Mutations In The Zinc Finger Domain Of Human and Mouse KLF1 Cause Congenital Dyserythropoietic Anemia (CDA) Via Promiscuous DNA Binding and Ectopic Target Gene Expression

Krûppel-like factor-1 (KLF1) is an essential erythroid-specific transcription factor^{1, 2}. A number of studies have shown up to ∼700 genes are poorly expressed when KLF1 is absent^3-6. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice, and partly responsible for congenital dyserythropoietic anemia type IV (CDA-IV) observed in humans with dominant mutations in the DNA-binding domain of KLF1⁷. Recently an ENU-generated mouse model of neonatal anemia, ‘nan’, was also reported to harbour a mutation in the second zinc-finger of KLF1⁸. Remarkably, the ‘nan’ mutation (E339D) resides at exactly the same amino acid which results in human CDA IV (i.e. E325 in humans). Unlike loss of function point mutations in KLF1, this mutation leads to a more severe phenotype than the KLF1 null allele, suggesting it is an unusual dominant mutation⁹.

To investigate how this mutation might cause disease, we introduced tamoxifen-inducible versions of KLF1 and KLF1^nan into an erythroid cell line derived from Klf1^-/- fetal liver cells¹⁰. We performed ChIP-seq to determine genome occupancy site preferences for KLF1 and KLF1^nan. We identified about 4-fold the number of binding sites within the genome for KLF1^nan versus KLF1; many of these are ectopic or promiscuous. Using de novo motif discovery¹¹, we find KLF1^nan binds a slightly degenerate CACC box element (CCMNGCCC) in comparison with wild type KLF1 (CCMCRCCC). This specificity is novel with respect to known TFs, so we think it represents specificity not normally present in mammals. The degenerate motif is consistent with models of how the second zinc finger of KLF1 specifically interacts with the 9bp consensus binding site^12,13. We also isolated nascent RNA from wild type and mutant cells, to identify primary transcriptional targets of KLF1 and aberrant targets of the KLF1^nanmutation. We performed primary transcript RNA-seq and validation using RT-PCR of pre-processed nuclear transcripts. Together the RNA-seq and ChIP-seq studies have provided a novel explanation for how mutations in KLF1 result in dominant anemia in mice and man. This mechanism, whereby a transcription factor DNA-binding domain mutation leads to promiscuous binding, activation of an aberrant transcriptional program and subsequent derailing of co-ordinated differentiation, is novel.

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