Abstract
The calbindin-D9K gene encodes a vitamin D-induced calcium-binding protein that is expressed as a marker of small intestine differentiation. We have shown that 4580 base pairs of its 5' DNA regulatory region can target reporter transgene expression in the intestine and cause this transgene to respond like the endogenous gene to vitamin D active metabolite and that the homeoprotein Cdx2 is bound to the TATA box in the intestine. We now show that the 4580 base pairs construct confers a differentiated pattern of reporter transgene expression in the intestine and that cooperation between the proximal promoter and a distal element located in an opened chromatin structure is responsible for the intestinal expression and vitamin D responsiveness of the transgene. Gel shift and footprinting assays using duodenal nuclear extracts indicate that this distal element contains a Cdx2-binding site. Finally, a mutation in this distal Cdx2-binding site dramatically decreases intestinal expression in transgenic mice. This report, using an in vivo approach, demonstrates the crucial role of Cdx2 for the transcription of an intestinal gene.
Highlights
The calbindin-D9K gene encodes a vitamin D-induced calcium-binding protein that is expressed as a marker of small intestine differentiation
We have shown that 4580 base pairs of its 5 DNA regulatory region can target reporter transgene expression in the intestine and cause this transgene to respond like the endogenous gene to vitamin D active metabolite and that the homeoprotein Cdx2 is bound to the TATA box in the intestine
We show that the 4580 base pairs construct confers a differentiated pattern of reporter transgene expression in the intestine and that cooperation between the proximal promoter and a distal element located in an opened chromatin structure is responsible for the intestinal expression and vitamin D responsiveness of the transgene
Summary
Construction of Hybrid Genes and Transgenic Mice—The plasmid (9K/Ϫ4400-CAT) has been previously described [15]. The transgene 9K/Ϫ117-CAT was isolated by HindIII digestion of the plasmid 9K/Ϫ4580-CAT This fragment contains the promoter region (beginning at Ϫ117), the first exon, the first intron, and the beginning of the second exon in front of the CAT gene. Frozen sections (7 m) were cut with a cryostat and incubated with the anti-CAT antibody (dilution, 1:1000) at room temperature for 1 h. The sections were rinsed in phosphatebuffered saline, incubated with anti-rabbit IgG-digoxigenin plus F(abЈ) fragment (dilution, 1:400; Boehringer Mannheim) for 30 min at room temperature, and rinsed three times with phosphate-buffered saline. The DNA fragments used as probes for footprinting assays were prepared by subcloning polymerase chain reaction products from the Ϫ3537 to Ϫ3281 region of 9K/Ϫ4580-CAT and Ϫ4580Cdxmut-CAT plasmids to generate wild type (WT HS1) and mutated probe (MUT HS1). The radiolabeled DNA was added [14]
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