A mutation in zinc finger domain of human KLF1 causes congenital dyserythropoietic anaemia (CDA) via promiscuous DNA binding and ectopic target gene expression — ASN Events

A mutation in zinc finger domain of human KLF1 causes congenital dyserythropoietic anaemia (CDA) via promiscuous DNA binding and ectopic target gene expression (#228)

Melissa Ilsley 1 , Kevin Gillinder 1 , Stephen Huang 1 , Charles Bell 2 , Graham Magor 1 , Andrew Perkins 1
  1. University of Queensland, Woolloongabba, QLD, Australia
  2. Peter MacCallum Cancer Research Institute, Melbourne, VIC, Australia

Krüppel-like factor 1 (KLF1) is an erythroid-specific transcription factor that is responsible for coordinating nearly all aspects of erythropoiesis. KLF1 consists of three C2H2-type zinc-fingers through which it binds to DNA at 9bp CACCC-box motifs (CCM CRC CCN). C2H2-type zinc-fingers (ZFs) consist of two antiparallel β-sheets and an α-helix, stabilised by a coordinating zinc ion. The α-helix of each ZF contacts the major groove of DNA, with specific residues binding to three nucleotides on the G-rich strand. Binding is mediated through -1, +3 and +6 amino acid residues of the ZF relative to the start of the α-helix. Since 2010, four unrelated patients diagnosed with a variant of Congenital Dyserythropoietic Anaemia (CDA - typeIV), a rare autosomal-dominant inherited erythrocyte disorder, have been shown to carry identical mutations at the +3 position in the second ZF of KLF1 (c.973G>A; E325K). Unlike loss-of-function mutations in KLF1, this mutation leads to a more severe phenotype than a KLF1 null allele, suggesting it is an unusual dominant mutation.

ChIP-seq analysis of cell lines developed to model CDA mutation in Klf1 identified ectopic DNA-binding events genome wide. Next-generation sequencing of newly-synthesised RNA (4sU-RNA-seq) reveals the dramatic transcriptional consequences of this ectopic binding. CDA-Klf1 binds and activates a large number of non-erythroid genes which derails erythroid differentiation and leads to red blood cell destruction (haemolysis). De novo motif discovery of ChIP-seq reveals that CDA-Klf1 prefers to bind to an altered CACC motif containing a central T at the nucleotide contacted by the mutated residue (CCM CTC CCN). Specificity for this altered motif was confirmed by biophysical measurements of in vitro DNA-binding affinity using recombinant zinc finger domains. Together, these results shed new light on the mechanisms by which missense mutations in DNA-binding domains of transcription factors can lead to autosomal dominant diseases.

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