Abstract
The erythroid Krüppel-like factor (EKLF) is essential for the transcription of betamaj globin in erythroid cells. We show here that RNA for this transcription factor did not alter during erythropoietin-induced differentiation of J2E cells; however, EKLF protein content decreased and was inversely related to globin production. This unexpected result was also observed during chemically induced maturation of two murine erythroleukemia cell lines. To explore the role of EKLF in erythroid terminal differentiation, an antisense EKLF construct was introduced into J2E cells. As a consequence EKLF RNA and protein levels fell by approximately 80%, and the cells were unable to manufacture hemoglobin in response to erythropoietin. The failure to produce hemoglobin was due to reduced transcription of not only globin genes but also key heme enzyme genes. However, numerous other genes, including several erythroid transcription factors, were unaffected by the decrease in EKLF. Although hemoglobin synthesis was severely impaired with depleted EKLF levels, morphological maturation in response to erythropoietin continued normally. Moreover, erythropoietin-induced proliferation and viability were unaffected by the decrease in EKLF levels. We conclude that EKLF affects a specific set of genes, which regulates hemoglobin production and has no obvious effect on morphological changes, cell division, or viability in response to erythropoietin.
Highlights
The erythroid Kruppel-like factor (EKLF) is essential for the transcription of maj globin in erythroid cells
We conclude that a threshold concentration of EKLF is essential for epo-stimulated transcription of certain genes associated with hemoglobin synthesis and that this transcription factor plays no significant role in other aspects of erythroid terminal differentiation
In this manuscript we have shown that below a threshold concentration of EKLF, epo is unable to stimulate hemoglobin synthesis
Summary
The importance of EKLF to each facet of erythroid differentiation could be assessed in this system. In this manuscript we show that as J2E cells differentiated, EKLF protein fell whereas globins were synthesized. Reduction of EKLF levels before exposure to epo via an antisense EKLF construct blocked hemoglobin synthesis due to reduced transcription of globin and some heme enzyme genes. Lowering EKLF levels had no effect on transcription of numerous other genes and had no impact on epo-induced cell division, viability, or morphological maturation. We conclude that a threshold concentration of EKLF is essential for epo-stimulated transcription of certain genes associated with hemoglobin synthesis and that this transcription factor plays no significant role in other aspects of erythroid terminal differentiation
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