Patrick G. Gallagher

A human beta-spectrin gene promoter directs high level expression in erythroid but not muscle or neural cells.

β-Spectrin is an erythrocyte membrane protein that is defective in many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. It is expressed not only in erythroid tissues but also in muscle and brain. We wished to determine the regulatory elements that determine the tissue-specific expression of the β-spectrin gene. We mapped the 5′-end of the β-spectrin erythroid cDNA and cloned the 5′-flanking genomic DNA containing the putative β-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, a β-spectrin gene erythroid promoter with two binding sites for GATA-1 and one site for CACCC-related proteins was identified. All three binding sites were required for full promoter activity; one of the GATA-1 motifs and the CACCC-binding motif were essential for activity. The β-spectrin gene promoter was able to be transactivated in heterologous cells by forced expression of GATA-1. In transgenic mice, a reporter gene directed by the β-spectrin promoter was expressed in erythroid tissues at all stages of development. Only weak expression of the reporter gene was detected in muscle and brain tissue, suggesting that additional regulatory elements are required for high level expression of the β-spectrin gene in these tissues.

Sequences Downstream of the Erythroid Promoter Are Required for High Level Expression of the Human α-Spectrin Gene

α-Spectrin is a membrane protein critical for the flexibility and stability of the erythrocyte. We are attempting to identify and characterize the molecular mechanisms controlling the erythroid-specific expression of the α-spectrin gene. Previously, we demonstrated that the core promoter of the human α-spectrin gene directed low levels of erythroid-specific expression only in the early stages of erythroid differentiation. We have now identified a region 3′ of the core promoter that contains a DNase I hypersensitive site and directs high level, erythroid-specific expression in reporter gene/transfection assays. In vitro DNase I footprinting and electrophoretic mobility shift assays identified two functional GATA-1 sites in this region. Both GATA-1 sites were required for full activity, suggesting that elements binding to each site interact in a combinatorial manner. This region did not demonstrate enhancer activity in any orientation or position relative to either the α-spectrin core promoter or the thymidine kinase promoter in reporter gene assays. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of this region and occupancy by GATA-1 and CBP (cAMP-response element-binding protein (CREB)-binding protein). These results demonstrate that a region 3′ of the α-spectrin core promoter contains a GATA-1-dependent positive regulatory element that is required in its proper genomic orientation. This is an excellent candidate region for mutations associated with decreased α-spectrin gene expression in patients with hereditary spherocytosis and hereditary pyropoikilocytosis.

Genome-wide detection of a TFIID localization element from an initial human disease mutation

Eukaryotic core promoters are often characterized by the presence of consensus motifs such as the TATA box or initiator elements, which attract and direct the transcriptional machinery to the transcription start site. However, many human promoters have none of the known core promoter motifs, suggesting that undiscovered promoter motifs exist in the genome. We previously identified a mutation in the human Ankyrin-1 (ANK-1) promoter that causes the disease ankyrin-deficient Hereditary Spherocytosis (HS). Although the ANK-1 promoter is CpG rich, no discernable basal promoter elements had been identified. We showed that the HS mutation disrupted the binding of the transcription factor TFIID, the major component of the pre-initiation complex. We hypothesized that the mutation identified a candidate promoter element with a more widespread role in gene regulation. We examined 17 181 human promoters for the experimentally validated binding site, called the TFIID localization sequence (DLS) and found three times as many promoters containing DLS than TATA motifs. Mutational analyses of DLS sequences confirmed their functional significance, as did the addition of a DLS site to a minimal Sp1 promoter. Our results demonstrate that novel promoter elements can be identified on a genome-wide scale through observations of regulatory disruptions that cause human disease.