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 Krü ppellike 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 wildtype 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 (ϳ 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 ␤-globin gene but results in a disease phenotype even with the wildtype 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. (Blood. 2011;118(11):3137-3145) IntroductionErythroid Krüppel-like factor (EKLF; KLF1) is an erythroid specific transcription factor essential for ␤-globin expression, the switch from fetal to adult globin and definitive erythropoiesis. 1,2 The role of EKLF in ␤-globin expression has been extensively studied. 3 EKLF null mice die in utero around embryonic day 14 to 15 because of failure of ␤-globin expression during fetal liver erythropoiesis. 4,5 Similarly, ␤-globin expression is absent in EKLF null mice containing a human ␤-locus transgene, whereas ␥-globin is increased. 6 Furthermore, accumulating evidence shows that EKLF also regulates many other erythroid genes and hence plays a critical and central role in erythropoiesis. 2,[7][8][9][10] Regulated by post-translational modifications, such as acetylation, phosphorylation, and sumoylation, EKLF modulates both chromatin remodeling and transcriptional activity via interaction with other proteins and complexes. 11-14 EKLF is composed of 362 amino acids, which form a proline-rich amino terminal domain, thought to be the site of interaction with chromatin, and 3 highly conserved Krüppel-like zinc fingers (ZF1-ZF3) at the carboxy terminus, responsible for recognition of the consensus NCNCNCCCN promoter sequence. 15,16 All of the mutations explored in this study fall within the second ZF domain, a key site of interaction with DNA promoter regions. Although the exact mechanism by which EKLF regulates gene expression is not yet fully elucidated, elegant data point toward EKLF as a coordinator of the 3-dimensional genomic architecture. 17,18 In regard to the globin switch, EKLF is thought to play a central role in 3-dimensional chromatin loopi...

Compound heterozygosity for KLF1 mutations is associated with microcytic hypochromic anemia and increased fetal hemoglobin

“…Surprisingly, compound heterozygotes of the KLF1 mutations c.809C4A (p. (Ser270Ter)) and c.994A4C (p.(Lys332Gln)), both of which affect the ZF domain, did not produce severe anemia or a β-thalassemia phenotype, suggesting that the c.994A4C (p.(Lys332Gln)) mutation has little impact on the transactivation capacity of KLF1. 13,17 Furthermore, the HS2 − γ prom/ Renilla − β prom/Firefly reporter assay demonstrated that the compound c.994A4C (p.(Lys332Gln)) mutations in the Sardinian family had less effect than the mutations described in the present study. (Supplementary Figure S4).…”

“…14 Studies suggest that the amount and type of mutant KLF1 protein produced are responsible for different RBC phenotypes. [15][16][17][18] Further research is required to determine the relationship between the various KLF1 mutations and the severity of clinical phenotypes.…”

Neomorphic effects of the neonatal anemia (Nan-Eklf) mutation contribute to deficits throughout development

“…In the present case, the most direct connection is with CDA type IV (Iolascon et al, 2012), which is caused by a mutation in human KLF1 at the same amino acid (Arnaud et al, 2010;Jaffray et al, 2013;Singleton et al, 2011). The change is non-conservative (E to K), so although not all characteristics of the Nan mouse may apply, individuals with CDA exhibit altered globin switching and membrane deficits that are highly reminiscent of the present model (discussed by Siatecka et al, 2010b).…”

“…However, some mutations lead to anemias (Arnaud et al, 2010;Huang et al, 2015;Jaffray et al, 2013;Singleton et al, 2011;Viprakasit et al, 2014; reviewed by Perkins et al, 2016). The human KLF1 mutation (E325K) in congenital dyserythropoietic anemia (CDA) (Arnaud et al, 2010;Jaffray et al, 2013;Singleton et al, 2011) is at the same amino acid as that seen in the mouse Nan mutant (Heruth et al, 2010;Siatecka et al, 2010b), albeit a different substitution. Nan is inherited in a semi-dominant fashion: homozygotes die in utero at E10-11, while heterozygous Nan/+ mice exhibit lifelong severe anemia that is characterized by reticulocytosis, splenomegaly, altered globin expression and cell membrane defects (Lyon, 1983;Siatecka et al, 2010b;White et al, 2009).…”

Extrinsic and intrinsic control by EKLF (KLF1) within a specialized erythroid niche

“…This variant is altered within the second zinc finger (E325K), changing its cognate binding specificity and probably converting it to an interfering protein (Singleton et al, 2011). In this context, it is interesting to compare these observations to that of the Nan mouse, which harbors a mutation at the same site but with a different substitution (E339D) (Heruth et al, 2010;Siatecka et al, 2010).…”