haematologica | 2011 96(5) EDITORIALS & PERSPECTIVES 635 Erythroid phenotypes associated with KLF1 mutations Joseph Borg, 1,2 George P. Patrinos, 3 Alex E. Felice 2,4 and Sjaak Philipsen 5 1 Department of Applied Biomedical Science, Faculty of Health Sciences, University of Malta; 2 Laboratory of Molecular Genetics, Department of Physiology & Biochemistry, University of Malta, Malta; 3 University of Patras, Department of Pharmacy, University Campus, Patras, Greece; 4 Thalassaemia Clinic, Section of Pathology, Mater Dei Hospital, Msida, Malta; 5 Erasmus MC, Department of Cell Biology; Netherlands Consortium for Systems Biology; Rotterdam, The Netherlands. E-mail: j.philipsen@erasmusmc.nl doi:10.3324/haematol.2011.043265 (Related Original Article on page 767) E rythroid Krüppel-Like Factor (KLF1; previously known as EKLF) is an essential erythroid-specific transcription fac- tor that was first identified by Miller and Bieker in 1993. 1 It binds the CACCC motif, an important DNA binding site in the regulatory elements of many erythroid genes including the HBB (β-globin) gene. Mutations in the β-globin CACC box which prevent KLF1 binding are a cause of β-thalassemia. 2 KLF1 has three zinc finger domains, which mediate sequence- specific binding to DNA and are, therefore, essential for activa- tion of KLF1 target genes (Figure 1). Functions of KLF1: studies in mice Mouse KLF1 null mutants displayed grossly normal erythro- poiesis at the embryonic stage when hematopoiesis takes place in the yolk sac, but they rapidly developed a very severe form of anemia at the fetal stage, when the site of hematopoiesis has shifted to the fetal liver. KLF1 null mutants failed to activate expression of β-globin, which is a fetal/adult globin in the mouse. Thus, inactivation of KLF1 causes lethal β-thalassemia. Remarkably, the expression of embryonic β-like globin genes, εy and βh1, and the α-like globin genes, embry- onic ζ and α1/α2, appeared to be normal. 3-4 This suggested that KLF1 has a role in fetal-to-adult globin gene switching as it occurs in humans. To test this idea, transgenic mice carrying a complete human β-globin locus were used. 5-6 Such mice express human γ-globin at the early fetal stages while the switch to β-globin is completed at the late fetal stages. An aus- tere reduction in β-globin expression was observed in KLF1 null fetuses, while expression of γ-globin was not dependent on KLF1 and even extended in its absence 5-6 (Figure 2). These data supported a role for KLF1 in globin switching. However, the anemia of KLF1 null mutants was not rescued by expression of γ-globin. 7 Genome-wide gene expression profiling studies revealed a global role for KLF1 in the activation of erythroid- specific genes 8-10 including globins, membrane- and structural proteins, heme synthesis enzymes and many other proteins involved in red cell metabolism. This explained the particularly severe anemia of KLF1 null mutants (Figure 3). KLF1 mutations in humans – inhibitor of Lutheran antigen expression [In(Lu)] Mutations in human KLF1 were first reported in 2008 by Singleton and colleagues. 11 They described 9 different loss-of- function KLF1 mutations which were causative to the rare In(Lu) blood group (Figures 1 and 3). Gene expression profiling revealed a list of more than 650 putative KLF1 target genes which considerably overlapped with those reported in KLF1 null mouse studies. 8-11 These target genes included BCAM that carries the Lutheran blood group antigens and CD44 that car- ries the Indian blood group antigens (Figure 3). The expression of these blood group antigens is suppressed in the In(Lu) indi- viduals. 11 No other clinical features were reported. KLF1 mutations in humans – hereditary persistence of fetal hemoglobin (HPFH) The direct association of mutations in human KLF1 with hemoglobin regulation came from the study of a large family from Malta. 12 Ten out of 27 family members exhibited HPFH Figure 1. Mutations in human KLF1. All currently reported mutations are shown; the color code refers to the original publications as indicated. (A) Schematic drawing of the KLF1 gene. Exons: green = non-coding regions; red = coding regions, orange = zinc fingers (F1, F2, F3). Mutations between brackets are believed to be neutral sub- stitutions. (B) Amino acid sequence of the zinc fin- gers of KLF1 (top line). Bottom line: amino acids invariable between the zinc finger domains of all 17 human KLF transcription factors (KLF_all). Blue boxes highlight amino acids involved in coor- dination of the Zn atom; yellow boxes highlight amino acids directly involved in DNA binding. The arrows indicate amino acids that make base-spe- cific contacts with the DNA double helix. A B