Pancreatic β Cell-specific Transcription of thepdx-1 Gene
THE ROLE OF CONSERVED UPSTREAM CONTROL REGIONS AND THEIR HEPATIC NUCLEAR FACTOR 3β SITES*
- Kevin Gerrish‡,
- Maureen Gannon§,
- David Shih¶,
- Eva Henderson‡,
- Markus Stoffel¶,
- Christopher V. E. Wright§ and
- Roland Stein‡§‖
- From the ‡Department of Molecular Physiology and Biophysics and §Department of Cell Biology, Vanderbilt Medical Center, Nashville, Tennessee 37232 and ¶Laboratory of Metabolic Diseases, Rockefeller University, New York, New York 10021
Abstract
To identify potential transactivators ofpdx-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 criticalcis-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 β 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 β- and non-β 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 HNF3β. 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 HNF3β were inactivated. pdx-1 mRNA levels induced upon differentiation to embryoid bodies were down-regulated in homozygous null HNF3β cells. Together, these results suggest that the conserved sequences represented by areas I and II define the binding sites for factors such as HNF3β, which control islet β cell-selective expression of the pdx-1 gene.
Footnotes
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↵* This work was supported by National Institutes of Health Grants NIH RO1 DK50203 (to R. S.) and NIH R01 HD 28062 and NIH DK 42502 (to C. V. E. W.), Juvenile Diabetes Foundation International postdoctoral fellowship Grant 397019 (to M. G.), and in part by the Vanderbilt University Diabetes Research and Training Center Molecular Biology Core Laboratory (Public Health Service NIH Grant P60 DK20593).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) , , and .
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↵‖ To whom correspondence should be addressed. Tel.: 615-322-7026; Fax: 615-322-7236; E-mail: Roland.Stein@mcmail.vanderbilt.edu.
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↵2 M. Gannon and C. V. E. Wright, unpublished results.
- Abbreviations:
- GLUT2
-
glucose transporter type 2
- HSS
-
hypersensitive site
- HNF
-
hepatic nuclear factor
- bp
-
base pair(s)
- ES
-
embryonic stem
- EB
-
embryoid bodies
- kb
-
kilobase(s)
- Tk
-
thymidine kinase
- CAT
-
chloramphenicol acetyltransferase
- BHK
-
baby hamster kidney
- RSV
-
Rous sarcoma virus
- PCR
-
polymerase chain reaction
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- Received June 28, 1999.
- Revision received November 9, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
