Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In some HS patients, mutations in the ankyrin promoter have been hypothesized to lead to decreased ankyrin mRNA synthesis. The ankyrin erythroid promoter is a member of the most common class of mammalian promoters which lack conserved TATA, initiator or other promoter cis elements and have high G+C content, functional Sp1 binding sites and multiple transcription initiation sites. We identified a novel ankyrin gene promoter mutation, a TG deletion adjacent to a transcription initiation site, in a patient with ankyrin-linked HS and analyzed its effects on ankyrin expression. In vitro, the mutant promoter directed decreased levels of gene expression, altered transcription initiation site utilization and exhibited defective binding of TATA-binding protein (TBP) and TFIID complex formation. In a transgenic mouse model, the mutant ankyrin promoter led to abnormalities in gene expression, including decreased expression of a reporter gene and altered transcription initiation site utilization. These data indicate that the mutation alters ankyrin gene transcription and contributes to the HS phenotype by decreasing ankyrin gene synthesis via disruption of TFIID complex interactions with the ankyrin core promoter. These studies support the model that in promoters that lack conserved cis elements, the TFIID complex directs preinitiation complex formation at specific sites in core promoter DNA and provide the first evidence that disruption of TBP binding and TFIID complex formation in this type of promoter leads to alterations in start site utilization, decreased gene expression and a disease phenotype in vivo.

The anion exchanger protein 1 (AE1; band 3) is an abundant erythrocyte transmembrane protein that regulates chloride-bicarbonate exchange and provides an attachment site for the erythrocyte membrane skeleton on the cytoplasmic domain. We analyzed the function of the erythroid AE1 gene promoter by using run-on transcription, RNase protection, transient transfection, and transgenic mouse assays. AE1 mRNA was transcribed at a higher level and maintained at a higher steady-state level than either ankyrin or ␤-spectrin in mouse fetal liver cells. When linked to a human ␥-globin gene, two different AE1 promoters directed erythroidspecific expression of ␥-globin mRNA in 18 of 18 lines of transgenic mice. However, variegated expression of ␥-globin was observed in 14 of 18 lines. While there was a significant correlation between transgene copy number and the amount of ␥-globin mRNA in all 18 lines, the transgene mRNAs initiated upstream of the start site of the endogenous AE1 mRNA. Addition of the insulator element from 5HS4 of the chicken ␤-globin cluster to the AE1/␥-globin transgene allowed position-independent, copy-number-dependent expression at levels similar to the AE1 transcription rate in six of six lines of transgenic mice. The mRNA from the insulated AE1/␥-globin transgene mapped to the start site of the endogenous AE1 mRNA, and ␥-globin protein was expressed in 100% of erythrocytes in all lines. We conclude that the chicken ␤-globin 5HS4 element is necessary for full function of the AE1 promoter and that position effect variegation is associated with RNA transcription from the upstream start sites.

No abstract