Identification of Novel STAT5 Target Genes in Human T-cells
As a major growth factor of T-cells, interleukin-2 (IL-2) promotes, among other biological effects, cell proliferation and anti-apoptosis. These biological consequences are thought to occur via IL-2 activation of signal transduction pathways, such as the Ras/ERK, PI3K, and JAK/STAT pathways, which transmit signals from the IL-2R to the nucleus, resulting in gene expression. Under normal circumstances, these pathways work to promote T-cell clonal expansion, which is critical to the successful mounting of an immune response. However, infection of T-cells by human T-cell leukemia, type I (HTLV-1) can lead to constitutive activation of these signaling cascades, and, ultimately, IL-2-independent T-cell growth. Much of the effects of HTLV-1 have been attributed to a viral oncoprotein called Tax, which affects cellular and viral transcriptional regulation, cell cycle control, DNA repair, and apoptosis. The work presented in this dissertation was aimed at investigating the signaling pathways activated by IL-2 in primary human T-cells and Tax-immortalized human T-cells. IL-2-induced gene expression, and its regulation by the transcription factor STAT5, was also studied in both cell types. The data demonstrate that IL-2 activates the Ras/ERK, PI3K, and JAK/STAT pathways in primary human T-cells to promote cell proliferation, and that significant crosstalk exists between them in the form of PI3K 2!MEK/ERK 2!STAT3. Interestingly, IL-2 activation of all these pathways was not observed in Tax-immortalized cells as only STAT5 activation was detected. A systematic computational approach that coupled microarray analysis to transcription factor binding site prediction was subsequently performed to evaluate STAT5-regulated gene expression in response to IL-2 stimulation in both cell types. Primary human T-cells exhibited numerous genes modulated by the growth factor, of which several were predicted to be regulated by STAT5 due to possession of a putative STAT5 binding site. Many genes were also found to be regulated by IL-2 in Tax-immortalized cells. Interestingly, several of them, including the cytokines il-5, il-9, and il-13, were unique to these cells and discovered to be STAT5 targets. Taken together, the results presented here provide an increased understanding of the molecular mechanisms induced by IL-2 stimulation in primary human T-cells and Tax-immortalized cells, as well as demonstrate how bioinformatics provides valuable tools for scientific discovery.