with SIFl sequence within the 5 / promoter Suh et al. (1994) used a
multimerised double-stranded SIFl oligonucleotide to screen an adult mouse jejunum cDNA expression library. The cDNA isolated by this procedure was
identical to mouse Cdx2 {caudal-type homeobox) which had been cloned earlier in the same year by James et al. (1994). Cdx2 belongs to a homeobox gene family, the first member of which was cloned by its homology to the
Drosophila caudal gene. RNase protection assays and in situ hybridisation show that Cdx2 is expressed throughout the length of the adult mouse
hCAl mCAl ACTAACATGA -215 O ' hCAl mCAl hCAl mCAl hCAl mCAl hCAl mCAl GATA-1 ********
GT G A A A C A G GACTTGGCTTTATAAAATCTAG GA.TC TGATAA PTCTCTTTTACAATATAAGAAAATTAAGCAATT
1 M i l l Ml II II M M M i l l II 1 GGAAAACAAGACCAGGTTTAATAATATCTATGATTl M i l l TGATAT Ml M i l l 1 II I I II II II M i l l rTCATTTTTATATTACAATAACATAAAACAATT Apol * * * * * * * * Asal GATA-1 TATA * * * * * * +1 T G ATGAAA T A A T G G C C T A T T A A A A G C A A . ATAAGTTTCTATAAAAACGCCCAAGCAGGGATTTAAGGCATCTCCT l l l l l l l l I II m i l l I I II I I I I l l l l l l l l l l M i l l I I I M i l l Ml II
TGATGAAACAGTGAACTATTACACGGAAGAC AAGCTTCTATAAAA. . GCCCATGGTGTCACTTAAGATCTCTGCT
!-► * * * GCATGCACAGTTGCAGTTAGTTATTCCAGGTATTATTTTTGTTTTCAGAAAAAGAAAACTCAGTAGAAGATAATG Ml I I l l l l l l l l l l Ml II Ml M M I Ml Ml II II II I I I Ml M M GCAGGTGTAAGTGCAGTTAGTCATTTCACATATCATTTCTCTTTACAGGAATCACAACCTAAATAAGAGAAAATG -141
GAAACTAACATAGCCCTTGTAGAATTTTTTACAACACCTTTTTTTTAGATATGTGTACTTCC PGATAA SCAGAGA -66
1 II II II Ml 1 l l l l l l l l Ml M M II II Ml M M M I C A T G C T G A C C T A C C C C . . AATAGACATTTTACAATACCATTTTATTTTATGTGTATACTTCC M M 1 IGATGA Ml II 3CAAAGT + 1 + 9 + 84
F ig u re 3 .1 8 Sequence comparison o f human (hCAJ) and mouse { mCAl \ Fraser et al., 1989) CA l colon promoters. The region o f CPl ( -5 9 to -1 4 7 b p ) and CP2 (-1 2 2 to -2 2 4 b p ) are underlined and overlap by 26bp. Vertical lines indicate sequence identity. The transcription start sites, +1, are indicated ( f ^ ) . The
Ap o \ and Rsa\ restriction enzym es used to digest CPl are shown. Asterisks indicate the position o f two Cdx2 binding sites, the methionine codon and the TATA box. Two potential GATA-1 sites are boxed. Sequences were compared using the GAP computer program.
intestine where its expression in the mouse is restricted to the intestinal epithelium. Transcript levels are greater in the colon than in the small
intestine (James & Kazenwadel, 1991; James et a l, 1994). In agreement with these findings a recent report has shown that human CDX2 is also expressed exclusively in the adult intestine (Mallo et al., 1997). Cdx2 was found to rra«5-activate both the SI and LPH genes in co-transfection assays (Suh et a l, 1994; Troelsen et a l, 1997).
To determine whether Cdx2 is the protein involved in the C O Fl/D N A complex, ‘supershift’ assays were performed using a mouse Cdx2 antibody (a gift from P. Traber). The binding of an antibody to the protein/DNA complex further retards the migration of the protein/DNA complex during
electrophoresis resulting in a ‘supershift’. The C O Fl complex was
supershifted when the Cdx2 antibody was added to the binding reaction (Figure 3.19) supporting the view that the C O Fl protein is Cdx2.
Substitution of antibody with pre-immune serum did not lead to a supershift of the C O Fl/D N A complex (not shown). The supershift was not complete and this may be ascribed to the relatively low specificity of this antibody; W estern blotting of proteins extracted from various cell lines showed binding to a number of proteins of different molecular weights (not shown). The antibody was in very limited supply but nevertheless it was possible to demonstrate that the proportion of the CO Fl complex supershifted was related to the amount of the Cdx2 antibody added to the binding mix. In addition, an increase in the incubation time of antibody with the protein/DNA mix enhanced the proportion of supershifted complex at the expense of the unbound CO Fl complex (Figure 3.19).
3 .3 Expression of C d x l , Cdx2 and C A l in regions of the in te stin e
Cdx2 is a member of a gene family of which two other members, C dxl
and Cdx4 have been identified. During mouse development Cdxl is
expressed in the endoderm and later in the intestinal epithelium where levels increase between 17 and 18 dpc (Hu et a l, 1993). C dxl expression persists into adult life where it is confined to the intestinal epithelium (James &
Ab 0 1 . 0 1 .5 0 5 . 0 ❖ * C O F l 32P C P 017
F ig u re 3 .1 9 Supershift assay with labelled CPO 17, protein extracts from H Tl 15 cells and an antibody to mouse Cdx2. 0, no antibody; 1.0, 1.5 and 5.0 indicates the amount o f antibody (pi ) added to the binding reaction. Binding times were 30 minutes for the 5pl sample and 15 minutes for the 1.0 and 1.5pl samples. Arrows indicates the COFl
complex. * indicates the supershifted complex; P, probe without protein or antibody.
Kazenwadel, 1991). Cdx4 is expressed in the endodermal lining of the gut at 8.5 to 9.5 dpc (Gamer & Wright, 1993) however, the expression pattern of
Cdx4 has not been reported in adult tissues. The DNA binding domains of Cdx2, C dxl and Cdx4 show high levels of similarity (see Figure 5.3) such that it seems likely that these proteins may bind to the same or a similar DNA element. C d xl, like Cdx2, is expressed throughout the intestine and can also be considered as a factor with a role in the regulation of C Al via the CPO 17 motif.
In order to investigate this possibility, the expression patterns of C dxl, Cdx2 and C A l were examined in the various cell lines used in EMSAs and mouse tissues. Tissue used to prepare RNA included the duodenum, small intestine (jejunum and ileum), caecum and colon. RNA was reverse
transcribed and amplified by the reverse transcription polymerase chain reaction (RT-PCR) using gene-specific primer pairs. The reverse transcriptase reaction was primed from random hexamer oligonucleotides so that the same cDNA sample could be used for PCRs using different sets of primers. This allowed a direct comparison of the products and a rough estimation of the relative levels of RNA. Amplified products were assessed using ethidium bromide staining. Primer pairs were designed such that each primer was situated in a different exon so that the products generated by amplification of cDNA could be distinguished from those generated by amplification of
genomic DNA. RT-PCR o f the ubiquitously expressed phosphoglucomutase 1 (P G M l) gene (Whitehouse et a l, 1992) was carried out on the same cDNA samples in order to test the integrity of the cDNA samples. The P G M l primer pair generated a 420bp product and the relative amounts of PCR product produced for the different cDNA samples were similar (Figure 3.20A).
Cdx2 was expressed along the entire length of the intestine with similar levels in the adult small and large intestine and in a fetal 14dpc intestine sample.
C dxl is also expressed throughout the intestine but shows more varied expression in different regions. Low levels of C dxl mRNA were seen in the fetal small intestine. The highest levels of expression were found in adult caecum with lower levels in the colon, duodenum and small intestine (Figure
G
Ë B
Figure 3.20 RT-PCR amplification o f samples from cell lines used in EM SA studies and from adult and fetal mouse intestine. Marker, is a 1 kb D N A ladder (Gibco, BRL); bp, the size o f fragments in the 1 kb ladder; 14.5dpc, fetal intestine sample at 14.5dpc and negative, no D N A control. Panel A: with PG M 7-specific primers, product size is 420bp. Panel B: with mouse C<ix2-specific primers, product size is 230bp.
3.21 A). This is similar to the distribution reported by James & Kazenwadel (1991) who found highest levels of C dxl in the colon and markedly lower levels in the small intestine. The levels of Cdxl mRNA appeared to be considerably lower than those seen for Cdx2, except in the caecum (Figure 3.20B). Results from analysis of the 14dpc fetal intestine sample were consistent with findings of Hu et al. (1993) and James et al. (1994) in that both C dxl and Cdx2 are expressed at this time of gestation, but at low levels.
By RT-PCR C A l mRNA appeared to be moderately abundant in the caecum and colon but was not present in the adult duodenum, small intestine, or fetal intestine samples (Figure 3.22). These results are in agreement with previous studies using Southern (Sowden et al., 1993) and Northern (Fleming
et a l; 1995) analyses and semiquantitative RT-PCR (Wang et a l, 1994). RT-PCR was also carried out using mRNA derived from cell lines of intestinal origin LIM1215, HT115 and Caco-2 and from non-intestinal cell lines HEL and HeLa. CD Xl and CDX2 were expressed in LIM1215 and H T l 15 but not in, HEL or HeLa cell lines; CDX2 was expressed in Caco-2 cells but C D Xl was not (Figures 3.20B and 3.2IB). The fact that C dxl is not expressed in Caco-2 colon-derived cells is of interest since the band shift seen with extracts of these cells and the C A l, S I (Traber et a l, 1992) and LPH
(Troelsen et a l, 1994b) Cdx binding elements must be due to the binding of Cdx2 and not C dxl (Figure 3.13).
C Al mRNA was amphfied from LIM1215 and H Tl 15 samples and not in HEL, HeLa or Caco-2 (Figure 3.22). These results are consistent with the findings of Sowden et al. (1993) who showed that the LIM1215 and H T l 15 cell lines express C A l and that Caco-2 cells lack C A l. The observation that
C A l is not expressed in Caco-2 colon cells was perhaps surprising in view of the finding that several intestinal genes, Cdx2 (Drummond et a l, 1996), SI
(Chantret et a l, 1994) and L P H l (Prof. D. Swallow, UCL; personal communication) are known to be expressed in these cells. However, it is thought that Caco-2 cells have a fetal/small intestinal phenotype and may thus not express C A l.
0> G
I lî
W) u u % B bp 506 298 220F ig u re 3.21 RT-PCR amplification o f samples from cell lines used in EM SA studies and from adult and fetal mouse intestine. Marker, is a Ikb D N A ladder (Gibco,BRL); bp, the size o f fragments in the Ikb ladder;
14.5dpc, fetal intestinesample at 14.5dpc and negative, no D N A control.