Regulation of the mouse and human beta-globin genes by Kruppel like transcription factors KLF1 and KLF2

Kruppel-like factors KLF1 and KLF2 are closely related transcription factors with three zinc finger domains in their carboxy-termini. KLF1 (erythroid Kruppel-like factor, or EKLF) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic beta- globin genes. KLF1 and KLF2 have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double knockout (KO) mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, and double KO embryos are more anemic and die sooner than either single KO. We have shown that KLF1 and KLF2 positively regulate the human epsilon-(embryonic) and gamma-globin (fetal) genes during embryonic erythropoiesis. Chromatin immunoprecipitation assays (ChIP) show that KLF1 and KLF2 bind to the promoters of the human epsilon- and gamma-globin genes, the mouse embryonic Ey- and Bh1-globin genes, and also to the beta-globin locus control region (LCR) in mouse embryonic erythroid cells. ChIP assays show that KLF1 but not KLF2 ablation results in abnormal histone modifications in the beta-globin locus in mouse embryonic erythroid cells. H3K9Ac and H3K4me3, which correlate with open chromatin and active transcription, are both reduced in KLF1-/- primitive erythroid cells. Human CD34+ hematopoietic stem cells obtained from umbilical cord blood were in vitro differentiated along the erythroid lineage. ChIP assays indicate that both KLF1 and KLF2 bind to the promoter of gamma-globin gene in this fetal erythroid model. KLF1 knockdown in these cells affects mainly adult beta-globin gene expression. However, the decrease in beta- globin gene expression in KLF1 knockdown also affects the ratio of gamma- to beta-globin in these cells. H3K9Ac and H3K4me3 were decreased only at the beta-globin gene which coincides with lower recruitment of RNA polymerase II and its active form, RNA polymerase II phospho-serine 2. In conclusion, we showed using mouse primitive erythroid cells and cord blood definitive cells that KLF1 and KLF2 coordinate the regulation of the mouse and human beta-globin genes by direct binding to the promoters and LCR in the beta-globin locus. In conclusion, cord blood hematopoietic cells could serve as a complimentary system in addition to the transgenic mouse models to study the regulation of gamma-globin gene expression.