Volume 115, Issue 2, Pages 381-387 (August 1998) Misexpression of the pancreatic homeodomain protein IDX-1 by the Hoxa-4 promoter associated with agenesis of the cecum  R.Scott Heller, Doris A. Stoffers, Mehboob A. Hussain, Christopher P. Miller, Joel F. Habener  Gastroenterology  Volume 115, Issue 2, Pages 381-387 (August 1998) DOI: 10.1016/S0016-5085(98)70204-5 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Diagram of the DNA plasmid containing the Hoxa-4 promoter driving IDX-1 DNA expression. The Hoxa-4 promoter (4.1 kb) was fused with 0.64 kb of the rat β-globin slice sequence, the full-length IDX-1 cDNA, in which an internal EcoRI site had been changed without altering the coding sequence of the protein, and the 0.52-kb rat β-globin poly(A) signal. Gastroenterology 1998 115, 381-387DOI: (10.1016/S0016-5085(98)70204-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Gross analysis of wild-type and transgenic mice. (A) Two 4-week-old nontransgenic (left) and transgenic H30 (right) mice. At 4 weeks of age, the transgenic mice are visibly smaller than wild-type mice. Already at this age, the eyelid abnormality is visible. (B and C) Closer examination of older (B) wild-type and (C) H30 mouse. Eyelid epithelium revealed a thicker, inflamed eyelid in the H30 mice. (D and E) Upon histological sectioning of the eyelid epithelium (stained with hematoxylin), it was observed that H30 mice had a greatly increased number of hair follicles. Gastroenterology 1998 115, 381-387DOI: (10.1016/S0016-5085(98)70204-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Linear-range PCR of genomic DNA prepared from Hoxa-4/IDX-1 transgenic mice. The copy number of transgenes in mouse strains 30 and 47 with cecal agenesis are considerably higher than in strains 29 and 70, which do not have cecal agenesis. Gastroenterology 1998 115, 381-387DOI: (10.1016/S0016-5085(98)70204-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Analysis of the lower gastrointestinal tract of wild-type and transgenic mice. (A) Complete absence of the cecum in H30 mice. Gross evaluation of the gastrointestinal tract of wild-type (WT) and H30 mice revealed complete absence of the cecum (arrow in wild-type). (B and C) Lower-magnification photographs of the luminal surface of the ileal-cecal-colonic region revealed a greatly reduced number of epithelial folds in transgenic mice compared with wild-type (the interfold distances are indicated between the arrows). (D and E) Histological section of the ileal-cecal region revealed a normal ileal cecal valve (data not shown), but (E) the epithelium located distal to the valve of H30 mice had a mixture of cecal-like epithelium (short wide villi) and a few very long villi characteristic of the ileum. Gastroenterology 1998 115, 381-387DOI: (10.1016/S0016-5085(98)70204-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Interactions of IDX-1 with Cdx-2 inhibit transactivation of the sucrase-isomaltase promoter. (A) Electromobility shift assay of Cdx-2 interaction with proximal G1 promoter element of the glucagon gene that binds Cdx-2. Nuclear extracts were prepared from Cos-7 cells transfected with Cdx-2 expression vector. Nuclear extracts (10 μg) of Cos cells were incubated with 32P-labeled oligonucleotides composed of the G1 element in the proximal promoter of the rat glucagon gene22 either alone or with GST, GST–IDX-1, or 1 μL of preimmune (Pi) or 1 μL of anti IDX-1 antiserum (diluted 1:5), after which DNA-protein complexes were resolved by polyacrylamide gel electrophoresis and autoradiography. Two complexes of Cdx-2 are formed with the G1-containing oligonucleotide probe. (B) Addition of increasing amounts of GST–IDX-1 leads to an increase in the formation of a higher-molecular-weight Cdx-2/IDX-1 complex. Asterisk indicates nonspecific complex. (C) GST-pulldown assay showing in vitro protein-protein interaction of Cdx-2 and IDX-1. In vitro–translated Cdx-2 is pulled down by GST–IDX-1, but not by GST-C/EBPβ. The known interaction of C/EBPβ and CHOP was used as positive control for the assay.30 (D) Functional inhibition of the transactivating properties of Cdx-2 on the sucrase-isomaltase promoter by IDX-1. BHK-21 cells were cotransfected with 5 μg of −100–base pair sucrase-isomaltase footprint 1 (SIF1)–luciferase reporter plasmid and 1.5 μg of Cdx-2 and IDX-1 expression vectors. Transfected DNA was kept constant by adding empty expression vector to a total of 8 μg. Gastroenterology 1998 115, 381-387DOI: (10.1016/S0016-5085(98)70204-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Interactions of IDX-1 with Cdx-2 inhibit transactivation of the sucrase-isomaltase promoter. (A) Electromobility shift assay of Cdx-2 interaction with proximal G1 promoter element of the glucagon gene that binds Cdx-2. Nuclear extracts were prepared from Cos-7 cells transfected with Cdx-2 expression vector. Nuclear extracts (10 μg) of Cos cells were incubated with 32P-labeled oligonucleotides composed of the G1 element in the proximal promoter of the rat glucagon gene22 either alone or with GST, GST–IDX-1, or 1 μL of preimmune (Pi) or 1 μL of anti IDX-1 antiserum (diluted 1:5), after which DNA-protein complexes were resolved by polyacrylamide gel electrophoresis and autoradiography. Two complexes of Cdx-2 are formed with the G1-containing oligonucleotide probe. (B) Addition of increasing amounts of GST–IDX-1 leads to an increase in the formation of a higher-molecular-weight Cdx-2/IDX-1 complex. Asterisk indicates nonspecific complex. (C) GST-pulldown assay showing in vitro protein-protein interaction of Cdx-2 and IDX-1. In vitro–translated Cdx-2 is pulled down by GST–IDX-1, but not by GST-C/EBPβ. The known interaction of C/EBPβ and CHOP was used as positive control for the assay.30 (D) Functional inhibition of the transactivating properties of Cdx-2 on the sucrase-isomaltase promoter by IDX-1. BHK-21 cells were cotransfected with 5 μg of −100–base pair sucrase-isomaltase footprint 1 (SIF1)–luciferase reporter plasmid and 1.5 μg of Cdx-2 and IDX-1 expression vectors. Transfected DNA was kept constant by adding empty expression vector to a total of 8 μg. Gastroenterology 1998 115, 381-387DOI: (10.1016/S0016-5085(98)70204-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Interactions of IDX-1 with Cdx-2 inhibit transactivation of the sucrase-isomaltase promoter. (A) Electromobility shift assay of Cdx-2 interaction with proximal G1 promoter element of the glucagon gene that binds Cdx-2. Nuclear extracts were prepared from Cos-7 cells transfected with Cdx-2 expression vector. Nuclear extracts (10 μg) of Cos cells were incubated with 32P-labeled oligonucleotides composed of the G1 element in the proximal promoter of the rat glucagon gene22 either alone or with GST, GST–IDX-1, or 1 μL of preimmune (Pi) or 1 μL of anti IDX-1 antiserum (diluted 1:5), after which DNA-protein complexes were resolved by polyacrylamide gel electrophoresis and autoradiography. Two complexes of Cdx-2 are formed with the G1-containing oligonucleotide probe. (B) Addition of increasing amounts of GST–IDX-1 leads to an increase in the formation of a higher-molecular-weight Cdx-2/IDX-1 complex. Asterisk indicates nonspecific complex. (C) GST-pulldown assay showing in vitro protein-protein interaction of Cdx-2 and IDX-1. In vitro–translated Cdx-2 is pulled down by GST–IDX-1, but not by GST-C/EBPβ. The known interaction of C/EBPβ and CHOP was used as positive control for the assay.30 (D) Functional inhibition of the transactivating properties of Cdx-2 on the sucrase-isomaltase promoter by IDX-1. BHK-21 cells were cotransfected with 5 μg of −100–base pair sucrase-isomaltase footprint 1 (SIF1)–luciferase reporter plasmid and 1.5 μg of Cdx-2 and IDX-1 expression vectors. Transfected DNA was kept constant by adding empty expression vector to a total of 8 μg. Gastroenterology 1998 115, 381-387DOI: (10.1016/S0016-5085(98)70204-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Interactions of IDX-1 with Cdx-2 inhibit transactivation of the sucrase-isomaltase promoter. (A) Electromobility shift assay of Cdx-2 interaction with proximal G1 promoter element of the glucagon gene that binds Cdx-2. Nuclear extracts were prepared from Cos-7 cells transfected with Cdx-2 expression vector. Nuclear extracts (10 μg) of Cos cells were incubated with 32P-labeled oligonucleotides composed of the G1 element in the proximal promoter of the rat glucagon gene22 either alone or with GST, GST–IDX-1, or 1 μL of preimmune (Pi) or 1 μL of anti IDX-1 antiserum (diluted 1:5), after which DNA-protein complexes were resolved by polyacrylamide gel electrophoresis and autoradiography. Two complexes of Cdx-2 are formed with the G1-containing oligonucleotide probe. (B) Addition of increasing amounts of GST–IDX-1 leads to an increase in the formation of a higher-molecular-weight Cdx-2/IDX-1 complex. Asterisk indicates nonspecific complex. (C) GST-pulldown assay showing in vitro protein-protein interaction of Cdx-2 and IDX-1. In vitro–translated Cdx-2 is pulled down by GST–IDX-1, but not by GST-C/EBPβ. The known interaction of C/EBPβ and CHOP was used as positive control for the assay.30 (D) Functional inhibition of the transactivating properties of Cdx-2 on the sucrase-isomaltase promoter by IDX-1. BHK-21 cells were cotransfected with 5 μg of −100–base pair sucrase-isomaltase footprint 1 (SIF1)–luciferase reporter plasmid and 1.5 μg of Cdx-2 and IDX-1 expression vectors. Transfected DNA was kept constant by adding empty expression vector to a total of 8 μg. Gastroenterology 1998 115, 381-387DOI: (10.1016/S0016-5085(98)70204-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions