Abstract
Erythroid Krüppel-like factor (EKLF) is a red cell-specific transcription factor whose activity is critical for the switch in expression from fetal to adult beta-globin during erythroid ontogeny. We have examined its own regulation using a number of approaches. First, the EKLF transcription unit is in an open chromatin configuration in erythroid cells. Second, in vivo transfection assays demonstrate that the more distal of the two erythroid-specific DNase-hypersensitive sites behaves as an enhancer. Although this conserved element imparts high level transcription to a heterologous promoter in all lines examined, erythroid specificity is retained only when it is fused to the proximal EKLF promoter, which contains an important GATA site. Third, extensive mutagenesis of this enhancer element has delimited its in vivo activity to a core region of 49 base pairs. Finally, in vitro footprint and gel shift assays demonstrate that three distinct DNA binding activities in erythroid cell extracts individually interact with three short sequences within this core enhancer element. These analyses reveal that high level erythroid expression of EKLF relies on the interplay between conserved proximal and distal promoter elements that alter chromatin structure and likely provide a target for genetic control via extracellular induction pathways.
Highlights
Erythroid Kruppel-like factor (EKLF) is a red cellspecific transcription factor whose activity is critical for the switch in expression from fetal to adult -globin during erythroid ontogeny
This prediction was verified by the genetic ablation of EKLF in mice, which leads to a profound -thalassemia, incomplete definitive erythropoiesis, and embryonic death at the time of the switch [5,6,7]
Previous analyses had focused on the proximal EKLF promoter and revealed that the GATA site at Ϫ60 and the CCAAT element at Ϫ45 are important for activity in erythroid cells [21]
Summary
Vol 273, No 39, Issue of September 25, pp. 25031–25040, 1998 Printed in U.S.A. Chromatin Structure and Transcriptional Control Elements of the Erythroid Kruppel-like Factor (EKLF) Gene*. In vitro footprint and gel shift assays demonstrate that three distinct DNA binding activities in erythroid cell extracts individually interact with three short sequences within this core enhancer element These analyses reveal that high level erythroid expression of EKLF relies on the interplay between conserved proximal and distal promoter elements that alter chromatin structure and likely provide a target for genetic control via extracellular induction pathways. Its dual pattern of embryonic expression (i.e. within the yolk sac and fetal liver) makes it an attractive target for these experiments, as these two tissues, erythropoietic, may regulate expression of their tissue-specific genes by distinct mechanisms Such considerations arise from experiments that disrupt critically important erythroid genes, yet do not affect both primitive and definitive red cell populations (e.g. c-myb [11], AML1/CBFA2 [12, 13], CBFB (14 –16), and the erythropoietin receptor [17, 18]). Transcriptional Control of the EKLF Gene conjunction with the tissue-specific proximal EKLF promoter, plays a critical role in generating high levels of expression
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