Conserved Sequences in a Tissue-Specific Regulatory Region of the pdx-1 Gene Mediate Transcription in Pancreatic β Cells: Role for Hepatocyte Nuclear Factor 3β and Pax6

Abstract

Pancreas duodenum homeobox 1 (PDX-1) is absolutely required for pancreas development and the maintenance of islet β-cell function. Temporal and cell-type-specific transcription of the pdx-1 gene is controlled by factors acting upon sequences found within its 5′-flanking region. Critical cis-acting transcriptional control elements are located within a nuclease hypersensitive site that contains three conserved subdomains, termed areas I, II, and III. We show that area II acts as a tissue-specific regulatory region of the pdx-1 gene, directing transgene expression to a subpopulation of islet cells. Mutation of the area II hepatocyte nuclear factor 3 (HNF3) binding element in the larger area I- and area II- containing PstBst fragment also decreases PB^hsplacZ transgene penetrance. These two results indicate possible ontogenetic and/or functional heterogeneity of the β-cell population. Several other potential positive- and negative-acting control elements were identified in area II after mutation of the highly conserved sequence blocks within this subdomain. Pax6, a factor essential for islet α-cell development and islet hormone gene expression, was shown to bind in area II in vitro. Pax6 and HNF3β were also found to bind to this region in vivo by using the chromatin immunoprecipitation assay. Collectively, these data suggest an important role for both HNF3β and Pax6 in regulating pdx-1 expression in β cells.

We thank Eva Henderson and Min Guo for technical support and Klaus Kaestner at the University of Pennsylvania for allowing us to cite his data on the β-cell-specific knockout of the HNF3β gene prior to publication. The monoclonal Pax6 and Nkx2.2 antibodies were developed by Atsushi Kawakami and Thomas Jessell, respectively, and were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, Iowa. The Human Islet Core Lab at the Diabetes Research and Training Center, Washington University, St. Louis, Mo., provided the human islets.

This work was supported by grants from the NIH (RO1 DK50203 to R.S. and PO1 DK42502 to C.V.E.W.), a training grant in Molecular Endocrinology (5T32DK07563 to M.G.), and the Juvenile Diabetes Foundation (JDF 398212 to S.E.S.). The transgenic mice were developed with the assistance of the Transgenic/ES cell Shared Resource (NCIP30 CA68485 and NIH DK20593). Partial support was also provided from the Vanderbilt University Diabetes Research and Training Center Molecular Biology Core Laboratory (Public Health Service grant P60 DK20593 from the NIH).