The Transcription Regulation Unit applies a transcriptional approach to the study of mechanisms of signal transduction in activate T-cells. In the past year the lab has undertaken two new initiatives to examine the role of transcriptional targeting during T-cell activation. The first initiative employs a high-throughput reporter assay to assess the transcriptional targeting of combinations of T-cell growth factors, immuno-suppressants and other immuno-reactive agents during T-cell activation. This method, referred to as Profiling of Transcriptional Targets (PTT), allows a simultaneous assessment of the targeting of specific gene promoters and promoter elements though the use of a 96-well electroporation device. The high density yield of information is then examined for trends in promoter targeting of various agents by Pearson Correlation coupled to a principal component analysis. In this way a global profiling of the influence of drug combinations on transcriptional responses in T-cells can be achieved. This method also provides a molecular analysis of the similarities and/or dissimilarities in the responsiveness of different gene promoters to specific drug and mitogen combinations. PTT forms the basis for a pharmacological dissection of the mitogen and/or drug targeting of gene promoter clusters identified by gene expression studies including differential display, cDNA micro-arrays, and SAGE analysis. It all provides a powerful approach for the molecular dissection of components involved in molecular signaling pathways through high-throughput gene insertion strategies to provide rapid feedback on gene over-expresssion, ablation or depletion studies using dominant negative expression vectors, anti-sense vectors, and RNAi constructs. We are also adapting the system to develop a mammalian two-hybrid system to screen for signal-dependent protein-protein interactions and post-translational modifications. Finally the use of episomal verus non-episomal vectors in PTT system will allow us to determine how signal transduction events influence chromatin structure in order to mediate cell-specific gene expression in activated T-cells. The second initiative is an extension of the work in the lab to define the in vivo formation of transcription factor complexes containing the transcriptional co-activator, p300, at the IL-2 gene. In order to define the molecular signal events that control the targeting of p300 and other transcriptional regulators to various promoter regions during T-cell activation we have combined the protein specificity of chromatin immuno-precipitation assay with potential for global analysis of micro-array technology. Genomic sequences, that are selectively immuno-precipitated after in vivo cross-linking to target proteins, are amplified and hybridized to micro-arrays containing known genomic promoter sequences. The assays is conducted over 2 hours at 15 minutes intervals after T-cell activation to assess simultaneously the dynamics of """"""""live"""""""" protein-DNA interactions during the T-cell activation process at multiple genes. We have successfully completed the proof -of-principle phase of this method. We are now utilizing this technique to define the possible existence of """"""""cis-regulatory element cluster codes"""""""" that define the association and retention times of co-regulator complexes with specific target genes. The goal of this work is to identify clearer and more effective molecular targets that will allow a selective disruption of abnormal cellular processes and responses by therapeutic intervention. Applications of this technique will enable the identification of common or shared in vivo protein targeting of the promoter elements of genes identified and clustered by gene expression studies. It will also help to define new regulatory regions of genes modulated during changes in cellular programming following signal transduction events. Finally this method will become a powerful tool to discover mechanisms of synergy and antagonism between downstream transcriptional regulatory factors and complexes.
