Functional analysis of a novel cis-acting regulatory region within the human ankyrin gene (ANK-1) promoter

Mol Cell Biol. 2010 Jul;30(14):3493-502. doi: 10.1128/MCB.00119-10. Epub 2010 May 17.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5' Untranslated Regions
  • Animals
  • Ankyrins / genetics*
  • Base Sequence
  • Binding Sites / genetics
  • Cell-Free System
  • Consensus Sequence
  • DNA / genetics
  • DNA / metabolism
  • DNA Primers / genetics
  • Gene Library
  • Humans
  • In Vitro Techniques
  • Mice
  • Mice, Transgenic
  • Molecular Sequence Data
  • Promoter Regions, Genetic*
  • Regulatory Sequences, Nucleic Acid*
  • Sequence Deletion
  • Sequence Homology, Nucleic Acid
  • TATA-Box Binding Protein / metabolism
  • Transcription Factor TFIID / metabolism
  • Transcription, Genetic


  • 5' Untranslated Regions
  • ANK1 protein, human
  • Ankyrins
  • DNA Primers
  • TATA-Box Binding Protein
  • Transcription Factor TFIID
  • DNA