Erythroid Kruppel-like factor (EKLF) is active in primitive and definitive erythroid cells and is required for the function of 5'HS3 of the beta -globin locus control region

Abstract

Disruption of the gene for transcription factor EKLF (erythroid Krü ppel-like factor) results in fatal anaemiacaused by severely reduced expression of the adult β-globin gene, while other erythroid-specific genes, including the embryonic ε-and fetal γ-globin genes, are expressed normally. Thus, EKLF is thought to be a stage-specific factor acting through the CACC box in the β-gene promoter, even though it is already present in embryonic red cells. Here, we show that a β-globin gene linked directly to the locus control region (LCR) is expressed at embryonic stages, and that this is only modestly reduced in EKLF -/-embryos. Thus, embryonic β-globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the locus control region, we analysed the expression of LCR-driven lacZ reporters. This shows that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5ЈHS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5ЈHS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Finally, we show that overexpression of EKLF results in an earlier switch from γ-to β-globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in β-globin gene activation.

In Vivo Formation of a Human β-Globin Locus Control Region Core Element Requires Binding Sites for Multiple Factors Including GATA-1, NF-E2, Erythroid Kruppel-like Factor, and Sp1

“…57,63,64). Recently, Sp1 has been shown to interact with an SWI/SNF-related complex, providing a direct link between this factor and nucleosomal remodeling (50). Our own data showing weak HS formation with an Sp1 site alone (Fig.…”

“…The fact that EKLF is able to bind to a portion of the HSFE in vitro that is required for LCR HS core formation raises the possibility that EKLF also recruits EKLF coactivator-remodeling complex 1 or another remodeling complex to the HS cores. EKLF has also been shown to interact with HS3 of the LCR (50), and in EKLF knockout mice HS3 (but not HS4) fails to form (51). The fact that HS4, on which our model HS is based, forms in these mice suggests that although the 5Ј site (which binds EKLF in vitro) is required for HS formation, the related factor, Sp1, is capable of forming the HS4 in the context of the full site.…”

Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the β-globin locus control region in vivo

“…The expression of other erythroid-specific genes examined, including the ␣-like and the embryonic/fetal ␤-like globins, is not affected showing that EKLF is essential for the activation of the adult ␤-globin promoter (Nuez et al 1995;Perkins et al 1995;Wijgerde et al 1996). In addition, we have demonstrated recently that the expression of 5ЈHS3-lacZ transgenes requires EKLF (Tewari et al 1998), and DNase I hypersensitive site analysis of the human ␤-globin locus in the EKLF null background showed strongly reduced sensitivity at the ␤-globin promoter and moderately reduced sensitivity at 5ЈHS3 . These data agree well with our observation that direct binding of mEKLF to the mutant fp 1-3 sequence results in elevated reporter gene expression and increased DNase I hypersensitivity at fp 1-3.…”

“…The analysis of knockout mice is a first step toward the solution of this problem (e.g., Nuez et al 1995;Perkins et al 1995;Marin et al 1997;Tewari et al 1998), but this approach can only provide indirect answers. Pioneering work in Drosophila has established that a DNA-binding specificity mutant can be used as a tool to demonstrate direct interaction of a transcription factor and a DNA target site in a multicellular organism (Schier and Gehring 1992).…”

Non-random subcellular distribution of variant EKLF in erythroid cells

“…Even though the most striking effect of EKLF ablation is the lack of adult β-globin expression in the definitive erythroid cell [11,13], later studies suggested that EKLF is also transcriptionally active within the primitive erythroid nucleus [53,54]. Recent studies more directly show that there are other targets, including some in the primitive cell, that are also negatively affected by the absence of EKLF [17][18][19][20].…”