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Am J Physiol Gastrointest Liver Physiol (August 14, 2003). doi:10.1152/ajpgi.00203.2003
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Submitted on May 5, 2003
Accepted on August 9, 2003

The Enterocyte Differentiation Marker Intestinal Alkaline Phosphatase Is a Target Gene Of The Gut-enriched Kruppel-like Factor

Brian F. Hinnebusch1, Aleem Siddique1, J. Welles Henderson1, Madhu S. Malo1, Wenying Zhang1, Christopher P. Athaide1, Mario A. Abedrapo1, Xinming Chen2, Vincent W. Yang2, and Richard A. Hodin1*

1 Department of Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA
2 Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA

* To whom correspondence should be addressed. E-mail: rhodin{at}partners.org.

Introduction: We have examined the role that the transcription factor, gut-enriched Kruppel-like factor (KLF4 or GKLF) plays in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). Methods: A yeast one-hybrid screen was used to identify proteins interacting with a previously identified cis-element (IF-III) located within the human IAP gene promoter. DNA-protein interactions were determined using electrophoretic mobility-shift assays (EMSA). Northern blotting was used to study RNA expression in human colon cancer RKO cells engineered to overexpress KLF4. Transient transfections with IAP-luciferase reporter constructs were used to characterize the mechanisms by which KLF4 activates IAP transcription. Results: The yeast-one hybrid screen and EMSA identified KLF4 as binding to IF-III. RKO cells induced to overexpress KLF4 demonstrated a corresponding dose-dependent increase in IAP expression, and EMSA with nuclear extract from these cells confirmed that KLF4 binds to the IF-III element. Transient transfections revealed that KLF4 transactivated the IAP gene largely via a critical segment in the IAP promoter that includes the IF-III cis-element. Mutant KLF4 constructs failed to fully activate IAP. Conclusions: We have identified the gut enterocyte differentiation marker IAP as a KLF4 target gene. IAP transactivation by KLF4 is likely mediated through a critical region located within the proximal IAP promoter region.




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