Mutations in the Second Linker of KLF1 Cause Congenital Non-Spherocytic Hemolytic Anemia Due to Global Reduction of In Vivo DNA-Binding Affinity

The mommeD45 mutation generates an amino acid transversion (H350R) within a conserved linker peptide between zinc fingers two and three of Klf1 (linker 2). Klf1^H350R/H350R mice have mild compensated microcytic anaemia ¹. Mice Carrying the H350R mutation were interbred with Klf1^+/- mice. Klf1^H350R/-mice have severe perinatal haemolytic anaemia, jaundice and marked splenomegaly. Haematological evaluation of these mice shows similar phenotypes to human patients who are compound heterozygotes for null and linker 2 mutations in KLF1². Analysis of Klf1^H350R/- fetal liver by flow cytometry showed an increase in circulating immature CD71+ Ter119+ erythroblasts. In the bone marrow, a marked reduction in mature (Cd71- Ter119+) red blood cells was observed. Flow cytometric analysis of the spleen from Klf1^H350R/- animals revealed an expansion of erythroid cells consistent with extramedullary erythropoiesis. ChIP-seq for Klf1 in 14.5DPC fetal liver from Klf1^H350R/H350R mice revealed no loss in specificity when compared to wildtype Klf1, but a global reduction in affinity. Affinity measurements of recombinant zinc finger domains in vitro will be presented. By RNA-seq, we observed significantly lower expression of Klf1 target genes in mice homozygous for the H350R mutation compared to mice carrying a wildtype allele. And this correlates with reduced DNA binding observed in ChIP-seq and in vitro assays. Previous studies of the linkers in C₂H₂ zinc finger transcription factors have revealed their necessity as structural and regulatory components for the C₂H₂ class of transcription factors. Our results show the second linker of Klf1 plays an indirect role in DNA-binding and does not act just as a spacer for the zinc fingers.

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