The overall goals of this proposal are to elucidate the mechanisms of pre-initiation complex (PIC) assembly in vivo and in vitro under the instruction of specific activator complexes and the molecular basis by which chromatin is modified to bring about transcriptional activation. We will also carry out in vivo experiments to dissect the spatial distribution and dynamics of the different components of the pre-initiation complex, histone acetyl-transferase, ATP-dependent chromatin remodelling complexes and RNA polymerase II in the nucleus in vivo. The research program proposed here relies on the close collaboration of seven groups, each of which has been previously working on different aspects of transcriptional regulation and each of which has traditionally used different techniques. Only the combination of the complementary expertise of each group, together with the combined efforts of all seven groups will allow us to reach our goals. The most pressing issues in the field are related to the fact that more than 100 proteins are believed to be involved in helping the RNA polymerase II to transcribe an eukaryotic gene in vivo. In addition these proteins are found in multiple sub-complexes. Therefore, the questions arise as to how the interaction of so many DNA-tethered activating complexes can trigger formation of the RIC and the consequent transcription of the gene. For example, are there gene- or tissue- specific PIC complexes assembled on the gene promoters? How do they assemble? Are all proteins in the complexes required for activation? Does the transcription complex form in a single, highly co-operative step or is recruitment a staged process? How does the local chromatin architecture affect or direct these effects? The participating seven groups have generated in the past sufficient basic knowledge and possess several valuable biochemical and genetic tools that will allow us to carry out the proposed research that is aimed to answer these important questions underlying specific gene activation. A number of short and long-term goals are envisaged concerning specific questions that are of common interest to the participating groups.

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