatin structure

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Only a very small fraction of a eukaryotic genome codes for proteins or is directly involved in regulating their synthesis. The function of the remainder of the genome remains largely unknown. It is now well established that the way in which the genome is packaged or folded into chromatin structures within the nucleus of the cell makes a major contribution towards regulating or controlling genetic activity. Within the nucleus the default state of chromatin folding is the 30 nm, higher-order chromatin fibre and the detailed way in which the DNA is packaged into this structure is a major determinant of the functional potential of the underlying DNA sequence. Our previous research strongly suggests that one factor which regulates how DNA is folded into the higher-order fibre is the long-range organisation of the DNA sequence itself. This happens because the DNA sequence influences the relative locations of the building blocks of chromatin, the nucleosomes. When these are regularly spaced along the DNA the resulting chain of nucleosomes can be folded into a very compact, inactive type of structure. Chains of nucleosomes which are less regularly spaced are less efficiently folded and are likely to incorporate regions of minor disruption which could provide access to the underlying sequence and thus be of functional importance. My research is directed towards measuring the locations of sites in the DNA sequence where nucleosomes prefer to bind. Thus, we measure an intrinsic property of the DNA sequence itself. We carry out this mapping on regions of the genome that contain very well characterised genes so that we can relate our findings about nucleosome positioning signals, and the kinds of chromatin structures they might dictate, in a relevant context. For some genes, we have also determined the locations of nucleosomes on the same DNA in the cell and we find that the in vitro and in vivo nucleosome positioning maps are related, confirming the involvement of DNA sequence in determining chromatin structure. Interestingly, most of the sequences that influence nucleosome positioning do not lie in the regions that code for proteins but are found in the sequences which interrupt and flank the genes. The correspondence between in vitro and in vivo nucleosome positioning has led us to consider whether the in vitro mapping datasets can be exploited to simulate chromatin organisation in detail using computational approaches. We have found that simulated chromatin structures are sensitive to nucleosome density and, perhaps most significantly, that at an appropriate nucleosome density the simulated chromatin 'structures' are even more closely related to the in vivo positioning state than is the original in vitro data. This latter observation underlines the value of the in vitro positioning data and strongly justifies this type of computational approach. For this proposal we intend to extend our in vitro nucleosome mapping by studying three entire gene regions, the human and chicken beta-globin domains and the human H19/Igf2 imprinted domain. In doing this we hope to demonstrate that the features and relationships we detect at gene level are also apparent, and perhaps even more clearly identified, at a more functionally relevant level of a chromatin organisation. We will also increase our database of nucleosome positioning sequences by an order of magnitude. A major focus of our proposal is to continue and expand our computational studies so that we can identify how the information we measure in vitro is translated into chromatin structure in the cell. Furthermore, the availability of such a large database of positioning sequences presents the prospect of actually identifying what features of the DNA sequence cause nucleosome positioning. If we knew this, we could develop algorithms that would 'read' DNA sequence in terms of chromatin structure, a goal which would undoubtedly expand our understanding of the genome and how it works.