title: Mutations in the second zinc finger of human EKLF reduce promoter affinity but give rise to benign and disease phenotypes.
abstract: 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 5 mutant EKLF proteins and quantitated their binding affinity to a range of EKLF-regulated genes. Missense mutants (R328H, R328L, and R331G) from persons with InLu phenotype did not bind DNA. Hence, as with the heterozygous loss of function nonsense (L127X, S270X, and K292X) and frameshift (P190Lfs and R319Efs) EKLF mutations, monoallelic loss of EKLF does not result in haploinsufficiency at all loci. 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 nonfunctional 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 classic 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.
authors.affiliation: Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant, Bristol, UK.
authors.forename: Belinda K
authors.initials: BK
authors.lastname: Singleton
authors.forename: Winnie
authors.initials: W
authors.lastname: Lau
authors.forename: Victoria S S
authors.initials: VS
authors.lastname: Fairweather
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Burton
authors.forename: Marieangela C
authors.initials: MC
authors.lastname: Wilson
authors.forename: Steve F
authors.initials: SF
authors.lastname: Parsons
authors.forename: Ben M
authors.initials: BM
authors.lastname: Richardson
authors.forename: Kongtana
authors.initials: K
authors.lastname: Trakarnsanga
authors.forename: R Leo
authors.initials: RL
authors.lastname: Brady
authors.forename: David J
authors.initials: DJ
authors.lastname: Anstee
authors.forename: Jan
authors.initials: J
authors.lastname: Frayne
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D004194:Disease / Q000235:genetics*; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D012720:Severity of Illness Index; D013379:Substrate Specificity / Q000235:genetics; D015533:Transcriptional Activation; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D051741:Kruppel-Like Transcription Factors; C080774:erythroid Kruppel-like factor