The disorderly study of ordered recruitment.

Activated transcription in eukaryotes requires the aid of numerous co-factors to overcome the physical barriers chromatin poses to activation, bridge the gap between activators and polymerase, and ensure appropriate regulation. S. cerevisiae has long been a model organism for studying the role of co-activators in the steps leading up to gene activation. Detailed studies on the recruitment of these co-activators have been carried out for more than a dozen promoters. Taking a step back to survey these results, however, suggests that there are few generalizations that could be used to guide future studies of uncharacterized promoters.

The transcriptional coactivators SAGA, SWI/SNF, and mediator make distinct contributions to activation of glucose-repressed genes.

The paradigm of activation via ordered recruitment has evolved into a complicated picture as the influence of coactivators and chromatin structures on gene regulation becomes understood. We present here a comprehensive study of many elements of activation of ADH2 and FBP1, two glucose-regulated genes. We identify SWI/SNF as the major chromatin-remodeling complex at these genes, whereas SAGA (Spt-Ada-Gcn5-acetyltransferase complex) is required for stable recruitment of other coactivators. Mediator plays a crucial role in expression of both genes but does not affect chromatin remodeling. We found that Adr1 bound unaided by coactivators to ADH2, but Cat8 binding depended on coactivators at FBP1. Taken together, our results suggest that commonly regulated genes share many aspects of activation, but that gene-specific regulators or elements of promoter architecture may account for small differences in the mechanism of activation. Finally, we found that activator overexpression can compensate for the loss of SWI/SNF but not for the loss of SAGA.

Artificial recruitment of mediator by the DNA-binding domain of Adr1 overcomes glucose repression of ADH2 expression.

The transcription factor Adr1 activates numerous genes in nonfermentable carbon source metabolism. An unknown mechanism prevents Adr1 from stably binding to the promoters of these genes in glucose-grown cells. Glucose depletion leads to Snf1-dependent binding. Chromatin immunoprecipitation showed that the Adr1 DNA-binding domain could not be detected at the ADH2 promoter under conditions in which the binding of the full-length protein occurred. This suggested that an activation domain is required for stable binding, and coactivators may stabilize the interaction with the promoter. Artificial recruitment of Mediator tail subunits by fusion to the Adr1 DNA-binding domain overcame both the inhibition of promoter binding and glucose repression of ADH2 expression. In contrast, an Adr1 DNA-binding domain-Tbp fusion did not overcome glucose repression, although it was an efficient activator of ADH2 expression under derepressing conditions. When Mediator was artificially recruited, ADH2 expression was independent of SNF1, SAGA, and Swi/Snf, whereas ADH2 expression was dependent on these factors with wild-type Adr1. These results suggest that in the presence of glucose, the ADH2 promoter is accessible to Adr1 but that other interactions that occur when glucose is depleted do not take place. Artificial recruitment of Mediator appears to overcome this requirement and to allow stable binding and transcription under normally inhibitory conditions.

Adr1 and Cat8 mediate coactivator recruitment and chromatin remodeling at glucose-regulated genes.

BACKGROUND: Adr1 and Cat8 co-regulate numerous glucose-repressed genes in S. cerevisiae, presenting a unique opportunity to explore their individual roles in coactivator recruitment, chromatin remodeling, and transcription. METHODOLOGY/PRINCIPAL FINDINGS: We determined the individual contributions of Cat8 and Adr1 on the expression of a cohort of glucose-repressed genes and found three broad categories: genes that need both activators for full derepression, genes that rely mostly on Cat8 and genes that require only Adr1. Through combined expression and recruitment data, along with analysis of chromatin remodeling at two of these genes, ADH2 and FBP1, we clarified how these activators achieve this wide range of co-regulation. We find that Adr1 and Cat8 are not intrinsically different in their abilities to recruit coactivators but rather, promoter context appears to dictate which activator is responsible for recruitment to specific genes. These promoter-specific contributions are also apparent in the chromatin remodeling that accompanies derepression: ADH2 requires both Adr1 and Cat8, whereas, at FBP1, significant remodeling occurs with Cat8 alone. Although over-expression of Adr1 can compensate for loss of Cat8 at many genes in terms of both activation and chromatin remodeling, this over-expression cannot complement all of the cat8Delta phenotypes. CONCLUSIONS/SIGNIFICANCE: Thus, at many of the glucose-repressed genes, Cat8 and Adr1 appear to have interchangeable roles and promoter architecture may dictate the roles of these activators.

A poised initiation complex is activated by SNF1.

Snf1, the yeast AMP kinase homolog, is essential for derepression of glucose-repressed genes that are activated by Adr1. Although required for Adr1 DNA binding, the precise role of Snf1 is unknown. Deletion of histone deacetylase genes allowed constitutive promoter binding of Adr1 and Cat8, another activator of glucose-repressed genes. In repressed conditions, at the Adr1-and Cat8-dependent ADH2 promoter, partial chromatin remodeling had occurred, and the activators recruited a partial preinitiation complex that included RNA polymerase II. Transcription did not occur, however, unless Snf1 was activated, suggesting a Snf1-dependent event that occurs after RNA polymerase II recruitment. Glucose regulation persisted because shifting to low glucose increased expression. Glucose repression could be completely relieved by combining the three elements of 1) chromatin perturbation by mutation of histone deacetylases, 2) activation of Snf1, and 3) the addition of an Adr1 mutant that by itself confers only weak constitutive activity.

Yeast mediator and its role in transcriptional regulation.

Activated eukaryotic transcription requires the components of the Mediator complex, which can act as both a positive and negative regulator of transcription. This review of the yeast Saccharomyces cerevisiae Mediator complex describes the role of Mediator and its effects on transcriptional regulation. One focal point of the review is to summarize new information regarding the negative effect of Mediator on transcription and suggest a possible mechanism that encompasses the latest results.

Evidence of transmission of Burkholderia cepacia, Burkholderia multivorans and Burkholderia dolosa among persons with cystic fibrosis.

Previous studies have identified specific Burkholderia cepacia complex strains that are common to multiple persons with cystic fibrosis (CF). Such so-called epidemic strains have an apparent enhanced capacity for inter-patient spread and reside primarily in Burkholderia cenocepacia (formerly B. cepacia complex genomovar III). We sought to identify strains from B. cepacia complex species other than B. cenocepacia that are similarly shared by multiple CF patients. We performed genotype analysis of 360 recent sputum culture isolates from 360 persons residing in 29 cities by using repetitive extragenic palendromic polymerase chain reaction (rep-PCR) and pulsed field gel electrophoresis. The results indicate that sharing of a common Burkholderia multivorans strain occurs relatively infrequently; however, several small clusters of patients infected with the same strain were identified. A cluster of seven patients infected with the same B. cepacia (genomovar I) strain was found. We also identified a large group of 28 patients receiving care in the same treatment center and infected with the same Burkholderia dolosa strain. These observations suggest that B. cepacia complex strains in species other than B. cenocepacia may be spread among CF patients.