Polycystic kidney disease (PKD) results from loss-offunction mutations in the PKD1 gene. There are also reports showing abnormally high levels of PKD1 expression in cystic epithelial cells. At present, nothing is known about the molecular mechanisms regulating the normal expression of the PKD1 gene or whether transcriptional disregulation of the PKD1 gene has a role in cyst formation. We have analyzed a 3.3-kb 5-proximal portion of the human PKD1 gene. Sequence analysis revealed the presence of consensus sequences for numerous transactivating factors, including four T-cell factor (TCF) binding elements (TBEs). Transcriptional activity of the 3.3-kb fragment and a series of deletion constructs was assayed in HEK293T cells. A 2.0-kb proximal promoter region containing one of the four TBEs (TBE1) was inducible up to 6-fold by cotransfection with ␤-catenin. ␤-catenin-mediated induction was inhibited by dominant-negative TCF and by deletion of the TBE1 sequence. 15-or 109-bp sequences containing the TBE1 site, when cloned upstream of a minimal promoter, were shown to respond to ␤-catenin induction. Gel shift assays confirmed that the TBE1 site is capable of forming complexes with TCF and ␤-catenin. To determine whether expression of the endogenous PKD1 gene responds to ␤-catenin, HT1080 cells were treated with LiCl, and HeLa cells were stably transfected with ␤-catenin. In both cases, endogenous PKD1 mRNA levels were elevated in response to these treatments. Taken together, these studies define an active PKD1 promoter region and suggest that the PKD1 gene is a target of the ␤-catenin/TCF pathway.

Human ␤-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves ␤-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Amino acid sequence and structural homology of hGUSB and Escherichia coli ␤-galactosidase active sites led us to propose that residues Glu

Viral promoters are widely utilized in commercial and customized vectors to drive expression of genes of interest including reporter, effector and transfection control, because of their high transcription efficiency in a variety of primary and transformed cell lines. However, we observed altered rate of transcription for these promoters under conditions such as presence of an effector protein. These variations in viral promoter driven expressions can potentially lead to incorrect conclusion, especially in comparative and quantitative experiments. We found significantly reduced viral promoter activity in cells overexpressing tumor suppressor protein p53, whereas markedly induced transcription in cells overexpressing MAP/ERK kinase kinase 1 (Mekk 1). Using deletion constructs generated from the CMV promoter, we found the transcription reduction by p53 is possibly mediated through the TATA motif present in proximal CMV promoter. The activation of the CMV promoter by Mekk1, on the other hand, is attributed to the proximal CRE binding site in the promoter. These findings may be of interest to investigators who use CMV (or other viral) promoter driven vectors for either comparative or quantitative gene expression, or effect on promoter activity.

Mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNF␣, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation.