Intestine-specific activity of the human guanylyl cyclase C promoter is regulated by Cdx2  Jason Park, Stephanie Schulz, Scott A. Waldman  Gastroenterology  Volume 119, Issue 1, Pages 89-96 (July 2000) DOI: 10.1053/gast.2000.8520 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Functional characterization of deletion mutants of the human GC-C gene promoter. Deletion mutants of the GC-C gene 5'-flanking region were linked to luciferase and cotransfected with the Renilla luciferase control plasmid pRL-TK into intestinal (T84, Caco2) and extraintestinal (HepG2, HeLa, HS766T) cell lines as described in Materials and Methods. Data are expressed as luciferase activity relative to the pGL3 Basic promoterless construct (Relative Activity). Each bar represents the mean ± SE of at least 3 independent transfections performed in duplicate. Gastroenterology 2000 119, 89-96DOI: (10.1053/gast.2000.8520) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 DNase I protection of the proximal human GC-C promoter. Footprinting reactions, as described in Materials and Methods, included the indicated microgram quantities of HepG2 or T84 nuclear extract (NE) and the −46 to −257 promoter fragment labeled at the 5'-end of the coding strand. A control digestion contained 60 μg of bovine serum albumin (BSA). Protected bases were identified by a Maxam–Gilbert sequencing reaction (G-A) of the labeled fragment.16 The sequence of FP1 is given. Arrowhead indicates DNase I hypersensitivity site at base −163. Gastroenterology 2000 119, 89-96DOI: (10.1053/gast.2000.8520) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Regulation of reporter gene expression by intestine-specific protected elements. FP1 and FP3 were deleted from the −835 luciferase construct by in vitro mutagenesis, and wild-type and deletion constructs were expressed in HepG2 and T84 cells, as described in Materials and Methods. Results are expressed as luciferase activity relative to a promoterless construct and represent the mean ± SE of 3 independent transfections performed in duplicate. Gastroenterology 2000 119, 89-96DOI: (10.1053/gast.2000.8520) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Intestinal specificity of FP1 probe EMSA. Nuclear extracts from intestinal or extraintestinal cells, or BSA (10 μg), were incubated with labeled FP1 for 30 minutes at room temperature before separation on a nondenaturing 6% polyacrylamide gel. Arrow and asterisk indicate the intestine-specific protein-FP1 complex. Gastroenterology 2000 119, 89-96DOI: (10.1053/gast.2000.8520) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 FP1 probe EMSA competition. Reaction conditions were similar to those in Figure 4, except (A) unlabeled competitors FP1, FP1-CCC, or (B) SIF1 were added in molar concentrations ranging from 25× to 250× greater than labeled FP1 probe, and were all incubated with 5 μg of T84 nuclear extract. Arrows and asterisks indicate the intestine-specific protein-FP1 complex. Gastroenterology 2000 119, 89-96DOI: (10.1053/gast.2000.8520) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 The FP1 intestine-specific complex is Cdx2. (A) T84 nuclear extract (NE in micrograms) or in vitro TNT Cdx2 (2 μL) was incubated with labeled FP1 or FP1-CCC. Control incubations contained a 2-μL aliquot of a TNT reaction performed without Cdx2 complementary DNA. The arrow indicates the intestine-specific protein-FP1 complex. (B) T84 nuclear extract (NE 5 μg) or in vitro TNT Cdx2 (2 μL) or control BSA (10 μg) was incubated with labeled FP1. Anti-Cdx1 or -Cdx2 antibody was included in the incubation where indicated. Arrows and asterisks indicate the intestine-specific protein-FP1 complex. Arrowheads indicate protein-FP1 complexes exhibiting decreased mobility in the presence of specific antibody. Gastroenterology 2000 119, 89-96DOI: (10.1053/gast.2000.8520) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Cdx2 binds directly to the GC-C Promoter. (A) Southwestern blot of 50 or 100 μg of T84, HepG2, or Caco2 nuclear extracts probed with labeled FP1B. The arrow indicates the protein bound by FP1B at approximately 40 kilodaltons as determined from the mobility of prestained molecular-weight markers (Bio-Rad). (B) Western blot of 100 μg of T84, HepG2, or HeLa nuclear extract or in vitro TNT Cdx2 using anti-Cdx2 antibody. The arrow indicates the  ̃40-kilodalton protein bound by anti-Cdx2 antibody, determined from the mobility of prestained molecular-weight markers (Bio-Rad). Gastroenterology 2000 119, 89-96DOI: (10.1053/gast.2000.8520) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Cdx2 binding element FP1 is required for GC-C reporter gene activation. Putative binding sites for Cdx2 and HNF-4α are indicated on the −835 construct. T84 and HepG2 cells were transfected with the −835 reporter construct from which FP1 was deleted or the construct containing the “CCC” mutation. Results are expressed as (Luciferase Activity of Mutant Construct / Luciferase Activity of Wild-type Construct) × 100, and represent the mean ± SE of 3 independent transfections performed in duplicate. The values expressed as relative luciferase activities are as follows: wild-type: FP1 deletion; “CCC” mutation: T84 (16.2 ± 2.7; 1.9 ± 0.3; 2.3 ± 0.1) and HepG2 (2.1 ± 0.1; 2.9 ± 0.3; 2.2 ± 0.1). Gastroenterology 2000 119, 89-96DOI: (10.1053/gast.2000.8520) Copyright © 2000 American Gastroenterological Association Terms and Conditions