Hematopoiesis is a complex developmental process through which pluripotent stem cells give rise to several types of terminally-differentiated blood cells, including the B and T lymphocytes responsible for adaptive immunity. The regulation of the hematopoietic pathways must be understood to elucidate the procedures. The objective of the research proposed in this application is to define the molecular events that control expression of the terminal doxynucleotidyl transferase (TdT) gene. This analysis will provide insight into the regulation of the earliest stages of lymphopoiesis, as TdT is one of the first genes activated following commitment of a hematopoietic stem cell to the B and T lymphoid lineages. An understanding of TdT gene expression will also provide insight into a key hematopoietic transition that occurs during mammalian ontogeny, as TdT is not expressed during lymphopoiesis in the fetus, but is efficiently expressed during adult lymphopoiesis. The TdT gene already has proved to be and excellent paradigm for the study of early lymphoid development, as one protein the investigators originally identified as a potential regulator of TdT transcription, called LyF-1 or more recently Ikaros, is now known from gene disruption experiments to be critical for an early stage of lymphopoiesis. The experiments proposed in this application will greatly extend the investigators' previous studies of TdT gene regulation. One of the principal goals will be to complete a biochemical analysis of the proteins encoded by the Ikaros gene. The investigators' previous studies have shown that this gene is quite complicated, in that it encodes several protein isoforms through alternative pre-mRNA splicing, with the isoforms apparently forming stable multimers within cells. A biochemical analysis of the Ikaros isoforms and isoform multimers will provide insight into their roles during TdT regulation and lymphocyte development. Although the Ikaros isoforms will be studied in detail, the primary focus will continue to be on a broad analysis of TdT gene regulation. This focus is essential for the investigators' long-term goals. Preliminary studies suggest that, in addition to the Ikaros proteins, an Ets-family member called Elf-1 may be critical to TdT transcription. Antisense and in vitro transcription approaches will be employed to determine the relative functions of Elf-1 and Ikaros proteins in regulation TdT expression. The in vitro assay will also be employed to identify, in an unbiased manner, other TdT regulatory proteins. Finally, extensive use will be made of a stable transfection assay that was recently developed for studies of the TdT promoter. With this assay, a detailed analysis of the promoter elements and distant control regions that regulate TdT expression in vivo can be pursued. Eventually, the investigators hope to use the knowledge generated during these studies to elucidate the molecular events that regulate early lymphoid development and the fetal/adult lymphopoietic transition.
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