Identifying regulatory networks by combinatorial analysis of promoter elements.

Several computational methods based on microarray data are currently used to study genome-wide transcriptional regulation. Few studies, however, address the combinatorial nature of transcription, a well-established phenomenon in eukaryotes. Here we describe a new approach using microarray data to uncover novel functional motif combinations in the promoters of Saccharomyces cerevisiae. In addition to identifying novel motif combinations that affect expression patterns during the cell cycle, sporulation and various stress responses, we observed regulatory cross-talk among several of these processes. We have also generated motif-association maps that provide a global view of transcription networks. The maps are highly connected, suggesting that a small number of transcription factors are responsible for a complex set of expression patterns in diverse conditions. This approach may be useful for modeling transcriptional regulatory networks in more complex eukaryotes.

The Swi/Snf family nucleosome-remodeling complexes and transcriptional control.

The Swi/Snf family of nucleosome-remodeling complexes has been shown to play important roles in gene expression throughout eukaryotes. Genetic and biochemical studies previously suggested that Swi/Snf activates transcription by remodeling nucleosomes, thereby permitting increased access of transcription factors for their binding sites. Recent studies have identified additional Swi/Snf biochemical activities and have suggested possible mechanisms by which Swi/Snf is targeted to specific promoters. Surprisingly, studies have also revealed that, besides being necessary for activation, Swi/Snf is required for transcriptional repression of some genes. These analyses have transformed our understanding of the function of the Swi/Snf family of complexes and suggest that they control transcription in diverse ways.

Whole-genome expression analysis of snf/swi mutants of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Snf/Swi complex has been previously demonstrated to control transcription and chromatin structure of particular genes in vivo and to remodel nucleosomes in vitro. We have performed whole-genome expression analysis, using DNA microarrays, to study mutants deleted for a gene encoding one conserved (Snf2) or one unconserved (Swi1) Snf/Swi component. This analysis was performed on cells grown in both rich and minimal media. The microarray results, combined with Northern blot, computational, and genetic analyses, show that snf2Delta and swi1Delta mutations cause similar effects on mRNA levels, that Snf/Swi controls some genes differently in rich and minimal media, and that Snf/Swi control is exerted at the level of individual genes rather than over larger chromosomal domains. In addition, this work shows that Snf/Swi controls mRNA levels of MATalpha-specific genes, likely via controlling transcription of the regulators MATalpha1 and MCM1. Finally, we provide evidence that Snf/Swi acts both as an activator and as a repressor of transcription, and that neither mode of control is an indirect effect of the other.

The nucleosome remodeling complex, Snf/Swi, is required for the maintenance of transcription in vivo and is partially redundant with the histone acetyltransferase, Gcn5.

Snf/Swi, a nucleosome remodeling complex, is important for overcoming nucleosome-mediated repression of transcription in Saccharomyces cerevisiae. We have addressed the mechanism by which Snf/Swi controls transcription in vivo of an Snf/Swi-dependent promoter, that of the SUC2 gene. By single-cell analysis, our results show that Snf/Swi is required for activated levels of SUC2 expression in every cell of a population. In addition, Snf/Swi is required for maintenance of SUC2 transcription, suggesting that continuous chromatin remodeling is necessary to maintain an active transcriptional state. Finally, Snf/Swi and Gcn5, a histone acetyltransferase, have partially redundant roles in the control of SUC2 transcription, suggesting a functional overlap between two different mechanisms believed to overcome repression by nucleosomes, nucleosome remodeling and histone acetylation.

Regions of RAG1 protein critical for V(D)J recombination.

The products of the recombination activating genes RAG1 and RAG2 are essential for activating V(D)J recombination, and thus are indispensable for the production of functional and diverse antigen receptors. To investigate the function of RAG1, we have tested a series of insertion and substitution mutation for their ability to induce V(D)J rearrangement on both deletional and inversional plasmid substrates. With these substrates we were also able to assess the effects of these mutations on both coding and signal joint formation, and to show that any one mutant affected all these reactions similarly. As defined previously, the core active regions of RAG1 and RAG2 permit the deletion of 40% and 25%, respectively, of well-conversed sequence. We show here that this "dispensable" region of RAG1 is not necessary for coding joint formation or recombination of an integrated substrate, and this portion is not functionally redundant with the "dispensable" region of RAG2. Recombination with these core regions is also still subject to the 12/23 joining rule. Further, the minimal essential core region of RAG1 can be located within an even smaller portion of the gene.