François Robert

We have determined how most of the transcriptional regulators encoded in the eukaryote Saccharomyces cerevisiae associate with genes across the genome in living cells. Just as maps of metabolic networks describe the potential pathways that may be used by a cell to accomplish metabolic processes, this network of regulator-gene interactions describes potential pathways yeast cells can use to regulate global gene expression programs. We use this information to identify network motifs, the simplest units of network architecture, and demonstrate that an automated process can use motifs to assemble a transcriptional regulatory network structure. Our results reveal that eukaryotic cellular functions are highly connected through networks of transcriptional regulators that regulate other transcriptional regulators.

Understanding how DNA binding proteins control global gene expression and chromosomal maintenance requires knowledge of the chromosomal locations at which these proteins function in vivo. We developed a microarray method that reveals the genome-wide location of DNA-bound proteins and used this method to monitor binding of gene-specific transcription activators in yeast. A combination of location and expression profiles was used to identify genes whose expression is directly controlled by Gal4 and Ste12 as cells respond to changes in carbon source and mating pheromone, respectively. The results identify pathways that are coordinately regulated by each of the two activators and reveal previously unknown functions for Gal4 and Ste12. Genome-wide location analysis will facilitate investigation of gene regulatory networks, gene function, and genome maintenance.

H2A.Z is an evolutionary conserved histone variant involved in transcriptional regulation, antisilencing, silencing, and genome stability. The mechanism(s) by which H2A.Z regulates these various biological functions remains poorly defined, in part due to the lack of knowledge regarding its physical location along chromosomes and the bearing it has in regulating chromatin structure. Here we mapped H2A.Z across the yeast genome at an approximately 300-bp resolution, using chromatin immunoprecipitation combined with tiling microarrays. We have identified 4,862 small regions--typically one or two nucleosomes wide--decorated with H2A.Z. Those "Z loci" are predominantly found within specific nucleosomes in the promoter of inactive genes all across the genome. Furthermore, we have shown that H2A.Z can regulate nucleosome positioning at the GAL1 promoter. Within HZAD domains, the regions where H2A.Z shows an antisilencing function, H2A.Z is localized in a wider pattern, suggesting that the variant histone regulates a silencing and transcriptional activation via different mechanisms. Our data suggest that the incorporation of H2A.Z into specific promoter-bound nucleosomes configures chromatin structure to poise genes for transcriptional activation. The relevance of these findings to higher eukaryotes is discussed.