Enterocyte differentiation marker intestinal alkaline phosphatase is a target gene of the gut-enriched Kruppel-like factor.
We have examined the role that the transcription factor gut-enriched Krüppel-like factor (KLF4 or GKLF) plays in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). 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 by using EMSA. Northern blot analysis 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. 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. We have identified the 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.
Hinnebusch
Brian F
BF
Deptartment of Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114, USA.
Siddique
Aleem
A
Henderson
J Welles
JW
Malo
Madhu S
MS
Zhang
Wenying
W
Athaide
Christopher P
CP
Abedrapo
Mario A
MA
Chen
Xinming
X
Yang
Vincent W
VW
Hodin
Richard A
RA
D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D015415:Biomarkers; D015152:Blotting, Northern; D019556:COS Cells; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D020895:Enterocytes / Q000502:physiology*; D006801:Humans; D007422:Intestines / Q000201:enzymology*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D008156:Luciferases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection
D015415:Biomarkers; D004268:DNA-Binding Proteins; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D011994:Recombinant Proteins; D014157:Transcription Factors; D008156:Luciferases; D000469:Alkaline Phosphatase