A Krüppel-like factor 1 (KLF1) mutation associated with severe congenital dyserythropoietic anemia alters its DNA-binding specificity

Kruppel-like factor 1 (KLF1/EKLF) is a transcription factor that globally activates genes involved in erythroid cell development. Various mutations are identified in the human KLF1 gene. The E325K mutation causes congenital dyserythropoietic anemia (CDA) type IV, characterized by severe anemia and non-erythroid-related symptoms. The CDA mutation is in the second zinc finger of KLF1 at a position functionally involved in its interactions with DNA. The molecular parameters of how CDA-KLF1 exerts its biological effects have not been addressed. Here, using an in vitro selection strategy we determined the preferred DNA-binding site for CDA-KLF1. Binding to the deduced consensus sequence is supported by in vitro gel shifts and by in vivo functional reporter gene studies. Two significant changes compared to WT binding are observed: G is selected as the middle nucleotide and the 3-portion of the consensus sequence is more degenerate. As a consequence CDA-KLF1 did not bind the WT consensus sequence. However, activation of ectopic sites is promoted. Continuous activation of WT target genes occurs if they fortuitously contain the novel CDA site nearby. Our findings provide a molecular understanding of how a single mutation in the KLF1 zinc finger exerts an effects on erythroid physiology in CDA type IV.