Mutations in the second zinc finger of human EKLF reduce promoter affinity but give rise to benign and disease phenotypes

Mutations in the human erythroid Krüppel-like factor (EKLF) can lead to either anemia or the benign InLu phenotype. To elucidate the relationship between these mutations and the differing phenotypes we prepared recombinant forms of wild type and five mutant EKLF proteins and quantitated their binding affinity to a range of EKLF-regulated genes. Missense mutants (R328H, R328L and R331G) from individuals with InLu phenotype did not bind DNA. Hence, as with the heterozygous loss of function nonsense (L127X, S270X, K292X) and frameshift (P190Lfs and R319Efs) EKLF mutations, monoallelic loss of EKLF does not result in haploinsufficiency. In contrast, K332Q has a slightly reduced DNA binding affinity (~ 2 fold) for all promoters examined, but exhibits a phenotype only in a compound heterozygote with a non-functional allele. E325K also has a reduced, but significant, binding affinity particularly for the β-globin gene but results in a disease phenotype even with the wild type allele expressed, although not as a classical dominant-negative mutant. E325K protein may therefore actively interfere with EKLF-dependent processes by destabilizing transcription complexes, providing a rational explanation for the severity of the disease phenotype. Our study highlights the critical role of residues within the second EKLF zinc finger domain.