Cdx2 is a caudal-related homeodomain transcription factor that is expressed in complex patterns during mouse development and at high levels in the intestinal epithelium of adult mice. Cdx2 activates transcription of intestinal gene promoters containing specific binding sites. Moreover, Cdx2 has been shown to induce intestinal differentiation in cell lines. In this study, we show that Cdx2 is able to bind to two well defined enhancer elements in the HoxC8 gene. We then demonstrate that Cdx2 is able to activate transcription of heterologous promoters when its DNA binding element is placed in an enhancer context. Furthermore, the ability to activate enhancer elements is cell-line dependent. When the Cdx2 activation domain was linked to the Gal4 DNA binding domain, the chimeric protein was able to activate Gal4 enhancer constructs in an intestinal cell line, but was unable to activate transcription in NIH3T3 cells. These data suggest that there are cell-specific factors that allow the Cdx2 activation domain to function in the activation of enhancer elements. We hypothesize that either a co-activator protein or differential phosphorylation of the activation domain may be the mechanism for intestinal cell line-specific function of Cdx2 and possibly in other tissues in early development.

Intestinal phospholipase A/lysophospholipase (IPAL) is an intestine-specific brush-border enzyme expressed during development and along the intestinal crypt-villus axis in a pattern similar to another well characterized brush-border enzyme, sucrase-isomaltase (SI). A tissue-specific DNase I hypersensitive site was identified in chromatin from intestinal nuclei immediately upstream from the transcriptional start site of the IPAL gene. Footprinting analysis showed that two DNA elements within the IPAL promoter were protected by intestinal nuclear proteins. The IPAL-FP1 element was shown to be a monomer binding site for Cdx1 and Cdx2, intestine-specific homeobox proteins. Moreover, this site was important for transcriptional activation of the promoter in intestinal cell lines via interaction with Cdx proteins. Nuclear proteins from both liver and intestine interacted with the IPAL-FP2 element, forming a complex consistent with binding to HNF1. Cdx and HNF1 binding sites have also been shown to be the two major regulatory elements responsible for transcriptional activation of the SI gene promoter, which directs intestine-specific transcription in transgenic mice. These findings suggest that enterocyte genes that are expressed in similar developmental patterns may be regulated by the interaction of common DNA elements and their associated transcription factors.

We have developed a site-specific chemical modification technique to incorporate a photoreactive azidophenacyl (APA) group at designated internal positions along the RNA phosphodiester backbone. Using this technique, we have analyzed interactions of the 5' splice site (5'SS) RNA within the spliceosome. Several crosslinked products can be detected within complex B using the derivatized 5'SS RNAs, including U6 snRNA, hPrp8p, and 114-, 90-, 70-, 54-, and 27-kDa proteins. The 5'SS RNAs derivatized at intron positions +4 to +8 crosslink to U6 snRNA, confirming the previously reported pairing interaction between these sequences. hPrp8p and p70 are crosslinked to the 5'SS RNA when the APA is placed within the 5' exon. Finally, a set of unidentified proteins, including p114, p54, and p27, is detected with the 5'SS RNA derivatized at intron positions +4 to +8. Introduction of the bulky APA group near the 5'SS junction (positions -2 to +3) strongly interferes with complex B formation and thus no APA crosslinks are observed at these positions. Together with our earlier observation that hPrp8p crosslinks to the GU dinucleotide at the 5' end of the intron, these results suggest that the inhibitory effect of APA results from steric hindrance of the hPrp8p:5'SS interaction. Unexpectedly, thio-modifications within the region of the 5'SS RNA that is involved in base pairing to U6 snRNA strongly stimulate complex B formation.