Hematopoiesis is a complex developmental process through which pluripotent stem cells in mammalian fetal liver or adult bone marrow give rise to several types of terminally differentiated blood cells, including cells in this pathway are likely to play significant roles in the onset of leukemias and immunodeficiencies. To understand the basis for these defects, the regulatory mechanisms for hematopoiesis need to be understood at the molecular level. The primary objective of the research described in this application is to identify and characterize proteins that regulate gene expression during early stages of lymphocyte differentiation. Towards this end, the transcriptional control of the terminal deoxynucleotidyltransferase (TdT) gene is being investigated. This gene is expressed specifically in both early B and early T cells and its transcriptional control mechanisms appear unique when compared with the control mechanisms for the immunoglobulin genes. The analysis currently focuses on a DNA-binding protein, called LyF-1, that has been implicated in the regulation of the TdT gene and of several other lymphoid genes. LyF-1 has been purified from cultured lymphocytes. The purified protein will be used to characterize the biochemical properties of LyF-1 and to obtain LyF-1 amino acid sequence. The amino acid sequence will lead to isolation of the LyF-1 gene. The nucleotide sequence of the gene will be determined and its DNA-binding domain defined, potentially leading to the isolation of related genes. The regulation of LyF-1 expression will also be examined. Knowledge of the activation pathway for LyF-1 will further our understanding of the activation pathways for the TdT gene and for LyF-1 will further our understanding of the activation pathways for the TdT gene and for other lymphoid genes. In addition to the extensive analysis of LyF-1, two other potential regulators of TdT transcription will be characterized. The TdT-DBE element is located downstream of the TdT transcription start site. Through an interaction with a putative DNA-binding protein, NF-TdT-DBE, this element strongly activates TdT transcription in vitro. NF-TdT-X is a protein that has been shown to compete with LyF-1 for binding to the TdT promoter and, therefore, may be a negative regulator of TdT transcription. The proteins and DNA sequence elements responsible for these activities will be analyzed in more detail to determine their roles in TdT regulation. Eventually, this systemic analysis of TdT transcription should allow precise TdT regulation to be recapitulated with purified proteins in a cell-free transcription reaction. These experiments will help us reach our long-term goals, which are 1) to understand the regulation of early stages of lymphocyte differentiation at the molecular level, and 2) to elucidate defects in the immunodifferentiation program that lead to leukemogenesis and immunodeficiency.
