Drummond, Felicity Jane; (1997) Carbonic anhydrase 1: A study of colon-specific gene regulation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The function of carbonic anhydrase in the colon is principally that of electroneutral sodium chloride reabsorption and short chain fatty acid uptake. Three carbonic anhydrase genes are expressed in the intestine and carbonic anhydrase 1 (CA1) is specific to the colon. The CA1 gene has two promoters which act in a mutually exclusive manner, generating high levels of CA1 in colon epithelial cells and in erythroid cells. This thesis describes an investigation of the regulation of human CA1 in the colon, with a focus on a region of the colon promoter marked by a colon-specific DNaseI hypersensitive site. Using a panel of deletion fragments and DNA/protein binding assays, an element which binds a colon-specific factor has been identified. In addition, it has been shown that mutation of the core motif of this element, 5'-TTTTACAA-3', abolishes protein binding. Comparison with transcription factor binding sites identified in other intestinal genes, suggests that the protein binding to the CA1 motif is the homeodomain transcription factor, Cdx2. This finding is supported by results from DNA/protein binding and immuno- supershift assays. Two Cdx2 binding sites have been identified in both the human and mouse CA1 colon promoters. The biological significance of Cdx2 binding to these sites has been investigated by co-transfection studies in cultured cells. Expression of Cdx2 in cells transfected with CA1 colon promoter/reporter gene constructs led to an upregulation of reporter gene expression. This activation was both orientation and distance independent; indicating that these elements behave as enhancer sequences in the CA1 gene promoter. However, there is evidence to suggest that only the 3' Cdx2 binding site is functional in vivo. These studies have also revealed that Cdx2 may be involved in complex regulatory mechanisms, since it seems that Cdx2 can also bind to TATA boxes of gene promoters, including the CA1 promoter. The human homologue of Cdx2 has been cloned from a human jejunum cDNA library and the sequence compared with rodent Cdx2. The homeodomains of Cdx2 and other caudal-type proteins have also been compared. The human CDX2 gene has been assigned to chromosome 13q12- 13.

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