“Krüppeling Erythropoiesis”: KLF1 function in development and disease

Abstract

KLF1 is a three-fingered C2H2 transcription factor essential for the differentiation of definitive erythroid cells. KLF1 regulates the expression of many genes involved in this process and loss of Klf1 results in perinatal death due to anaemia and severe -thalassaemia in mice. The three C2H2 zinc-fingers of KLF1 are interconnected by two linker peptides. The first linker that connects the first and second zinc finger of KLF1 contains the conserved TGEKP sequence. The second linker connects the second and third zinc fingers and sequence TGHRP is more variant. In human patients, there have been multiple mutations identified in the 2 nd linker of KLF1. These mutations result in non-spherocytic haemolytic anaemia (NSHA) with microcytosis, and high foetal haemoglobin. While we understand pathology resulting from KLF1 mutations in these patients, very little is known about the precise molecular function of the 2 nd linker mutation, and in general, linker mutations across all C2H2 proteins. C2H2 zinc fingers are the largest class of DNA-binding proteins in the mammalian genome. They are characterized by the presence of two anti-parallel -sheets and an -helix (fold). Two cysteine and two histidine residues in the -fold coordinate a zinc molecule and are highly conserved across the family giving the zinc fingers their identity. Additionally, the -helix is directly involved in DNA contact through 3 amino acid residues at positions-1, +3 and +6 relative to the start of the helix. The linker peptide between each finger allows flexibility so that the -helix of each zinc-finger can contact DNA, while providing additional stability to these interactions through forming hydrogen bonds with the preceding -helix. This mechanism is known as c-capping and closes the 'frayed' c-terminus end of the -helix upon contacting DNA. Our laboratory recently identified the mommeD45 mutation from a mouse ENU mutagenesis screen for modifiers of an alpha-globin transgene. The mutation results in a transversion of the histidine to an arginine (H350R) residue in the second linker of KLF1. The mutation appears relatively benign in the heterozygous state. However homozygous D45 mice develop a mild anemic phenotype, including splenomegaly and microcytosis. To generate a model of NSHA in mice, like that observed in human patients, I crossed the mommeD45 mouse to a Klf1 heterozygous loss of function mouse to produce compound