The erythroid Kruppel-like factor (EKLF) is essential for chromatin remodeling a id transcriptional activation of the β-globin gene. We have recently defined separable domains of EKLF that are responsible for these activities (Brown et al., ms. in preparation). Previous studies have demonstrated that EKLF interacts with a human SWI-SNF compk x, E-RC1, to generate an open chromatin configuration at the β-globin promoter in vitro. To study the structural and functional consequences of the E-RC1/EKLF interaction, we used a series of amino terminal mutants of EKLF mutants with established activities at t le endogenous promoter. GST pulldown experiments demonstrated that EKLFA253-362 a mutant that contains the DNA-binding domain alone, was sufficient for interaction w th hBRGl, a critical component of the E-RC1 complex. Although this interaction was confirmed by co-immunopreciptation studies, DNase I sensitivity studies demonstratîd that an additional domain contained in the EKLFA221-362 mutant was required to reste re an open chromatin configuration at the endogenous promoter. To determine whether E-R< 1 was also involved in initiation of transcription, we studied a reporter construct contain! ig the β-globin promoter linked to the luciferase gene in SW13 cells, a hBRGl null cell line. Co-transfection of EKLFA221-362 or EKLFA253-362 with hBRGl failed to activité transcription in this assay. In contrast, both full-length EKLF and the EKLFA164-352 mutant activated transcription in a hBRGl -dependent manner. Moreover, enforced expression of hBRG 1 in an erythroid cell line resulted in enhanced EKLF-dependent β-globin promo 1er activity. Taken together, we conclude that hBRG 1 is required for both chromatin remodeli ng and transcriptional activation of the β-globin promoter. In addition, our findings sugg :st that interactions in addition to E-RC1 are required for maximal β-globin gene expression.
|Issue number||11 PART I|
|Publication status||Published - 1 Dec 2000|