Erythroid Kruppel-like factor (EKLF) is recruited to the gamma-globin gene promoter as a co-activator and is required for gamma-globin gene induction by short-chain fatty acid derivatives.

Pharmacological reactivation of developmentally silenced fetal globin expression is a promising targeted molecular approach to the treatment of the β-hemoglobinopathies, sickle cell anemia, and β-thalassemia. During human hematopoietic development, the first β-type globin gene expressed in blood cells formed within the yolk sac is the embryonic (e)-globin variant. As the primary site of hematopoiesis migrates to the fetal liver, it is paralleled by a switch to fetal (γ)-globin gene expression. A terminal switch in expression occurs, with adult (β)-globin gene expression predominating once hematopoiesis moves permanently to the bone marrow. Mice lack a fetal globin gene equivalent and display only a single switch, from embryonic (βH1, βH0, eγ)-globin genes in the yolk sac to the adult (β minor- and β major-) globin genes in the fetal liver, adult bone marrow, and spleen. The relative proportions of expression of the genes comprising the β-globin cluster and the temporal sequence of their expression are regulated by the interactions of both ubiquitous and erythroid-specific transcription factors and complexes with the cis-acting elements residing in the promoters of each of these genes, and the interactions of these promoter-protein complexes with the upstream β-globin locus control region (βLCR), designated the active chromatin hub (1-5). Agents capable of reactivating fetal globin gene expression in vitro or in vivo fall into several major groups, including cytotoxic/chemotherapeutic agents (e.g., hydroxyurea or azacytidine) and histone deacetylase (HDAC) inhibitors (e.g., the short-chain fatty acids [SCFAs] butyrate and valproate). More recently, a new class of SCFA derivatives has been developed, which efficiently reactivates γ-globin gene expression in vivo and in vitro in multiple species, but which does not possess any HDAC-inhibitory activity, and certain of these compounds are now entering clinical trials for the hemoglobinopathies [(6-11) and S.P. Perrine, M.S. Boosalis, D.V. Faller, unpublished data]. These compounds have been shown to relieve active repression of the silenced γ-globin promoter by selectively displacing a repressor complex containing HDAC3 and NCoR (11), but the way in which these agents then facilitate transcriptional activation of the γ-globin promoter has not yet been fully elucidated. The erythroid Kruppel-like factor (EKLF/KLF1) gene is a erythroid cell-specific transcription factor, containing a DNA-binding domain (DBD) located at its C-terminus, composed of three ‘Kruppel-like’ C2H2 zinc finger motifs, and a proline-rich transactivation domain at its N-terminus (12, 13). EKLF preferentially binds to, and activates, the CACCC motif located in the adult β-globin promoter (12-14). EKLF is essential for adult β-globin gene transcription, as mice homozygous null for EKLF die at E14.5–E15, the time of hemoglobin switching, because of a severe deficiency in β-globin production (15, 16). EKLF also binds to the βLCR and β-globin promoters, and is required for direct interactions between the βLCR and the β-globin gene in humans, or the β major (βmaj) gene in mice (17, 18). EKLF is critical for the recruitment of a number of transcription factors or co-activators to the β-globin gene promoter in hematopoietic progenitor cells, including p45, CBP, and SWI/SNF complexes, which then contribute to locus chromatin activation and β-globin gene potentiation (19). Mammalian SWI/SNF complexes consist of ~15 subunits and contain either BRM or BRG1 as the core ATPase, along with diverse BRG1-associated factors (BAFs). SWI/SNF complexes can serve as coactivators or co-repressors depending upon the constituent BAFs (20-22), induce the partial unwrapping of DNA from the nucleosome, and can promote both octamer sliding and transfer to neighboring DNA (23). SWI/SNF is required for the developmental regulation of the human β-globin locus (24-26). EKLF recruits an erythroid-specific BRG1-containing SWI/SNF chromatin remodeling complex to the β-globin locus (27). This EKLF-BRG1 interaction appears to be crucial for EKLF transcriptional activity. The absence of EKLF leads to reduced DNase I hypersensitive site (HS) formation at the mouse βmaj and human β-globin promoters (19, 28), and mice expressing a mutant hypomorphic BRG1 exhibit abnormal definitive erythroid cell differentiation, which resembles the phenotype observed in EKLF-knockout mice (29). While required for the adult switch to high-level β-globin expression, EKLF is not essential for early hematopoietic differentiation. Yet, the CACCC-binding EKLF protein is expressed promiscuously in early hematopoietic progenitor cells and cell lines (19, 30). The human fetal (γ) and embryonic (e) and the murine embryonic (ey) globin gene promoters have distal core CACCC sites (at −145 and −114, respectively) that are very similar to the adult β-type promoter proximal core CACCC site (at −90). EKLF has been reported to transactivate the human embryonic globin gene e through the embryonic/fetal CACCC site (31). In addition, EKLF occupies the eγ2- and βh1-globin promoters in primitive murine erythroid cells (32). Furthermore, the CACCC sites in the human γ-globin and the murine ey-globin promoters appear essential for maximal induction or reactivation by SCFAs (33-35). We demonstrate herein that EKLF is required for activation of the γ-globin gene by a new class of SCFA derivatives, which do not have intrinsic HDAC-inhibitory activity. EKLF is actively and selectively recruited to the γ-globin gene promoter by exposure to these compounds. The SWI/SNF complex chromatin-modifying core ATPase BRG1 is required for γ-globin induction by these SCFA derivatives, and is actively recruited to the γ-globin promoter in an EKLF-dependent fashion. This is the first description of a necessary role for EKLF and BRG1 in γ-globin gene expression.