Functional Analysis of a Novelcis-Acting Regulatory Region within the Human Ankyrin Gene (ANK-1) Promoter

Abstract

The characterization of atypical mutations in loci associated with diseases is a powerful tool to discover novel regulatory elements. We previously identified a dinucleotide deletion in the human ankyrin-1 gene (ANK-1) promoter that underlies ankyrin-deficient hereditary spherocytosis. The presence of the deletion was associated with a decrease in promoter function both in vitro and in vivo establishing it as a causative hereditary spherocytosis mutation. The dinucleotide deletion is located in the 5 untranslated region of the ANK-1 gene and disrupts the binding of TATA binding protein and TFIID, components of the preinitiation complex. We hypothesized that the nucleotides surrounding the mutation define an uncharacterized regulatory sequence. To test this hypothesis, we generated a library of more than 16,000 ANK-1 promoters with degenerate sequence around the mutation and cloned the functional promoter sequences after cell-free transcription. We identified the wild type and three additional sequences, from which we derived a consensus. The sequences were shown to be functional in cell-free transcription, transient-transfection, and transgenic mouse assays. One sequence increased ANK-1 promoter function 5-fold, while randomly chosen sequences decreased ANK-1 promoter function. Our results demonstrate a novel functional motif in the ANK-1 promoter.Hereditary spherocytosis (HS; OMIM 182900) is a dominant inherited hemolytic anemia that affects approximately 1/2,500 people of all races worldwide (1, 17, 18). Typically, HS patients have mild symptoms, which can be exacerbated by viral infections (19). These symptoms include elevated reticulocyte counts and smaller, spherical erythrocytes on a blood smear and are accompanied by an abnormal osmotic fragility (13,19,23). The majority of HS mutations have been found in the genes encoding the erythrocyte membrane skeleton proteins ankyrin-1 (ANK-1; ϳ60%) and Band 3 (SLC4A1; ϳ20%) (1,19). Virtually all of the described HS mutations cause a functional deficiency of erythrocyte skeleton proteins, either by premature termination and/or amino acid substitutions in regions critical for the protein-protein interactions that stabilize the erythrocyte membrane skeleton (17, 18). In the 10 to 20% of patients in whom no mutations have been detected in the coding region of the membrane skeleton protein genes, the causative mutations are proposed to be in cis-acting regulatory regions resulting in decreased transcription of mRNA resulting in haploinsufficiency (12,17,18).Support for this hypothesis has come from our previous analysis of a German patient with a severe form of HS (20). The patient was shown to have two mutations in the ANK-1 gene. The first was a 20-bp deletion in exon 6, leading to premature termination, presumably inherited from the father. The second mutation was a deletion of a TG dinucleotide in the 5Ј untranslated region of the ANK-1 gene located at position Ϫ72/73 relative to the ATG initiation codon (12,20) or ϩ 12/13 from the transcriptional start site (TSS) ...

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

“…The significant enrichment of the DLS motif in promoter sequences compared with control data implicated the DLS in widespread transcriptional regulatory processes in the genome, but did not address the many complexities associated with motif searches in promoter sequences. Moreover, this consensus motif was based on experimental data derived from a small number of examples (24) in a stringent in vitro assay system, which could underestimate the nucleotide diversity allowable at promoters other than ANK -1.

Figure 1.Development of a DLS motif pattern for promoter analyses.

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“…The Tesc promoter contained the initial ‘GC’ that was found to be most active in previous in vitro assays (24). Mutation of the Tesc DLS resulted in the greatest loss of function in this analysis, consistent with our previous experimental evidence that a motif with a G at position one bound TFIID most efficiently (24).

Figure 5.Transient transfection analysis of four promoters containing DLS sequences in K562 cells.

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“…Combinations of motifs or the absence of any features in a promoter were determined through intersecting the genomic coordinates. Binding sites were mapped within the region of the expected functional location allowing some padding for small windows to accommodate imprecise TSS mapping, for example TATA motifs (−49, −20), DPE from ( 24 , 34 ), Inr at (−15, 15), BRE at (−44, −18), CCAAT at (−108, 9) and Sp1 (−80, −40). DLS motifs were initially assessed in the range from (−100 to +40) and later expanded to (−550 to +100).…”

“…We have previously reported that the human ankyrin-1 ( ANK -1) promoter has a high G + C content (77%) and a broad initiation pattern containing multiple, closely spaced TSSs ( 24 ). Furthermore, the gene utilizes several alternative promoters to confer tissue specificity to its transcript isoforms ( 25 ).…”

“…As a result, the choice of initiation site utilized in transgenic mice and cell-free transcription assays became altered ( 27 ). Further experimental analyses of the DNA at the deletion site identified a consensus motif that bound TFIID and was essential for transcription in vitro and in vivo ( 24 ). One particular version of the TFIID localization sequence (DLS) motif conferred a 5-fold increase over the wild-type DLS sequence when assayed in transgenic mice, illustrating the potential for modulation of regulatory activity through site-specific variation in this binding motif.…”