Specialized cell types in the body are produced from undifferentiated cells through a process of chromatin mediated genome reprogramming whereby the pattern of gene expression unique to each cell type is established and epigenetically maintained. However, because of inherent technical difficulties in studying individual cell types in isolation, our knowledge of the mechanisms of cellular differentiation remains extremely limited. The goals of the work proposed herein are to develop a simple and widely applicable method for the isolation of RNA and chromatin from specific cell types, and to use this system to investigate the mechanisms by which the two epidermal cell types of the Arabidopsis thaliana root (hair cells and nonhair cells) are generated from common progenitor cells. These goals will be achieved through the following specific aims: (1) Development of an affinity-based method for the isolation of nuclei from specific cell types. Coexpression of the E. coli biotin ligase enzyme (BirA) along with a nuclear envelope protein containing the BirA recognition peptide (BLRP) will produce biotin labeled nuclei that can be isolated from cell extracts using streptavidin-coated beads. BirA will be expressed constitutively, while the BLRP-tagged nuclear membrane protein will be expressed in root hair cells in one transgenic line, and in non-hair cells in a separate line. (2) Isolation of hair cell and non-hair cell nuclei for genome-wide expression analysis and chromatin immunoprecipitation-microarray (ChlP-chip) analysis of histone modifications throughout the genome in each cell type. Gene expression studies will provide a qualitative and quantitative description of the transcriptome of each cell type and will reveal the genes that are expressed exclusively in one type or the other, while ChlP-chip analysis will yield insight into the epigenetic processes underlying the transcriptional profile of each cell type. (3) Use reverse genetics to identify genes that are required for the specification of each cell type. By manipulating genes expressed exclusively in one cell type or the other, we can fully dissect how the hair cell and non-hair cell identities are specified. Understanding the mechanisms by which cell fate is established and maintained is not only a central problem in developmental biology, but is also relevant to understanding the development of cancer.
This research will yield general insight into how specialized cells are generated from stem cells, and how they maintain their specialized state. The technology developed in this research can also be applied directly to the study of gene activity in cells that are susceptible to cancerous changes, and to tumors derived form those cells. In this way, the changes that lead to cancer can be determined, paving the way for the development of new therapies.
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