Klf1 mutations in the second linker cause congenital non-spherocytic hemolytic anemia due to global reduction of <em>in vivo</em> DNA-binding affinity — ASN Events
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  3. Klf1 mutations in the second linker cause congenital non-spherocytic hemolytic anemia due to global reduction of in vivo DNA-binding affinity

Klf1 mutations in the second linker cause congenital non-spherocytic hemolytic anemia due to global reduction of in vivo DNA-binding affinity (#187)

Stephen Huang 1 , Kevin Gillinder 1 , Emma Whitelaw 2 , Andrew Perkins 1
  1. University of Queensland, Woolloongabba, QLD, Australia
  2. La Trobe University, Melbourne, VIC, Australia

Mutations of KLF1 in humans are becoming more apparent in the population as sequencing technology becomes more accessible to the clinic. Recent discoveries have shown that some anemias can be attributed to Klf1 mutations. We previously identified and characterized a mutation in the DNA binding domain of Klf1 (mommeD45). The mommeD45 mutation generates an amino acid transversion (H350R) within a conserved linker between zinc fingers two and three of Klf1. Klf1H350R/H350R mice have mild compensated microcytic anaemia which models the phenotype in a recently described human family. Mice Carrying the H350R mutation were interbred with Klf1+/- mice. Klf1H350R/- mice have severe perinatal haemolytic anaemia and marked splenomegaly. Haematological evaluation of these mice shows similar phenotypes to human patients with the same mutation and genetic composition (compound heterozygous for Klf1 mutations). Analysis of Klf1H350R/- by flow cytometry showed an increase in circulating immature red blood cells. In the bone marrow, a lack of mature red blood cells was observed. Flow cytometric analysis of the spleen from Klf1H350R/- animals revealed an expansion of erythroid cells. ChIP-seq of Klf1 in 14.5DPC fetal liver from Klf1H350R/H350R mice revealed no loss in specificity when compared to wild type Klf1, but a global reduction in affinity. Affinity measurements of recombinant zinc finger domains in vitro will be presented. The expression of Klf1 target genes was determined by RNA-seq. We observed lower expression of important set of Klf1 target genes in mice carrying the mommeD45 mutation compared to mice carrying a wild type allele. The reduced expression correlates with reduced binding observed in ChIP-seq and in vitro studies. 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 thus far show that the second linker of Klf1 has a role in maintaining the integrity of Klf1 function and does not act just as a spacer for the zinc fingers.

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