Divergent Effects of KLF1 Variants: Gain and Loss of DNA Binding Determine the Severity of Erythropoietic Disruption

Abstract

Mutations in transcription factor (TF) DNA-binding domains (DBDs) have been implicated in a wide range of human diseases. One such TF, KLF1, plays a central role in erythropoiesis, the process by which hematopoietic stem cells differentiate into erythrocytes (red blood cells). Mutations in the KLF1 DBD are associated with a spectrum of blood disorders, ranging from relatively benign conditions, such as the Lutheran inhibitor blood type and hereditary persistence of fetal hemoglobin, to severe hemolytic anemias, including congenital dyserythropoietic anemia type IV. Although specific KLF1 variants have been linked to distinct clinical phenotypes, the extent to which these mutations differentially reshape KLF1 genomic occupancy and downstream transcriptional programs remains unclear. Addressing this gap requires an integrative analysis of how variant-specific alterations in DNA binding influence gene regulatory outcomes. This study investigates five clinically diverse KLF1 DBD variants (p.A298P, p.H299Y, p.R328H, p.E325D, and p.E325K) to elucidate the molecular mechanisms underlying disrupted erythropoiesis. Combined analyses of genomic occupancy, DNA sequence affinity, gene expression, and chromatin state reveal distinct mechanistic modes of variant KLF1 dysfunction: gain- and loss-of-function. Variants associated with milder clinical phenotypes (p.A298P, p.H299Y, and p.R328H) primarily reduce DNA-binding affinity, resulting in decreased occupancy at regulatory elements and underexpression of genes essential for erythrocyte development and differentiation. In contrast, variants linked to more severe phenotypes (p.E325D and p.E325K) modify DNA-binding affinity and specificity, enabling disruption of canonical KLF1 regulatory networks and ectopic occupancy at novel genomic sites that drive transcriptional programs opposing normal erythropoiesis. Collectively, these results establish a framework for understanding the mechanisms of KLF1 DBD variant perturbations, and clarify how changes in DNA binding, chromatin state, and gene expression drive diverse blood disorders.