Role of KLF4 and KLF12 in γ-Globin Gene Regulation.

Abstract 4075

Poster Board III-1010

Kruppel-like factors (KLFs) are a family of Cys₂His₂ zinc-finger DNA binding proteins that regulate gene expression through CACCC/GC/GT box binding in gene promoters. The CACCC element is critical for the developmental regulation of the human γ-globin and β-globin genes and studies are being done to ferret out various factors that bind this region and modulate gene activity. We recently identified two Kruppel-like factors, KLF4 and KLF12 whose expression levels decreased based on microarray-based gene profiling, concomitantly with decreased γ-globin expression during erythroid maturation. Decreased expression of both factors was further confirmed using quantitative PCR (qPCR) analysis. KLF4 and KLF12 mRNA levels decreased 56-fold and 16-fold respectively by day 28 compared to levels in day 7 erythroid progenitors. We next determined if KLF4 and KLF12 bind the γ-globin CACC box by electrophoretic mobility shift assay (EMSA) using nuclear proteins extracted from K562 cells and a [γ-³²P] labeled γ-CACC probe located between -155 to -132 relative to the γ-globin gene cap site. Three DNA-protein complexes were observed. The specificity of these interactions was confirmed by competition reactions in which preincubation with excess unlabelled γ-CACC oligonucleotide effectively abolished the formation of all DNA/protein complexes; addition of nonspecific oligonucleotide had no effect on binding activity. Addition of polyclonal KLF4 or KLF12 antibodies to the EMSA reaction resulted in a marked decrease in intensity of all DNA-protein complexes suggesting both KLF4 and KLF12 are present. Additional studies were performed to determine the effect of the known fetal hemoglobin inducer hemin on KLF gene expression in K562 cells. Hemin stimulated γ-globin transcription while increasing KLF4 and KLF12 66-fold and 4-fold respectively (p<0.05). Hemin treatment in KU812 erythroleukemia cells which actively transcribe both γ- and β-globin, also produced a 10-fold increase (p<0.05) in KLF4; KLF12 levels were not changed. Our preliminary data suggest these KLFs might play a role in γ-globin regulation. siRNA mediated gene silencing studies are underway to determine if KLF4 and/or KLF12 play a direct role in γ-globin gene regulation. This mechanism could provide important molecular targets for fetal hemoglobin reactivation. This will be highly significant towards developing therapeutic strategies for hemoglobinopathies like sickle cell anemia and β-thalassemia.