Cell differentiation is a orchestrated by changes in the expressed genetic program at distinct developmental stages. Hematopoiesis represents a dynamic example of this process maintaining formed multi- lineage elements of the blood throughout life. Pluripotent hematopoietic stem cells give rise to subsets of progenitor cells with progressively restricted fates. The molecular basis of lineage commitment is poorly understood and appears to be directed, in part, by cytokines and micro- environmental factors. We will examine the hematopoietic cascade to identify changes in gene expression that initiate differentiation and lineage commitment. We will determine how the development status of the organism influences lineage commitment during times of stress. To address the influence of 'master genes' in guiding differentiation, we will examine the role of the ets family of transcription factors in hematopoiesis. Members of this family play an essential role in the function of terminally differentiated hematopoietic cells such as T and B cells. They may play a similar role in early hematopoiesis. We will survey cells at various steps for known and novel ets family members and test their role in differentiation by blocking ets function with deletion constructs lacking activation domains. We will also examine mechanisms of ets binding and activation using the T cell Beta chain enhancer as a model. Cytokines also influence lineage commitment. As characterized by the ability of G-CSF to differentiate early myeloid progenitor cells into mature granulocytes. To dissect the complex genetic steps associated with differentiation, we will identify target genes induced by G-CSF stimulation of myeloid progenitor cells. Two strategies, differential display PCR and cDNA substraction techniques, will be used to isolate genes activated after cytokine stimulation. Developmental status may influence the ability of the hematopoietic compartment to amplify lineage commitment in response to stimuli. G-CSF gene regulation is altered in the human newborn and is responsible for an increased risk of infection. To understand the molecular basis of cytokine gene dysregulation in early development we will localize defects in the transcriptional and post-transcriptional regulation of the G-CSF gene in term and preterm neonates. Identification of pivotal genetic steps directing hematopoietic differentiation will lead to identification of gene defects associated with disorders of differentiation and allow the development of strategies to restore effective hematopoiesis.
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