New Insights into the Mechanism of Dominant Anemia Caused By Zinc Finger Mutations in KLF1

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 KLF1 [7]. Recently an ENU-generated mouse model of neonatal anemia, ‘nan’, was also reported to harbour a mutation in the second zinc-finger of KLF1 [8]. Remarkably, the ‘nan’ mutation (E339D) resides at exactly the same amino acid which results in human CDA IV (= 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 mutation [9].

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 cells [10]. We performed ChIP-seq to determine differences in genome occupancy in vivo, and identified novel sites occupied by EKLF-E339D but not by wild type KLF1. Using de novo motif discovery [11], 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 any known TFs, so we think it represents a sequence specificity not normally encoded in mammals. Ectopic binding to non-erythroid gene promoters is accompanied by aberrant gene expression as determined by 4sU labelling and deep sequencing of tamoxifen-induced primary nuclear RNAs. We find a 4-fold greater number of genes induced by KLF1-nan compared with wild type KLF1 which is consistent with degenerate genome occupancy. We compared the KLF1-nan dependent genes with RNA-seq performed in primary fetal liver for KLF1^+/nan versus KLF1^+/- mice. We confirmed aberrant binding using EMSA and surface plasmon resonance (SPR) using recombinant GST-Klf1 zinc finger domains expressed in E.coli. The degenerate motif is consistent with structural models of how the second zinc finger of KLF1 specifically interacts with its binding site [12, 13]. We are undertaking structural studies to confirm this modelling. Together RNA-seq, ChIP-seq and SPR studies have provided a novel explanation for how mutations in KLF1 result in dominant anemia in mice and man. To our knowledge 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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