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Erythroid Kruppel-Like Factor and the Cell Cycle: A Role beyond Globin Gene Regulation

Erythropoiesis, the process of producing mature erythrocytes from the haematopoietic stem cells (HSCs) that reside in the bone marrow, is tightly regulated at both the cell and molecular level by a well defined set of extracellular cytokine signals and intracellular transcription factors. Diseases affecting erythropoiesis are among the most commonly inherited conditions and result from disturbances to the cellular and molecular events that normally regulate this process. Erythroid Kruppel-like factor (EKLF/KLF1) is a transcription factor that is essential for erythropoiesis. EKLF is the founding member of the Kruppel-like factor family of transcription factors that bind to GC rich CACC-Box elements within gene promoters and activate transcription. The β-like globin genes are critical targets of EKLF through its binding at sites within the proximal promoters and the upstream locus control region (LCR) enhancer. Mice lacking EKLF die prior to birth by E16 with a phenotype that closely resembles the human disease thalassaemia. Thalassaemia is due to mutations in the α or β-globin genes, leading to globin chain imbalance, red cell destruction and ineffective erythropoiesis. However, restoration of expression of γ-globin (a β-like gene) failed to prevent embryonic lethality in EKLF knockout mice and suggested that additional target genes were critical to erythropoiesis. This thesis describes investigation into the transcriptional network of EKLF and an in depth analysis of previously uncharacterised phenotypes present in the EKLF knockout mouse. I have identified a suite of target genes for EKLF that include critical components of the cells cycle. I have also tested the hypothesis that EKLF is able to function in vivo as a tumour suppressor gene. Additionally, I report a role for EKLF in the determination of cell fate within the haematopoietic system and describe the development of a new approach to globally understanding erythroid transcription factor function. A previously performed microarray transcriptional profiling study provided a set of potential target genes for EKLF. I have expanded on this study by identifying that the cell cycle genes p18INK4c, and E2f2 are direct transcriptional targets of EKLF, where binding of EKLF occurs at the promoter and a novel intronic enhancer region, respectively. I have also described a previously undiscovered cell cycle phenotype of aberrant entry into S-phase in EKLF -/- erythroid cells that is directly related to abrogated expression of E2f2. The Kruppel-like factor family of genes have been implicated as players in the tumour process. By constructing a model for the loss of EKLF within HSCs in vivo, I have tested whether EKLF is functional as a tumour suppressor. The loss of EKLF in vivo was found to be insufficient to generate erythroleukaemia, however did result in erythroid hyperplasia, extramedullary haematopoieis and a mild macrocytic anaemia. In addition to regulation of erythropoiesis, EKLF performs a critical role in the lineage choice for a megakaryocyte-erythroid progenitor (MEP) between the megakaryocytic and erythroid lineages. This thesis describes that in the absence of EKLF, MEPs fail to commit properly to either lineage and proceed along a promiscuous pathway sharing the hallmarks of both megakaryocytes and erythroid cells. A detailed molecular mechanism for this phenotype remains undetermined, but is likely to involve interactions with the megakaryocyte transcription factor Fli1 and other members of the Kruppel-like factor family, such as BKLF (KLF3). While the transcriptional mechanisms that drive erythropoiesis have been slowly discovered, the development of chromatin immunoprecipitation (ChIP) assays and next generation DNA sequencing technology has presented the potential to rapidly enhance the progression of these studies. In this thesis I describe the development of ChIP-seq using Applied Biosystems SOLiD technology, an approach to rapidly identify binding sites for erythroid transcription factors in an unbiased genome wide approach. The work described in this thesis has expanded the transcriptional network of EKLF to include critical components of the cell cycle and has suggested many additional target genes from ChIP-seq requiring validation. As one of the major transcription factor players during erythropoiesis, EKLF performs many critical functions that include the regulation of the cell cycle, lineage selection and erythroid development. I suggest that current and future studies of EKLF function will influence our understanding of erythropoiesis and refine our understanding of human conditions such as thalassaemia and erythroleukaemia.