Previous studies have suggested that transcription elongation results in changes in chromatin structure. Here we present studies of Saccharomyces cerevisiae Spt6, a conserved protein implicated in both transcription elongation and chromatin structure. Our results show that, surprisingly, an spt6 mutant permits aberrant transcription initiation from within coding regions. Furthermore, transcribed chromatin in the spt6 mutant is hypersensitive to micrococcal nuclease, and this hypersensitivity is suppressed by mutational inactivation of RNA polymerase II. These results suggest that Spt6 plays a critical role in maintaining normal chromatin structure during transcription elongation, thereby repressing transcription initiation from cryptic promoters. Other elongation and chromatin factors, including Spt16 and histone H3, appear to contribute to this control.

Transcription by RNA polymerase II in Saccharomyces cerevisiae and in humans is widespread, even in genomic regions that do not encode proteins. The purpose of such intergenic transcription is largely unknown, although it can be regulatory. We have discovered a role for one case of intergenic transcription by studying the S. cerevisiae SER3 gene. Our previous results demonstrated that transcription of SER3 is tightly repressed during growth in rich medium. We now show that the regulatory region of this gene is highly transcribed under these conditions and produces a non-protein-coding RNA (SRG1). Expression of the SRG1 RNA is required for repression of SER3. Additional experiments have demonstrated that repression occurs by a transcription-interference mechanism in which SRG1 transcription across the SER3 promoter interferes with the binding of activators. This work identifies a previously unknown class of transcriptional regulatory genes.

We have identified waved 3 (wa3), a novel recessive mutation that causes abnormalities of the heart and skin. The cardiac defect results in a severe and rapidly progressive dilated cardiomyopathy. We identified the gene mutated in these mice, which we call NFkB interacting protein1 (Nkip1), using positional cloning. Nkip1 is expressed in skin, heart and vascular endothelium and shares homology with a small family of proteins that play a role in the regulation of transcription factors. A C-terminal fragment of this protein was previously identified as the RelA associated inhibitor (RAI). We show that the full-length protein is larger than previously described, and we confirm that it interacts with NFkB in vivo. Expression analysis of genes known to be regulated by NFkB revealed that Intercellular adhesion molecule 1 (Icam1) expression is consistently elevated in mutant mice. This result suggests that wa3 mutant mice represent a potentially important model for the analysis of the role of inflammatory processes in heart disease.

The Spt-Ada-Gcn5-acetyltransferase (SAGA) complex of Saccharomyces cerevisiae is a multifunctional coactivator complex that has been shown to regulate transcription by distinct mechanisms. Previous results have shown that the Spt3 and Spt8 components of SAGA regulate initiation of transcription of particular genes by controlling the level of TATA-binding protein (TBP/Spt15) associated with the TATA box. While biochemical evidence exists for direct Spt8-TBP interactions, similar evidence for Spt3-TBP interactions has been lacking. To learn more about Spt3-TBP interactions in vivo, we have isolated a new class of spt3 mutations that cause a dominant-negative phenotype when overexpressed. These mutations all cluster within a conserved region of Spt3. The isolation of extragenic suppressors of one of these spt3 mutations has identified two new spt15 mutations that show allele-specific interactions with spt3 mutations with respect to transcription and the recruitment of TBP to particular promoters. In addition, these new spt15 mutations partially bypass an spt8 null mutation. Finally, we have examined the level of SAGA-TBP physical interaction in these mutants. While most spt3, spt8, and spt15 mutations do not alter SAGA-TBP interactions, one spt3 mutation, spt3-401, causes a greatly increased level of SAGA-TBP physical association. These results, taken together, suggest that a direct Spt3-TBP interaction is required for normal TBP levels at Spt3-dependent promoters in vivo.

Spt3 of Saccharomyces cerevisiae is required for the normal transcription of many genes in vivo. Past studies have shown that Spt3 is required for both mating and sporulation, two events that initiate when cells are at G1/START. We now show that Spt3 is needed for two other events that begin at G1/START, diploid filamentous growth and haploid invasive growth. In addition, Spt3 is required for normal expression of FLO11, a gene required for filamentous growth, although this defect is not the sole cause of the spt3Δ/spt3Δ filamentous growth defect. To extend our studies of Spt3's role in filamentous growth to the pathogenic yeast Candida albicans, we have identified the C. albicans SPT3 gene and have studied its role in C. albicans filamentous growth and virulence. Surprisingly, C. albicans spt3Δ/spt3Δ mutants are hyperfilamentous, the opposite phenotype observed for S. cerevisiae spt3Δ/spt3Δ mutants. Furthermore, C. albicans spt3Δ/spt3Δ mutants are avirulent in mice. These experiments demonstrate that Spt3 plays important but opposite roles in filamentous growth in S. cerevisiae and C. albicans.