Megakaryocytes and erythroid cells are thought to derive from a common progenitor during hematopoietic differentiation. Although a number of transcriptional regulators are important for this process, they do not explain the bipotential result. We now show by gain-and loss-offunction studies that erythroid Krü ppellike factor (EKLF), a transcription factor whose role in erythroid gene regulation is well established, plays an unexpected directive role in the megakaryocyte lineage. EKLF inhibits the formation of megakaryocytes while at the same time stimulating erythroid differentiation. Quantitative examination of expression during hematopoiesis shows that, unlike genes whose presence is required for establishment of both lineages, EKLF is uniquely down-regulated in megakaryocytes after formation of the megakaryocyte-erythroid progenitor. Expression profiling and molecular analyses support these observations and suggest that megakaryocytic inhibition is achieved, at least in part, by EKLF repression of Fli-1 message levels. IntroductionHematopoiesis is the process by which a self-renewing population of stem cells provide a continuous replenishment of differentiated blood cells by generating progeny with sequentially altered gene expression patterns. [1][2][3] Identification of these cells has relied on selective enrichment by cell-surface markers combined with culture and in vivo cellular assays that enable detection of cells at specific stages of differentiation. Although stem cells are multipotent, individual steps of subsequent differentiative decisions are performed by a series of simpler, even bipotential, decisions whereby one cell type gives rise to 2 or 3 descendants of differing character. 4 This has led to a commonly accepted pattern of parent and progeny relations, 2 although variations of it have recently been suggested 5 (but see Forsberg et al 6 ).A large number of genetic, cellular, and gene expression studies point to the critical importance of cytokine pathways 7 and expression patterns of transcription factors 1,[8][9][10] for establishing and maintaining steady state numbers of lymphoid, myeloid, and erythroid cells that, at the same time, can respond quickly to changes in the organismal environment and increase or decrease the cellularity of specific blood cell types. The megakaryocyte and erythrocyte lineages are proposed to derive from a common precursor, the megakaryocyte-erythroid progenitor (MEP) 4,11,12 (reviewed in Pang et al 13 ). Strikingly, these 2 lineages share a number of commonalities with respect to transcription factors that are absolutely required (eg, GATA1,14,15 FOG1, 16 SCL,17, ). At the same time, the protein partners that form with these factors as differentiation proceeds can be significantly different between lineages. 20 However, because these factors are all positively required for both lineages, we are still left with an incomplete picture of how these lineages are differentially established during hematopoiesis. 13 Erythroid Krüppel-like factor (EKLF; KLF1 21 ) is ...

The erythroblastic island provides an important nutritional and survival support niche for efficient erythropoietic differentiation. Island integrity is reliant on adhesive interactions between erythroid and macrophage cells. We show that erythroblastic islands can be formed from single progenitor cells present in differentiating embryoid bodies, and that these correspond to erythro-myeloid progenitors (EMPs) that first appear in the yolk sac of the early developing embryo. Erythroid Krüppel-like factor (EKLF; KLF1), a crucial zinc finger transcription factor, is expressed in the EMPs, and plays an extrinsic role in erythroid maturation by being expressed in the supportive macrophage of the erythroblastic island and regulating relevant genes important for island integrity within these cells. Together with its well-established intrinsic contributions to erythropoiesis, EKLF thus plays a coordinating role between two different cell types whose interaction provides the optimal environment to generate a mature red blood cell.

Objective-Targeted regulation of β-like globin genes was studied using designer zinc finger transcription factors containing the DNA binding domain of the red cell specific transcription factor EKLF fused to repression domains.Methods-Globin gene expression was analyzed after introduction of the modified transcription factors into cell lines, embryonic stem cells and transgenic mice.Results-As would be predicted, when introduced transiently into cells these transcription factors were effective in repressing the adult β-globin promoter CACCC element which is the natural target for EKLF. In murine erythroleukemia cells repression of the adult β-globin gene was accompanied by a reactivation of the endogenous embryonic βH1-globin gene. Studies in differentiated embryonic stem cells and transgenic mice confirmed the reactivation of embryonic gene expression during development.Conclusion-Our studies support a competition model for β-globin gene expression and underscore the importance of EKLF in the embryonic/fetal to adult globin switch. They also demonstrate the feasibility of designer zinc finger transcription factors in the study of transcriptional control mechanisms at the β-globin locus and as potential gene therapy agents for sickle cell disease and related hemoglobinopathies.

A large volume of laboratory and human epidemiological studies have shown that high doses of ionizing radiation engender significant health risks. In contrast, the health risks of low level radiation remain ambiguous and have been the subject of intense debate. To reduce the uncertainty in evaluating these risks, research advances in cellular and molecular biology are being used to characterize the biological effects of low dose radiation exposures and their underlying mechanisms. Radiation type, dose rate, genetic susceptibility, cellular redox environment, stage of cell growth, level of biological organization and environmental parameters are among the factors that modulate interactions among signaling processes that determine short-and long-term outcomes of low dose exposures. Whereas, recommended radiation protection guidelines assume a linear dose-response relationship in estimating radiation cancer risk, in vitro and in vivo investigations of phenomena such as adaptive responses and non-targeted effects, namely bystander effects and genomic instability, suggest that low dose/low fluence-induced signaling events act to alter linearity of the dose-response relation as supported by the biophysical argument. The latter predicts that increases in dose simply increase the probability that a given cell in a tissue will be intersected by an electron track, and by corollary, each unit of radiation, no matter how small would increases risk. These predictions assume that similar molecular events mediate both low and high dose radiobiological effects, and the cumulative risk from two sequential radiation exposures can never be less than one alone. KeywordsLow dose; adaptive response; LET; Dose-rate Using normal human or rodent cells maintained in culture and a variety of biological endpoints, studies have shown that exposure to low dose/low linear energy transfer (LET) radiation delivered at low dose-rates (≤ 10 cGy from 137 Cs or 60 Co γ rays delivered at ≤ 0.2 cGy/h), triggers signaling events that protect cells from endogenous oxidative damage or damage due to a subsequent challenge dose of ionizing radiation