Role of Mediator in regulating Pol II elongation and nucleosome displacement in Saccharomyces cerevisiae.

Mediator is a modular multisubunit complex that functions as a critical coregulator of RNA polymerase II (Pol II) transcription. While it is well accepted that Mediator plays important roles in the assembly and function of the preinitiation complex (PIC), less is known of its potential roles in regulating downstream steps of the transcription cycle. Here we use a combination of genetic and molecular approaches to investigate Mediator regulation of Pol II elongation in the model eukaryote, Saccharomyces cerevisiae. We find that ewe (expression without heat shock element) mutations in conserved Mediator subunits Med7, Med14, Med19, and Med21-all located within or adjacent to the middle module-severely diminish heat-shock-induced expression of the Hsf1-regulated HSP82 gene. Interestingly, these mutations do not impede Pol II recruitment to the gene's promoter but instead impair its transit through the coding region. This implies that a normal function of Mediator is to regulate a postinitiation step at HSP82. In addition, displacement of histones from promoter and coding regions, a hallmark of activated heat-shock genes, is significantly impaired in the med14 and med21 mutants. Suggestive of a more general role, ewe mutations confer hypersensitivity to the anti-elongation drug 6-azauracil (6-AU) and one of them-med21-impairs Pol II processivity on a GAL1-regulated reporter gene. Taken together, our results suggest that yeast Mediator, acting principally through its middle module, can regulate Pol II elongation at both heat-shock and non-heat-shock genes.

Expression of the Pkd1 gene is momentously regulated by Sp1.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease that is caused by a mutation of a single gene inherited from either parent. Mutations in the Pkd1 gene result in the formation of multiple fluid-filled cysts in kidneys. In previous studies, the functional regulatory sequences of Pkd1 promoter region were detected by the use of comparative genome analysis. METHODS: To investigate the transcriptional regulation of the Pkd1 gene, the Pkd1 promoter was isolated. This promoter contains three Sp1-binding sites. Two of the sites which are found in a 300 bp fragment (-127 to +157) were mutated. An electrophoretic mobility shift assay (EMSA) was performed to determine which transcription factors are bound to Pkd1. RESULTS: Based on studies using a luciferase assay, the Sp1-A site (the nearest Sp1 to the ATG start codon) is more important for activation of Pkd1. The result of EMSA showed that Sp1 transcription factor binds with Pkd1 promoter regions. CONCLUSIONS: Two of the Sp1 sites were found in a proximal promoter region of Pkd1 (-127 to +157). Sp1 sites affect an important role in the activation of the gene. Especially, the Sp1-A site is more important for expression of Pkd1.

Gene expression profiling of rewarding effect in methamphetamine treated Bax-deficient mouse.

Methamphetamine is an illicit drug that is often abused and can cause neuropsychiatric and neurotoxic damage. Repeated administration of psychostimulants such as methamphetamine induces a behavioral sensitization. According to a previous study, Bax was involved in neurotoxicity by methamphetamine, but the function of Bax in rewarding effect has not yet been elucidated. Therefore, we have studied the function of Bax in a rewarding effect model. In the present study, we treated chronic methamphetamine exposure in a Bax-deficient mouse model and examined behavioral change using a conditioned place preference (CPP) test. The CPP score in Bax knockout mice was decreased compared to that of wild-type mice. Therefore, we screened for Bax-related genes that are involved in rewarding effect using microarray technology. In order to confirm microarray data, we applied the RT-PCR method to observe relative changes of Bcl2, a pro-apoptotic family gene. As a result, using our experiment microarray, we selected genes that were associated with Bax in microarray data, and eventually selected the Tgfbr2 gene. Expression of the Tgfbr2 gene was decreased by methamphetamine in Bax knockout mice, and the gene was overexpressed in Bax wild-type mice. Additionally, we confirmed that Creb, FosB, and c-Fos were related to rewarding effect and Bax using immunohistochemistry.

Inactivation of Mxi1 induces Il-8 secretion activation in polycystic kidney.

The Mxi1 proteins are biochemical and biological antagonists of c-myc oncoprotein. It has been reported that the overexpression pattern of c-myc might be similar to a molecular feature of early and late stages of human autosomal dominant polycystic kidney disease. We identified the cyst phenotype in Mxi1-deficient mice aged 6-12 months using H&E staining. Some chemokines containing a protein domain similar to human IL-8, which is associated with the inflammatory response, were subsequently selected from the up-regulated genes. We confirmed the expression level of these chemokines and measured protein concentrations of IL-8 using ELISA in the Mxi1-knockdown cells. IL-8 was found to be significantly increased in Mxi1-knockdown cells. We found that p38 MAP kinase activation was involved in the signal transduction of the Mxi1-inactivated secretion of IL-8. Therefore, we could suggest that the inactivation of Mxi1 leads to the inflammatory response and has the potential to induce polycystic renal disease.

The gene expression profiling in murine cortical cells undergoing programmed cell death (PCD) induced by serum deprivation.

PCD (programmed cell death) is important mechanism for development, homeostasis and disease. To analyze the gene expression pattern in brain cells undergoing PCD in response to serum deprivation, we analyzed the cDNA microarray consisting of 2,300 genes and 7 housekeeping genes of cortical cells derived from mouse embryonic brain. Cortical cells were induced apoptosis by serum deprivation for 8 hours. We identified 69 up-regulated genes and 21 down-regulated genes in apoptotic cells. Based on the cDNA microarray data four genes were selected and analyzed by RT-PCR and northern blotting. To characterize the role of UNC-51-like kinase (ULK2) gene in PCD, we investigated cell death effect by ULK2. And we examined expression of several genes that related with PCD. Especially GAPDH was increased by ULK2. Theses findings indicated that ULK2 is involved in apoptosis through p53 pathway.