Pancreatic beta cell-specific transcription of the pdx-1 gene. The role of conserved upstream control regions and their hepatic nuclear factor 3beta sites

To identify potential transactivators of pdx-1, we sequenced approximately 4.5 kilobases of the 5' promoter region of the human and chicken homologs, assuming that sequences conserved with the mouse gene would contain critical cis-regulatory elements. The sequences associated with hypersensitive site 1 (HSS1) represented the principal area of homology within which three conserved subdomains were apparent: area I (-2694 to -2561 base pairs (bp)), area II (-2139 to -1958 bp), and area III (-1879 to -1799 bp). The identities between the mouse and chicken/human genes are very high, ranging from 78 to 89%, although only areas I and III are present within this region in chicken. Pancreatic beta cell-selective expression was shown to be controlled by mouse and human area I or area II, but not area III, from an analysis of pdx-1-driven reporter activity in transfected beta- and non-beta cells. Mutational and functional analyses of conserved hepatic nuclear factor 3 (HNF3)-like sites located within area I and area II demonstrated that activation by these regions was mediated by HNF3beta. To determine if a similar regulatory relationship might exist within the context of the endogenous gene, pdx-1 expression was measured in embryonic stem cells in which one or both alleles of HNF3beta were inactivated. pdx-1 mRNA levels induced upon differentiation to embryoid bodies were down-regulated in homozygous null HNF3beta cells. Together, these results suggest that the conserved sequences represented by areas I and II define the binding sites for factors such as HNF3beta, which control islet beta cell-selective expression of the pdx-1 gene.     

The role of hepatocyte nuclear factor-3 alpha (Forkhead Box A1) and androgen receptor in transcriptional regulation of prostatic genes

Androgens and mesenchymal factors are essential extracellular signals for the development as well as the functional activity of the prostate epithelium. Little is known of the intraepithelial determinants that are involved in prostatic differentiation. Here we found that hepatocyte nuclear factor-3 alpha (HNF-3 alpha), an endoderm developmental factor, is essential for androgen receptor (AR)-mediated prostatic gene activation. Two HNF-3 cis-regulatory elements were identified in the rat probasin (PB) gene promoter, each immediately adjacent to an androgen response element. Remarkably, similar organization of HNF-3 and AR binding sites was observed in the prostate-specific antigen (PSA) gene core enhancer, suggesting a common functional mechanism. Mutations that disrupt these HNF-3 motifs significantly abolished the maximal androgen induction of PB and PSA activities. Overexpressing a mutant HNF-3 alpha deleted in the C-terminal region inhibited the androgen-induced promoter activity in LNCaP cells where endogenous HNF-3 alpha is expressed. Chromatin immunoprecipitation revealed in vivo that the occupancy of HNF-3 alpha on PSA enhancer can occur in an androgen-depleted condition, and before the recruitment of ligand-bound AR. A physical interaction of HNF-3 alpha and AR was detected through immunoprecipitation and confirmed by glutathione-S-transferase pull-down. This interaction is directly mediated through the DNA-binding domain/hinge region of AR and the forkhead domain of HNF-3 alpha. In addition, strong HNF-3 alpha expression, but not HNF-3 beta or HNF-3 gamma, is detected in both human and mouse prostatic epithelial cells where markers (PSA and PB) of differentiation are expressed. Taken together, these data support a model in which regulatory cues from the cell lineage and the extracellular environment coordinately establish the prostatic differentiated response.     

Role of hepatocyte nuclear factor 1alpha and 1beta in the transcriptional regulation of human dipeptidyl peptidase IV during differentiation of Caco-2 cells

Caco-2 cells undergo differentiation to an enterocytic-like cell when maintained in a post-confluent state for 1-2 weeks. During this period Caco-2 cells begin to express high levels brush border membrane associated enzymes such as dipeptidyl peptidase IV. Using the dipeptidyl peptidase IV gene promoter in electrophoretic mobility shift assays, we have shown for the first time that levels of hepatocyte nuclear factor 1alpha increase three- to fourfold during Caco-2 cell differentiation. Transient cotransfection experiments with 3T3 cells using dipeptidyl peptidase IV promoter constructs and expression vectors containing hepatocyte nuclear factor 1alpha and beta show that the ratio of alpha and beta modulates reporter gene expression. These results suggest that the increase in levels of hepatocyte nuclear factor 1alpha that occur during intestinal cell differentiation, are important for expression of dipeptidyl peptidase IV and other intestinal proteins.