Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling.

The Wnt signal-transduction pathway induces the nuclear translocation of membrane-bound beta-catenin (Catnb) and has a key role in cell-fate determination. Tight somatic regulation of this signal is essential, as uncontrolled nuclear accumulation of beta-catenin can cause developmental defects and tumorigenesis in the adult organism. The adenomatous polyposis coli gene (APC) is a major controller of the Wnt pathway and is essential to prevent tumorigenesis in a variety of tissues and organs. Here, we have investigated the effect of different mutations in Apc on the differentiation potential of mouse embryonic stem (ES) cells. We provide genetic and molecular evidence that the ability and sensitivity of ES cells to differentiate into the three germ layers is inhibited by increased doses of beta-catenin by specific Apc mutations. These range from a severe differentiation blockade in Apc alleles completely deficient in beta-catenin regulation to more specific neuroectodermal, dorsal mesodermal and endodermal defects in more hypomorphic alleles. Accordingly, a targeted oncogenic mutation in Catnb also affects the differentiation potential of ES cells. Expression profiling of wildtype and Apc-mutated teratomas supports the differentiation defects at the molecular level and pinpoints a large number of downstream structural and regulating genes. Chimeric experiments showed that this effect is cell-autonomous. Our results imply that constitutive activation of the Apc/beta-catenin signaling pathway results in differentiation defects in tissue homeostasis, and possibly underlies tumorigenesis in the colon and other self-renewing tissues.

Tight control of transcription in Toxoplasma gondii using an alternative tet repressor.

Fusion of yellow fluorescent protein (YFP) to the N-terminus of the Escherichia coli Tn10 tet repressor (TetR) created a functional YFP-TetR repressor with the capacity of 88-fold repression of transcription when expressed in Toxoplasma gondii. As a test promoter we used the T. gondii ribosomal protein RPS13 promoter for which we provide experimental evidence of having a single major transcriptional start site, a condition favourable to the design of inducible expression systems. Integration of four tet operator (tetO) elements, 23-43 bp upstream of the RPS13 transcriptional start site, resulted in maximal repression of transcription (88-fold). Moreover, integration of these four tetO elements reduced the promoter activity only 20% in comparison with the wildtype promoter. Regulation was six-fold higher compared with an inducible expression system employing wildtype TetR. Importantly, only 0.1 microg/ml tetracycline was required for maximal induction demonstrating a higher affinity of tetracycline for YFP-TetR than for wildtype TetR which required 1 microg/ml tetracycline for maximal induction. The use of 0.1 microg/ml tetracycline allows prolonged continuous culturing of T. gondii for which levels of 1 microg/ml tetracycline are toxic. Our results show that YFP-TetR is superior to TetR for transcriptional regulation in T. gondii and we expect that its improved characteristics will be exploitable in other parasites or higher eukaryotes.

De novo ribosome biosynthesis is transcriptionally regulated in Eimeria tenella, dependent on its life cycle stage.

Protozoan parasites go through various developmental stages during their parasitic life, which requires the expression of different genes. To identify stage specific gene products in Eimeria tenella, a differential screening was performed comparing the intracellular schizont stage with the extracellular oocyst stage. De novo transcripts of 18S-5.8S-26S rRNA transcription units and of two ribosomal proteins (RPL5 and RPL23) were specifically identified in schizonts and were undetectable in oocysts. The stage specific transcription of pre-rRNAs (prior to processing) was confirmed with Northern blot analysis. Since the E. tenella genome contains a repeated gene cluster with an estimated 140 large rRNA transcription units, they all might be similarly regulated. Specific expression of RPL5 and RPL23 in E. tenella schizonts was also confirmed by Northern blotting. Furthermore, an analysis of the E. tenella EST database with 26,705 ESTs showed that 9.5% of all merozoite ESTs and only 0.2% of the sporozoite ESTs encoded ribosomal proteins (RPs). These ESTs encoded 69 different RPs, suggesting that most and possibly all RPs are differentially transcribed in E. tenella. Analysis of EST data from other Coccidia, such as Toxoplasma gondii, indicated a similar stage dependent transcription of RP genes. We conclude that ribosome biosynthesis is transcriptionally regulated in E. tenella and other Coccidia, such that rapidly growing parasite stages utilize much of their resources to de novo biosynthesis of ribosomes, and that "dormant" oocyst stages do not synthesize new ribosomes. The 50- to 100-fold reduction in transcription of RPs together with the reduced rRNA transcription prevents that unnecessary new ribosomes are synthesized in oocysts.