Long-Range Enhancers Are Required to Maintain Expression of the Autoantigen Islet-Specific Glucose-6-Phosphatase Catalytic Subunit–Related Protein in Adult Mouse Islets In Vivo

Long-Range Enhancers Are Required to Maintain Expression of the Autoantigen Islet-Specific Glucose-6-Phosphatase Catalytic Subunit–Related Protein in Adult Mouse Islets In Vivo Yingda Wang 1 , Brian P. Flemming 1 , Cyrus C. Martin 1 , Shelley R. Allen 1 , Jay Walters 2 , James K. Oeser 1 , John C. Hutton 2 and Richard M. O'Brien 1 1 Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 2 Barbara Davis Center for Childhood Diabetes, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado Address correspondence and reprint requests to Richard M. O'Brien, Department of Molecular Physiology and Biophysics, 761 PRB, Vanderbilt University Medical School, Nashville, TN 37232-0615. E-mail: richard.obrien{at}vanderbilt.edu Abstract OBJECTIVE —Islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP) is selectively expressed in islet β-cells and is a major autoantigen in both mouse and human type 1 diabetes. This study describes the use of a combination of transgenic and transfection approaches to characterize the gene regions that confer the islet-specific expression of IGRP. RESEARCH DESIGN AND METHODS —Transgenic mice were generated containing the IGRP promoter sequence from −306, −911, or −3911 to +3 ligated to a LacZ reporter gene. Transgene expression was monitored by 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside staining of pancreatic tissue. RESULTS —In all the transgenic mice, robust LacZ expression was detected in newborn mouse islets, but expression became mosaic as animals aged, suggesting that additional elements are required for the maintenance of IGRP gene expression. VISTA analyses identified two conserved regions in the distal IGRP promoter and one in the third intron. Transfection experiments demonstrated that all three regions confer enhanced luciferase reporter gene expression in βTC-3 cells when ligated to a minimal IGRP promoter. A transgene containing all three conserved regions was generated by using a bacterial recombination strategy to insert a LacZ cassette into exon 5 of the IGRP gene. Transgenic mice containing a 15-kbp fragment of the IGRP gene were then generated. This transgene conferred LacZ expression in newborn mouse islets; however, expression was still suppressed as animals aged. CONCLUSIONS —The data suggest that long-range enhancers 5′ or 3′ of the IGRP gene are required for the maintenance of IGRP gene expression in adult mice.