New approaches for in silico identification of cytokine-modified beta cell gene networks.

Beta cell dysfunction and death in type 1 diabetes mellitus (T1DM) is caused by direct contact with activated macrophages and T lymphocytes and by exposure to soluble mediators secreted by these cells, such as cytokines and nitric oxide. Cytokine-induced apoptosis depends on the expression of pro- and anti-apoptotic genes that remain to be characterized. Using microarray analyses, we identified several transcription factor and "effector" gene networks regulated by interleukin-1beta and/or interferon-gamma in beta cells. This suggests that beta cell fate following exposure to cytokines is a complex and highly regulated process, depending on the duration and severity of perturbation of key gene networks. In order to draw correct conclusions from these massive amounts of data, we need to utilize novel bioinformatics and statistical tools. Thus, we are presently performing in silico analysis for the localization of binding sites for the transcription factor NF-kappaB (previously shown to be pivotal for beta cell apoptosis) in 15 temporally related gene clusters, identified by time-course microarray analysis. In silico analysis is based on a broad range of computational techniques used to detect motifs in a DNA sequence corresponding to the binding site of a transcription factor. These computer-based findings must be validated by use of positive and negative controls, and by "ChIP on chip" analysis. Moreover, new statistical approaches are required to decrease false positive findings. These novel approaches will constitute a "proof of principle" for the integrated use of bioinformatics and functional genomics in the characterization of relevant cytokine-regulated beta cell gene networks leading to beta cell apoptosis in T1DM.