The DNA of eukaryotic organisms is pacl<aged into the nucleus by wrapping around histone proteins in a structure l<nown as chromatin. This pacl<aging can influence gene regulation with condensed chromatin regions containing genes and their associated regulatory elements that are inaccessible to transcriptional activators. The modulation of chromatin structure is therefore one of the key mediators of transcriptional regulation. The most basic unit of chromatin is the nucleosome, which consists of approximately 146 base pairs of DNA wrapped around an octamer of histone proteins. The precise positioning of nucleosomes in functional regulatory regions of the genome can directly influence the transcriptional regulation of nearby genes. ATP-dependent chromatin remodeling enzymes, post-translational modifications to histones and the DNA sequence itself have all been implicated in regulating the positioning of nucleosomes at specific regions of the genome. We propose to investigate the roles of each of these mechanisms in regulating the positioning of nucleosomes in the Drosophila melanogaster and human genomes and furthermore to begin to elucidate the relationship between chromatin and transcription factors in gene regulation. We will first investigate the basic questions of how chromatin remodeling enzymes are recruited to their targets and how ATP-dependent chromatin remodeling, histone modifications and DNA sequence interact to regulate nucleosome positioning by using cultured Drosophila cells. The insights obtained from these experiments in Drosophila cells will then be applied to analogous experiments in human hematopoietic cells, a system for which the network of transcription factors involved in lineage specification has been extensively studied. By integrating data on nucleosome positioning and chromatin remodeling with the already established transcription factor programs in these cells, I will be able to study the interactions between chromatin and transcription factors in regulating the transcriptional programs of cells during development. These studies will lead to a greater understanding of the mechanisms responsible for the organization of nucleosomes across the genome and further elucidate the role of chromatin in the regulation of transcription.
The scientific goal of this grant is to gain a better understanding of the mechanisms responsible for regulating the structure of chromatin and to furthermore explore how this regulation is involved in development. This research will lead to a greater understanding of developmental processes in general, with specific applications to hematopoietic development. Relevance These studies will further elucidate how components responsible for packaging the genome into the nucleus interact with other proteins to regulate gene expression. These insights will shed further light on gene regulation and developmental processes in general.
