Abstract 292: Validation of a Peripheral Blood Mononuclear Cell Gene Expression Network. A Systems Biological Approach to the Identification of Immunomodulatory Gene Targets.

Background: Reconstruction of intracellular networks underlying complex processes is a formidable challenge. We hypothesized that the human peripheral blood mononuclear cell (PBMC) molecular network can be delineated using a systems-biological/computer-driven approach. Method: We used a large PBMC microarray library, an information-theoretic, reverse-engineering algorithm (ARACNe, Algorithm for the Reconstruction of Accurate Cellular Networks) and Chromatin Immunoprecipitation (ChIP) assay to reconstruct and validate a putative gene interaction network. Results: Gene expression data from 285 focused-microarrays obtained from 98 heart transplant patients enrolled in the Cardiac Allograft Rejection Gene expression Observational (CARGO) study was analyzed by ARACNe to identify highly connected “hub” genes. Biological criteria identified Transcription Factor CREB and its subnetwork as important candidates involved in the regulation of the alloimmune response. ARACNe predicted 29 putative direct interactions (first neighbor genes) of CREB (Fig. 1A ). Eleven of these (Fig. 1A red nodes) were previously reported (p<0.001) and 18 were not (Fig 1A , pink nodes). Out of the non validated, 14 suitable primers were identified and 11 could be immunoprecipitated (Fig 1B ), demonstrating binding of CREB to their promoter in vivo. Overall, 22 (75%) inferred CREB targets were validated; a significantly higher fraction than randomly expected (p<0.001, Fisher’s exact). Conclusion Our results confirm the accuracy of ARACNe to reconstruct the PBMC transcriptional network. Systems biological approaches may be used to identify immunomodulatory pharmacological targets. Figure 1