MECHANISTIC PROBLEMS IN EUKARYOTIC GENE REGULATION

Northwestern University, Evanston IL

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Abstract

A fundamental requirement for gene regulation in eukaryotes--and hence for human health and development--is that gene regulatory proteins must be provided access to their DNA target sequences. However, the mechanisms by which such access is guaranteed and accomplished are not known. Existing ideas have well-established counter examples, or appear to violate fundamental physical principles, or leave essential questions unanswered. Our long-term goal is to develop a quantitative understanding of gene regulation in vivo. Our preliminary studies and their continuation, proposed here, begin our work toward this larger goal by investigating simple model systems in vitro. We recently developed a "site-exposure" model that would provide the needed access of regulatory proteins. In preliminary studies we established that nucleosomes do exhibit a dynamic property of site exposure. Moreover, we find that the site exposure model has a striking predictive capability: it quantitatively accounts for a wide range of published observations in vitro, and it provides physically plausible explantions for a wide range of phenomena in vivo. The goal of the studies proposed here is to further develop and explore the site exposure model: to characterize the site exposure process in vitro and to explore to what extent it can quantitatively or even quantitatively account for aspects of gene regulation in vivo. An important feature of our appoach is that we pose definte, testable, models for the phenomena under investigation, and we design and carry out experiments that specifically test those ideas. Four specific aims will be addresssed: (i) We will investigate experimental systems that more closely resemble the state of chromatin in vivo, and will allow us to quantitatively relate histone variants and posttranslational modifications to gene regulation. (ii) We will evaluate a prediction of our model which is that unrelated proteins will in general act cooperatively (synergistically) to facilitate each other's binding. (iii) We will analyze the structural consequences of the binding of regulatory proteins to nucleosomal DNA target sites. And (iv) We will test whether the site exposure model can quantitatively account for the roles of poly (dA:dT) elements at eukaryotic promoters.