Cdx binding determines the timing of enhancer activation in postnatal duodenum.
In mammalian intestine, adenosine deaminase (ADA) is expressed at high levels only along the villi of the duodenal epithelium. A duodenum-specific enhancer identified in the second intron of the human ADA gene controls this pattern of expression. This enhancer faithfully recapitulates this expression pattern in transgenic mice, when included in CAT reporter gene constructions. Multiple binding sites for PDX-1 and GATA factors were previously identified within the approximately 300-bp region that encompasses the enhancer. Mutation analyses demonstrated that binding of PDX-1 and of GATA-4 was absolutely essential for enhancer function. In the present study, we have identified additional enhancer binding sites for Cdx factors, for YY1, and for NFI family members. Detailed EMSA studies were used to confirm binding at these sites. This brings the number of confirmed binding sites within the enhancer to thirteen, with five different factors or family of factors contributing to the putative enhanceosome complex. Mutation analysis was utilized to examine the specific roles of the newly identified sites. Two sites were identified that bound both Cdx1 and Cdx2. Mutations were identified in these two sites that completely and specifically eliminated Cdx binding. In transgenic mice, these enhancer mutations dramatically changed the developmental timing of enhancer activation (delaying it by 2-3 weeks) without affecting other aspects of enhancer function. In the chromatin context of certain transgenic insertion sites, mutation of the two YY1 sites to specifically ablate binding caused a delay in enhancer activation similar to that observed with the Cdx mutations. No overt changes were observed from mutation of the NFI site.
Maier
Elizabeth A
EA
Department of Pediatrics, Division of Developmental Biology, University of Cincinnati College of Medicine and Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio 45229, USA.
Dusing
Mary R
MR
Wiginton
Dan A
DA
D000243:Adenosine Deaminase / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites; D000071616:CDX2 Transcription Factor; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D004386:Duodenum / Q000254:growth & development*; D004742:Enhancer Elements, Genetic; D050981:Erythroid-Specific DNA-Binding Factors; D050991:GATA4 Transcription Factor; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D025542:Neurofibromin 1 / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D019076:Transgenes; D050986:YY1 Transcription Factor
C491273:CDX1 protein, human; D000071616:CDX2 Transcription Factor; C491270:CDX2 protein, human; C491272:Cdx1 protein, mouse; C487514:Cdx2 protein, mouse; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050991:GATA4 Transcription Factor; D018398:Homeodomain Proteins; D025542:Neurofibromin 1; D015534:Trans-Activators; D014157:Transcription Factors; D050986:YY1 Transcription Factor; C494698:YY1 protein, human; C494699:Yy1 protein, mouse; C084010:pancreatic and duodenal homeobox 1 protein; D000243:Adenosine Deaminase