Jason S. Iacovoni

A new inducible protein expression system in fission yeast based on the glucose-repressed inv1 promoter

Studies of the fission yeast Schizosaccharomyces pombe have made major contributions towards understanding cell-cycle control and many other important aspects of cell biology. A series of pREP expression vectors that utilize the thiamine-repressible nmt1 promoter are used routinely to manipulate the expression of genes in fission yeast. A shortcoming of the nmt1 promoter is that it is very slowly induced following removal of thiamine from the growth medium, requiring approx. 16h for full induction. Invertase, an enzyme responsible for sucrose metabolism, is regulated transcriptionally by glucose derepression in S. pombe. Using the inv1 promoter, we have developed the pINV1 set of inducible protein expression vectors. A shift from glucose to sucrose-based culture medium leads to a very rapid induction of the inv1 promoter. Genes that are regulated by the inv1 promoter are fully induced within 1h of the shift to sucrose-based medium. The pINV1 vectors utilize a simple induction protocol and enable studies in fission yeast requiring tight and rapid regulation of protein synthesis.

Avian winged helix proteins CWH-1, CWH-2 and CWH-3 repress transcription from Qin binding sites.

The chicken winged helix proteins, CWH-1, CWH-2 and CWH-3, were isolated and identified by homology cloning using the winged helix sequence of the retroviral oncogene qin as a probe. The CWH proteins act as growth stimulators in chicken embryo fibroblasts and in this activity resemble the Qin protein. Qin is a transcriptional regulator that functions as a repressor, and its oncogenic potential is correlated with the ability to repress transcription. In this communication we show that CWH proteins are localized in the cell nucleus, recognize the Qin DNA binding site and also function as transcriptional repressors. The repression activity of CWH-3 was mapped to the region of amino acids 211 to 311, a domain that is homologous to the major repression domain of Qin.

Glutaredoxin is a direct target of oncogenic jun.

We have analysed differential gene expression in v-jun-transformed chicken embryo fibroblasts (CEF) compared to normal CEF by using the directional tag PCR subtraction method. From a first generation of putative Jun targets four clones were selected for study; they are upregulated in jun-transformed cells. Three of these clones showed homology to known genes: glutaredoxin, growth associated protein (GAP)-43/neuromodulin, and phenobarbital-induced cytochrome P450. The expression of these genes was analysed in fibroblasts transformed by various oncogenes. Expression of the glutaredoxin mRNA could be induced by a Jun-estrogen receptor chimaera in the absence of de novo protein biosynthesis. Based on this observation we conclude that glutaredoxin is a direct target of v-Jun.

v-Jun targets showing an expression pattern that correlates with the transformed cellular phenotype

Targets of the oncogenic transcription factor v-Jun in the murine cell line C3H 10T1/2 cells have been identified using DNA microarrays. Two targets, Akap12 and Marcks, are downregulated in transformed cells and are known tumor suppressor genes. Overexpression of either Akap12 or Marcks in v-Jun-transformed cells reverses the transformed phenotype and leads to the re-expression of the other tumor suppressor gene, suggesting that these two genes cooperate in the establishment of the nontransformed state. Reverted cells continue to express v-Jun at high levels and also re-express c-Jun, which is normally repressed by v-Jun. A panel of six cell lines has been generated to evaluate the expression levels of other v-Jun targets in 10T1/2 cells. With these cells, we find that the upregulated target Sprr1a has an expression pattern that correlates with the transformed phenotype.