EKLF-meidated transcription of erythroid genes

Desgardin, Aurelie D. (2010) EKLF-meidated transcription of erythroid genes. (PhD thesis), Kingston University, uk.bl.ethos.537584.


Erythroid Kruppel-like factor (EKLF) is an erythroid specific transcription factor that binds to the proximal [beta]-globin gene promoter and is essential for high level expression. In addition, EKLF binds to the far upstream enhancer commonly referred to as the Locus Control Region (LCR). The nature of these two events, their relationship to other events at the multigene [beta]-globin locus and the precise and required interaction of these cis-acting sequences remains unclear. Equally, the mode of action ofEKLF at other erythroid-specific gene loci that are not regulated by an LCR has not yet been reported. These targets include chaperones, membrane-bound proteins, and enzymes of the heme biosynthesis pathway that are essential for red blood cell function. To elucidate the role of EKLF at the multigene locus, and at other erythroid genes, we monitored the temporal EKLF-directed events across the [beta]-globin locus, at the AHSP and Dematin as well as the ALAD promoter using a unique 4-0H-Tamoxifen EKLF-inducible erythroid cell line (JH31), developed in the laboratory, chromatin immunoprecipitation (ChIP) studies, and DNasel hypersensitivity assays. We demonstrate here that EKLF is not required for priming of the [beta]-globin locus for expression. However, EKLF is essential for maximal erythroid factor occupancy, recruitment of chromatin-modifying enzymes, and effective recruitment of the RNA Polymerase II complex. We show that EKLF recruits these complexes first to the LCR prior to the [beta]-globin promoter, suggesting that the LCR serves as a docking element. Finally we provide evidence that the LCR/promoter interacting factor, Ldb-l, is recruited to the [beta]-promoter in an EKLF-dependent manner. We extend our observations to several EKLF-regulated genes outside the [beta]-globin cluster, demonstrating not only the kinetics of transcriptional activation, but also a previously unknown mechanism of chromatin remodeling that implicates histone eviction. Finally, we report a discrepancy between the roles of the histone acetyltransferases CBP and p300 at EKLF target gene promoters, challenging conservative notions of basic transcriptional events. Together, our observations deepen our understanding of the mechanisms of action of EKLF, and provide a platform for additional studies.

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