A novel Sp1-related cis element involved in intestinal alkaline phosphatase gene transcription.
We have used sodium butyrate-treated HT-29 cells as an in vitro model system to study the molecular mechanisms underlying intestinal alkaline phosphatase (IAP) gene activation. Transient transfection assays using human IAP-CAT reporter genes along with DNase I footprinting were used to localize a critical cis element (IF-III) corresponding to the sequence 5'-GACTGGGCGGGGTCAAGATGGA-3'. Deletion of the IF-III element resulted in a dramatic reduction in reporter gene activity, and IF-III was shown to function in the context of a heterologous (SV40) promoter in a cell type-specific manner, further supporting its functional role in IAP transactivation. Electrophoretic mobility shift assays revealed that IF-III binds Sp1 and Sp3, but these factors comprise only a portion of the total nuclear binding and appear to mediate only a small portion of its transcriptional activity. IF-III does not correspond to any previously characterized regulatory region from other intestine-specific genes. We have thus identified a novel, Sp1-related cis-regulatory element in the human IAP gene that appears to play a role in its transcriptional activation during differentiation in vitro.
Kim
J H
JH
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School and Harvard Digestive Diseases Center, Boston, Massachusetts 02215, USA.
Meng
S
S
Shei
A
A
Hodin
R A
RA
D000469:Alkaline Phosphatase / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D018983:DNA Footprinting; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D015500:Chloramphenicol O-Acetyltransferase; D000469:Alkaline Phosphatase