Volume 116, Issue 4, Pages 842-854 (April 1999) Transcriptional regulation of pig lactase-phlorizin hydrolase: Involvement of HNF-1 and FREACs  Nikolaj Spodsberg, Jesper T. Troelsen, Peter Carlsson, Sven Enerbäck, Hans Sjöström, Ove Norén  Gastroenterology  Volume 116, Issue 4, Pages 842-854 (April 1999) DOI: 10.1016/S0016-5085(99)70067-3 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 A schematic diagram outlining the relative positions of the cis elements (CE-LPH1a to 4) in the LPH upstream region in relation to the transcription start site and the names and the sequences of the oligonucleotides used in the EMSAs. The oligonucleotides CE1a to CE4 cover the CE-LPH1a to 4 cis elements. Positions relative to the transcription start site of the LPH gene are indicated. The numbers indicate base pair position relative to the transcription start site. For each oligonucleotide pair, one of the oligonucleotides was labeled with γ-[32P]adenosine triphosphate, purified using the Mermaid Kit (BIO 101), and annealed. They were purified on a 15% polyacrylamide gel. The SP-1 oligonucleotide39 was used as an unspecific competitor. Gastroenterology 1999 116, 842-854DOI: (10.1016/S0016-5085(99)70067-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 The indicated fragments of LPH upstream region were cloned in front of the rabbit β-globin gene as described in Materials and Methods (the numbers represent 5' positions relative to the transcriptional start site). Reporter gene expression was quantified by S1 protection analysis, and the quantified transcription of the LPH/β-globin constructs was related to the level of a reference plasmid (OVEC-REF) mRNA product. The resulting values were normalized to that of OVEC-LPH894 at 2 weeks after confluence, which was set to 100%. The three sets of columns represent measurements using total RNA from Caco-2 cells at confluence (□) and 1 (▩) and 2 weeks (■) after confluence. A two-sided paired t test was performed to test the statistical significance of the variations in reporter gene transcription between confluence and 2 weeks after confluence. The P value is the probability of finding the observed data given the null hypothesis that there is no difference between the “Confluence” and the “2 weeks after” data sets. Sample number varied between 3 and 8. Gastroenterology 1999 116, 842-854DOI: (10.1016/S0016-5085(99)70067-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 EMSAs with (A) CE2a, (B) CE2b, and (C) CE2c spanning the CE-LPH 2A, 2B, and 2C cis elements, respectively (see Figure 1). The nuclear extracts were prepared from differentiated Caco-2 cells (Caco-2 day 14) as described in Materials and Methods. In each EMSA, 8 μg of the nuclear extracts was used. The unlabeled competitor oligonucleotides are shown above the lanes. The specific (sc) and unspecific (us) complexes are indicated. (D) Supershifts of CE2a and CE2c using a 1:10 Tris-buffered saline plus bovine serum albumin dilution of an HNF-1α antiserum (lanes 3 and 6) or preimmune serum (lanes 2 and 5) were performed as described previously.44 The specific (sc), unspecific (us), and supershift complexes (ssc) are indicated. Gastroenterology 1999 116, 842-854DOI: (10.1016/S0016-5085(99)70067-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 EMSAs with (A) CE2a, (B) CE2b, and (C) CE2c spanning the CE-LPH 2A, 2B, and 2C cis elements, respectively (see Figure 1). The nuclear extracts were prepared from differentiated Caco-2 cells (Caco-2 day 14) as described in Materials and Methods. In each EMSA, 8 μg of the nuclear extracts was used. The unlabeled competitor oligonucleotides are shown above the lanes. The specific (sc) and unspecific (us) complexes are indicated. (D) Supershifts of CE2a and CE2c using a 1:10 Tris-buffered saline plus bovine serum albumin dilution of an HNF-1α antiserum (lanes 3 and 6) or preimmune serum (lanes 2 and 5) were performed as described previously.44 The specific (sc), unspecific (us), and supershift complexes (ssc) are indicated. Gastroenterology 1999 116, 842-854DOI: (10.1016/S0016-5085(99)70067-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 EMSAs with (A) CE2a, (B) CE2b, and (C) CE2c spanning the CE-LPH 2A, 2B, and 2C cis elements, respectively (see Figure 1). The nuclear extracts were prepared from differentiated Caco-2 cells (Caco-2 day 14) as described in Materials and Methods. In each EMSA, 8 μg of the nuclear extracts was used. The unlabeled competitor oligonucleotides are shown above the lanes. The specific (sc) and unspecific (us) complexes are indicated. (D) Supershifts of CE2a and CE2c using a 1:10 Tris-buffered saline plus bovine serum albumin dilution of an HNF-1α antiserum (lanes 3 and 6) or preimmune serum (lanes 2 and 5) were performed as described previously.44 The specific (sc), unspecific (us), and supershift complexes (ssc) are indicated. Gastroenterology 1999 116, 842-854DOI: (10.1016/S0016-5085(99)70067-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 EMSAs with (A) CE2a, (B) CE2b, and (C) CE2c spanning the CE-LPH 2A, 2B, and 2C cis elements, respectively (see Figure 1). The nuclear extracts were prepared from differentiated Caco-2 cells (Caco-2 day 14) as described in Materials and Methods. In each EMSA, 8 μg of the nuclear extracts was used. The unlabeled competitor oligonucleotides are shown above the lanes. The specific (sc) and unspecific (us) complexes are indicated. (D) Supershifts of CE2a and CE2c using a 1:10 Tris-buffered saline plus bovine serum albumin dilution of an HNF-1α antiserum (lanes 3 and 6) or preimmune serum (lanes 2 and 5) were performed as described previously.44 The specific (sc), unspecific (us), and supershift complexes (ssc) are indicated. Gastroenterology 1999 116, 842-854DOI: (10.1016/S0016-5085(99)70067-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 EMSAs with (A) CE1a and (B) CE1b spanning the CE-LPH 1a and CE-LPH 1b cis elements, respectively (Figure 1). In each EMSA, 8 μg of the nuclear extracts prepared from undifferentiated (Caco-2 day 1) or differentiated (Caco-2 day 14) Caco-2 cells was used. The unlabeled competitor oligonucleotides are shown above the lanes. The specific complexes (sc) are indicated. Gastroenterology 1999 116, 842-854DOI: (10.1016/S0016-5085(99)70067-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 EMSAs with (A) CE1a and (B) CE1b spanning the CE-LPH 1a and CE-LPH 1b cis elements, respectively (Figure 1). In each EMSA, 8 μg of the nuclear extracts prepared from undifferentiated (Caco-2 day 1) or differentiated (Caco-2 day 14) Caco-2 cells was used. The unlabeled competitor oligonucleotides are shown above the lanes. The specific complexes (sc) are indicated. Gastroenterology 1999 116, 842-854DOI: (10.1016/S0016-5085(99)70067-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 EMSAs with CE4 covering the CE-LPH 4 cis element (Figure 1). In each EMSA, 8 μg of the nuclear extracts prepared from differentiated Caco-2 cells (Caco-2 day 14) or from adult pig small intestine (Small intestine) or liver (Liver) was used. The unlabeled competitor oligonucleotides are shown above the lanes. The specific (sc) and unspecific (us) complexes are indicated. Other batches of the same liver nuclear extract were tested positive for CE-LPH 2 and Sp1 binding activities (data not shown). Gastroenterology 1999 116, 842-854DOI: (10.1016/S0016-5085(99)70067-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) EMSAs with CE3 covering the CE-LPH 3 cis element (Figure 1). In each EMSA, 8 μg of the nuclear extracts prepared from undifferentiated (Caco-2 day 1) or differentiated (Caco-2 day 14) Caco-2 cells was used. The unlabeled competitor oligonucleotides are shown above the lanes. The specific complexes A–D are indicated. (B) EMSAs with CE3 covering CE-LPH 3 cis element using 0.5 μg of the protein fragments HL 5 and HL 9 of the transcription factors FREAC-2 and FREAC-3 in each EMSA as noted above the lanes. The unlabeled competitor oligonucleotides are shown above the lanes. Gastroenterology 1999 116, 842-854DOI: (10.1016/S0016-5085(99)70067-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) EMSAs with CE3 covering the CE-LPH 3 cis element (Figure 1). In each EMSA, 8 μg of the nuclear extracts prepared from undifferentiated (Caco-2 day 1) or differentiated (Caco-2 day 14) Caco-2 cells was used. The unlabeled competitor oligonucleotides are shown above the lanes. The specific complexes A–D are indicated. (B) EMSAs with CE3 covering CE-LPH 3 cis element using 0.5 μg of the protein fragments HL 5 and HL 9 of the transcription factors FREAC-2 and FREAC-3 in each EMSA as noted above the lanes. The unlabeled competitor oligonucleotides are shown above the lanes. Gastroenterology 1999 116, 842-854DOI: (10.1016/S0016-5085(99)70067-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions