| Regulation of gene expression is fundamental to biological systems. Previous research into mechanisms of transcription regulation focused on a restricted number of model genes, often studied using in vitro assays. The availability of whole genome sequences allows genome-wide approaches to be applied to understanding transcription regulation in vivo. The first aim of this proposal is to develop assays that will allow determination of the localization of key components of the RNA polymerase II transcription apparatus, as well as other DNA-bound factors, in chromatin. To this end in vivo crosslinking of protein-DNA complexes will be combined with DNA microarray technology (ChIP-on-chip). Three critical improvements will be made on recently developed techniques. 1) To improve sensitivity, accuracy, and resolution, localization microarrays will be developed that encorporate approximately 13,000 70-mer oligonucleotides. 2) External control normalization procedures will be introduced that for the first time will allow the localization of globally-binding factors. 3) Presently available protein-tagging techniques will be improved by systematic evaluation of different tags (HA, myc, TAP) and development of new high-affinity tags. Implementation of this will allow mapping of the locations of globally-bound factors on the genome in vivo. This will then be applied to TFIID, SAGA and Tup1-Ssn6 complexes. These are major effectors of gene-specific regulation. The project will be carried out in the yeast Saccharomyces cerevisiae, serving as a test-bed for similar analyses in mammalian cells. We will follow the dynamic redistribution of these complexes during transcriptional reprogramming. Together with ongoing microarray expression analyses and through bioinformatic combination of different genome-wide datasources, this will result in: understanding transcription regulation pathways; links to regulation of diverse cellular processes; insight into the dynamics of transcription machinery assembly and functioning of large protein complexes. It is important to note that any cellular process involving DNA as a template can be studied by the proposed technology. |
