Transcriptional regulation of the squalene synthase gene (ERG9) in the yeast Saccharomyces cerevisiae.
The ergosterol biosynthetic pathway is a specific branch of the mevalonate pathway. Since the cells requirement for sterols is greater than for isoprenoids, sterol biosynthesis must be regulated independently of isoprenoid biosynthesis. In this study we explored the transcriptional regulation of squalene synthase (ERG9) in Saccharomyces cerevisiae, the first enzyme dedicated to the synthesis of sterols. A mutant search was performed to identify genes that were involved in the regulation of the expression of an ERG9-lacZ promoter fusion. Mutants with phenotypes consistent with known sterol biosynthetic mutations (ERG3, ERG7, ERG24) increased expression of ERG9. In addition, treatment of wild-type cells with the sterol inhibitors zaragozic acid and ketoconazole, which target squalene synthase and the C-14 sterol demethylase respectively, also caused an increase in ERG9 expression. The data also demonstrate that heme mutants increased ERG9 expression while anaerobic conditions decreased expression. Additionally, the heme activator protein transcription factors HAP1 and HAP2/3/4, the yeast activator protein transcription factor yAP-1, and the phospholipid transcription factor complex INO2/4 regulate ERG9 expression. ERG9 expression is decreased in hap1, hap2/3/4, and yap-1 mutants while ino2/4 mutants showed an increase in ERG9 expression. This study demonstrates that ERG9 transcription is regulated by several diverse factors, consistent with the idea that as the first step dedicated to the synthesis of sterols, squalene synthase gene expression and ultimately sterol biosynthesis is highly regulated. (+info)
Down-regulation of cyclin B1 gene transcription in terminally differentiated skeletal muscle cells is associated with loss of functional CCAAT-binding NF-Y complex.
The observation that cyclin B1 protein and mRNAs are down-regulated in terminally differentiated (TD) C2C12 cells, suggested us to investigate the transcriptional regulation of the cyclin B1 gene in these cells. Transfections of cyclin B1 promoter constructs indicate that two CCAAT boxes support cyclin B1 promoter activity in proliferating cells. EMSAs demonstrate that both CCAAT boxes are recognized by the trimeric NF-Y complex in proliferating but not in TD cells. Transfecting a dominant-negative mutant of NF-YA we provide evidence that NF-Y is required for maximal promoter activity. Addition of recombinant NF-YA to TD C2C12 nuclear extracts restores binding activity in vitro, thus indicating that the loss of NF-YA in TD cells is responsible for the lack of the NF-Y binding to the CCAAT boxes. Consistent with this, we found that the NF-YA protein is absent in TD C2C12 cells. In conclusion, our data demonstrate that NF-Y is required for cyclin B1 promoter activity. We also demonstrate that cyclin B1 expression is regulated at the transcriptional level in TD C2C12 cells and that the switch-off of cyclin B1 promoter activity in differentiated cells depends upon the loss of a functional NF-Y complex. In particular the loss of NF-YA protein is most likely responsible for its inactivation. (+info)
Activation domain-mediated targeting of the SWI/SNF complex to promoters stimulates transcription from nucleosome arrays.
The yeast SWI/SNF complex is required for the transcription of several yeast genes and has been shown to alter nucleosome structure in an ATP-dependent reaction. In this study, we show that the complex stimulated in vitro transcription from nucleosome templates in an activation domain-dependent manner. Transcription stimulation by SWI/SNF required an activation domain with which it directly interacts. The acidic activation domains of VP16, Gcn4, Swi5, and Hap4 interacted directly with the purified SWI/SNF complex and with the SWI/SNF complex in whole-cell extracts. The similarity of activation domain interactions and transcriptional stimulation between SWI/SNF and the SAGA histone acetyltransferase complex may account for their apparent overlapping functions in vivo. (+info)
Identification of proteins that interact with NF-YA.
We used the yeast two-hybrid system to show that the serum response factor (SRF) and zinc-fingers and homeobox 1 (ZHXI) proteins interact with the A subunit of nuclear factor-Y (NF-YA). GST pulldown assays revealed that both proteins interact specifically with NF-YA in vitro. Amino acids located between 272 and 564, a region that contains two homeodomains, are required for the interaction of ZHX1 with NF-YA. Two different domains of NF-YA, a glutamine-rich region and a serine/threonine-rich region, are necessary for the interactions with ZHX1 and SRF, respectively. (+info)
A nuclear factor Y (NFY) site positively regulates the human CD34 stem cell gene.
Proper regulation of the human CD34 gene requires a combinatorial action of multiple proximal and long-range, cis elements. This report shows that, like the murine CD34 5' untranslated region (UTR), the corresponding region of the human CD34 gene is necessary for optimal promoter activity. We localized the most critical element of this region to base pairs +48/+75. Through oligonucleotide competition and antibody supershift experiments in electrophoretic mobility shift assays, we found that this sequence contains a binding site (CCAAT box) for the transcription factor NFY (nuclear factor Y), a factor mediating cell type-specific and cell-cycle regulated expression of genes. Mutating this site led to a 5-fold decrease in CD34 promoter activity in transient transfection experiments. Interestingly, NFY binds adjacently to the earlier identified c-myb binding site. Here we show that both binding sites are important for CD34 promoter function: mutating either site alone decreased CD34 promoter-driven reporter gene activity 4-fold. We also show that the integrity of the c-myb binding site is necessary for stabilization of NFY binding to its site. Such cooperation between c-myb, which is expressed in early hematopoietic cells, and NFY, which is expressed in many cell types, might contribute to specific activation of CD34 in stem cells. The CCAAT box motif was also noted in the 5' UTR of the murine CD34 gene, however, NFY did not bind to this region. Thus, our results indicate that the functional similarities between the human and murine CD34 5' UTRs are achieved through different molecular mechanism(s). (+info)
Identification and characterization of basal and cyclic AMP response elements in the promoter of the rat GTP cyclohydrolase I gene.
5812 base pairs of rat GTP cyclohydrolase I (GTPCH) 5'-flanking region were cloned and sequenced, and the transcription start site was determined for the gene in rat liver. Progressive deletion analysis using transient transfection assays of luciferase reporter constructs defined the core promoter as a highly conserved 142-base pair GC-rich sequence upstream from the cap site. DNase I footprint analysis of this region revealed (5' --> 3') a Sp1/GC box, a noncanonical cAMP-response element (CRE), a CCAAT-box, and an E-box. Transcription from the core promoter in PC12 but not C6 or Rat2 cells was enhanced by incubation with 8-bromo-cyclic AMP. Mutagenesis showed that both the CRE and CCAAT-box independently contribute to basal and cAMP-dependent activity. The combined CRE and CCAAT-box cassette was also found to enhance basal transcription and confer cAMP sensitivity on a heterologous minimal promoter. The addition of the Sp1/GC box sequence to this minimal promoter construct inhibited basal transcription without affecting the cAMP response. EMSA showed that nuclear proteins from PC12 but not C6 or Rat2 cells bind the CRE as a complex containing activating transcription factor (ATF)-4 and CCAAT enhancer-binding protein beta, while both PC12 and C6 cell nuclear extracts were recruited by the CCAAT-box as a complex containing nuclear factor Y. Overexpression of ATF-4 in PC12 cells was found to transactivate the GTPCH promoter response to cAMP. These studies suggest that the elements required for cell type-specific cAMP-dependent enhancement of gene transcription are located along the GTPCH core promoter and include the CRE and adjacent CCAAT-box and the proteins ATF-4, CCAAT enhancer-binding protein beta, and nuclear factor Y. (+info)
Sterol regulation of human fatty acid synthase promoter I requires nuclear factor-Y- and Sp-1-binding sites.
To understand cholesterol-mediated regulation of human fatty acid synthase promoter I, we tested various 5'-deletion constructs of promoter I-luciferase reporter gene constructs in HepG2 cells. The reporter gene constructs that contained only the Sp-1-binding site (nucleotides -82 to -74) and the two tandem sterol regulatory elements (SREs; nucleotides -63 to -46) did not respond to cholesterol. Only the reporter gene constructs containing a nuclear factor-Y (NF-Y) sequence, the CCAAT sequence (nucleotides -90 to -86), an Sp-1 sequence, and the two tandem SREs responded to cholesterol. The NF-Y-binding site, therefore, is essential for cholesterol response. Mutating the SREs or the NF-Y site and inserting 4 bp between the Sp-1- and NF-Y-binding sites both resulted in a minimal cholesterol response of the reporter genes. Electrophoretic mobility-shift assays using anti-SRE-binding protein (SREBP) and anti-NF-Ya antibodies confirmed that these SREs and the NF-Y site bind the respective factors. We also identified a second Sp-1 site located between nucleotides -40 and -30 that can substitute for the mutated Sp-1 site located between nucleotides -82 and -74. The reporter gene expression of the wild-type promoter and the Sp-1 site (nucleotides -82 to -74) mutant promoter was similar when SREBP1a [the N-terminal domain of SREBP (amino acids 1-520)] was constitutively overexpressed, suggesting that Sp-1 recruits SREBP to the SREs. Under the same conditions, an NF-Y site mutation resulted in significant loss of reporter gene expression, suggesting that NF-Y is required to activate the cholesterol response. (+info)
Redirection of the respiro-fermentative flux distribution in Saccharomyces cerevisiae by overexpression of the transcription factor Hap4p.
Reduction of aerobic fermentation on sugars by altering the fermentative/oxidative balance is of significant interest for optimization of industrial production of Saccharomyces cerevisiae. Glucose control of oxidative metabolism in baker's yeast is partly mediated through transcriptional regulation of the Hap4p subunit of the Hap2/3/4/5p transcriptional activator complex. To alleviate glucose repression of oxidative metabolism, we constructed a yeast strain with constitutively elevated levels of Hap4p. Genetic analysis of expression levels of glucose-repressed genes and analysis of respiratory capacity showed that Hap4p overexpression (partly) relieves glucose repression of respiration. Analysis of the physiological properties of the Hap4p overproducer in batch cultures in fermentors (aerobic, glucose excess) has shown that the metabolism of this strain is more oxidative than in the wild-type strain, resulting in a significant reduced ethanol production and improvement of growth rate and a 40% gain in biomass yield. Our results show that modification of one or more transcriptional regulators can be a powerful and a widely applicable tool for redirection of metabolic fluxes in microorganisms. (+info)