Repression of the yeast HO gene by the MATalpha2 and MATa1 homeodomain proteins

The HO gene in Saccharomyces cerevisiae is regulated by a large and complex promoter that is similar to promoters in higher order eukaryotes. Within this promoter are 10 potential binding sites for the a1-α2 heterodimer, which represses HO and other haploid-specific genes in diploid yeast cells. We have determined that a1-α2 binds to these sites with differing affinity, and that while certain strong-affinity sites are crucial for repression of HO, some of the weak-affinity sites are dispensable. However, these weak-affinity a1-α2-binding sites are strongly conserved in related yeast species and have a role in maintaining repression upon the loss of strong-affinity sites. We found that these weak sites are sufficient for a1-α2 to partially repress HO and recruit the Tup1-Cyc8 (Tup1-Ssn6) co-repressor complex to the HO promoter. We demonstrate that the Swi5 activator protein is not bound to URS1 in diploid cells, suggesting that recruitment of the Tup1-Cyc8 complex by a1-α2 prevents DNA binding by activator proteins resulting in repression of HO.     

Regulation of human Cripto-1 gene expression by TGF-beta1 and BMP-4 in embryonal and colon cancer cells

Human Cripto-1 (CR-1) is a cell membrane protein that is overexpressed in several different types of human carcinomas. In the present study we investigated the mechanisms that regulate the expression of CR-1 gene in cancer cells. We cloned a 2,481 bp 5'-flanking region of the human CR-1 gene into a luciferase reporter vector and transfected NTERA-2 human embryonal carcinoma cells and LS174-T colon cancer cells to test for promoter activity. Activity of CR-1 promoter in both cell lines was modulated by two TGF-beta family members, TGF-beta1 and BMP-4. In particular, TGF-beta1 significantly up-regulated CR-1 promoter activity, whereas a dramatic reduction in CR-1 promoter activity was observed with BMP-4 in NTERA-2 and LS174-T cells. Changes in the CR-1 promoter activity following TGF-beta1 and BMP-4 treatments correlated with changes in CR-1 mRNA and protein expression in NTERA-2 and LS174-T cells. We also identified three Smad binding elements (SBEs) within the CR-1 promoter and point mutation of SBE1 (-2,197/-2,189) significantly reduced response of the CR-1 promoter to both TGF-beta1 and BMP-4 in NTERA-2 and LS174-T cells. Chromatin immunoprecipitation assay also demonstrated binding of Smad-4 to a CR-1 promoter DNA sequence containing SBE1 in LS174-T cells. Finally, BMP-4 inhibited migration of LS174-T cells and F9 mouse embryonal carcinoma cells by downregulation of CR-1 protein. In conclusion, these results suggest a differential modulation of CR-1 gene expression in embryonal and colon cancer cells by two different members of the TGF-beta family.     

Neuroectodermal and endodermal expression of the ascidian Cdx gene is separated by metamorphosis

Ascidians are a group of invertebrate chordates that exhibit a biphasic life history, with chordate-specific structures developing during embryogenesis (dorsal neural tube and notochord) and metamorphosis (pharyngeal gill slits and endostyle). Here we characterize the expression of a caudal/Cdx gene homologue, Hec-Cdx, from the ascidian Herdmania curvata. Vertebrate Cdx genes are expressed at gastrulation and in the posterior of the developing neural tube and endoderm. Hec-Cdx expression is initiated at the earliest stages of gastrulation, with peaks in RNA abundance occurring first during neurulation and tailbud extension and then in 3- to 5-day-old juveniles. Hec-Cdx is expressed in a pair of cells in the anterior lip of the blastopore in the late gastrula which form the most posterior portion of the neural plate. During tailbud formation expression is maintained in and solely restricted to these cells. During metamorphosis expression is localized to the intestine of the juvenile. These data, along with data for the H. curvata Otx gene, suggest that the evolution of the novel ascidian biphasic body plan was not accompanied by a deployment of these genes into new pathways but by a temporal separation of tissue-specific expression. Received: 10 October 1999 / Accepted: 1 November 1999