FKLF, a Novel Krüppel-Like Factor That Activates Human Embryonic and Fetal β-Like Globin Genes

The programmed expression of globin genes is tissue and developmental stage specific. In humans, five β-like globin genes (ɛ, Aγ, Gγ, δ, and β) form a cluster on the short arm of chromosome 11, and their expression is characterized by two major switches initially from embryonic (ɛ) to fetal (Aγ and Gγ) and subsequently to adult (δ and β) globin gene expression (39). Although a number of cis-acting elements of globin genes and corresponding trans-acting factors have been identified (9, 27), the precise molecular mechanisms of globin gene regulation are still unclear. Especially limited is the information on the trans-acting factors involved in the developmental control of fetal and embryonic globin genes. The CACCC (or GT) box is a cis-acting element found in a variety of genes expressed in erythroid and nonerythroid tissues. Each β-like globin gene (except the δ gene) has one or two CACCC boxes among the conserved promoter sequences. The importance of the CACCC box for β-globin gene transcription has been demonstrated by naturally occurring mutations which cause β thalassemia (20, 28, 29). A role of CACCC box in γ gene expression is supported by several pieces of evidence. First, CACCC box-deleted γ gene promoters display reduced activation of linked genes in K562 cells by transient (21, 45) and stable (6) transfection assays. Second, transgenic mice carrying a truncated Aγ promoter construct which lacks a functional CACCC box show decreased Aγ gene expression in all developmental stages (38). Third, in vivo footprinting studies have shown that the γ CACCC sequence shows significant protein binding in γ gene-expressing cells but not in β-gene-expressing cells (16). Collectively, these results suggest that the CACCC box is one of the key cis-acting elements for γ gene expression. Among the proteins binding to the CACCC boxes of the globin genes, Sp1, a ubiquitous (18) Kruppel-like zinc finger protein, and EKLF, an erythroid tissue-specific (24), Kruppel-like zinc finger protein, are well characterized. Sp1 is known to interact with the ɛ (47)-, γ (13)-, and β (15)-globin gene CACCC boxes. However, Sp1−/− mice express embryonic globin genes at a reduced but still significant level (22), suggesting that this ubiquitous protein is not sufficient, at least in the embryonic stage, for globin gene transcription. In contrast, EKLF binds to the proximal β gene CACCC box (5, 24) and plays a critical role in β-globin gene expression (26, 32). Studies of EKLF-deficient mice carrying human β-globin loci, however, revealed that the γ gene expression is not dependent on EKLF (31, 46). The specificity of interaction of EKLF with the β gene CACCC box is achieved by a 9-bp sequence (CCA CAC CCT), which can be recognized by the three zinc fingers of EKLF (24). The analogous sequence of the CACCC box of the γ-globin gene promoter is CTC CAC CCA, and it is also found in the mouse ɛy gene promoters. We assumed that a factor having Kruppel-type zinc finger structures such as Sp1 and EKLF exists and functions as an activator of the fetal globin gene promoter. Human fetal liver erythroid cells were screened for Sp1- or EKLF-type cDNAs by PCR (30), and a cDNA which encodes a novel Kruppel-type zinc finger protein, FKLF (embryonic/fetal β-like globin gene-activating Kruppel-like factor), was isolated. We show that the FKLF gene is preferentially expressed in erythroid cells and that it strongly activates ɛ- and γ-globin gene promoters.