Characterisation of Novel Hypomorphic and Null Mutations in Klf1 Derived from a Genetic Screen for Modifiers of a-Globin Transgene Variegation

Position-effect variegation of transgene expression is sensitive to the chromatin state. We previously reported a forward genetic screen in mice carrying a variegated a-globin GFP transgene to find novel genes encoding epigenetic regulators. We named the phenovariant strains "Mommes" for Modifiers of murine metastable epi-alleles. Here we report positional cloning of mutations in two Momme strains which result in suppression of variegation; i.e. an increased percentage of GFP+ circulating red blood cells. Both strains harbour point mutations in the erythroid specific transcription factor, Klf1. One (D11) generates a stop codon in the zinc finger domain. D11 homozygous mice die in utero of anaemia at 14.5DPC. The other (D45) generates an amino acid transversion (H350R) within a conserved linker between zinc fingers two and three. Homozygous MommeD45 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. Klf1^H350R/- mice have severe perinatal haemolytic anaemia and marked splenomegaly. Furthermore blood haemoglobin content, haematocrit and red blood cell size (MCV) were significantly reduced in Klf1^H350R/- mice compared to wildtype and D45 homozygous offspring of the same age. Analysis of Klf1^H350R/- 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 Klf1^H350R/- animals revealed an expansion of erythroid cells and a relative reduction in B and T Cells. Gel shifts assays of a recombinant Klf1 zinc finger protein with the H350R mutation showed normal binding to the b -globin promoter sequence but weak binding to the Alas2 intronic enhancer site. Furthermore b -globin gene expression was near normal whereas expression of other known Klf1 target genes was decreased. We will discuss how H350R disrupts function from ChIP-seq and RNA-seq in primary fetal liver tissue. 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 (at a subset of Klf1-occupied sites,) and does not act just as a spacer for the zinc fingers.