Erythroid Krüppel-like factor (EKLF or KLF1) is a transcription factor with roles in embryonic and adult erythropoiesis. KLF1 knockout mouse embryos die due to severe anemia. Dominant human mutations in KLF1 can cause hereditary persistence of fetal hemoglobin. We show that KLF1 positively regulates β-globin and Bcl11A gene expression using KLF1 knockdown in in vitro-differentiated CD34+ human umbilical cord blood cells. -globin expression appears dependent on KLF1; it is increased with modest KLF1 knockdown but not in cells with low KLF1. KLF2 mRNA amounts are usually increased in KLF1 knockdown. KLF1 knockdown in CD34+ cells results in reduced colony forming ability. Interestingly, the expression of certain proliferation and cell cycle genes is reduced due to KLF1 knockout in mouse or knockdown in human erythroid cells. In conclusion, KLF1 is an important regulator of the β-globin locus and has roles in proliferation and cell cycle.

Sickle cell disease and β-thalassemia affect millions of people worldwide. γ-globin is the fetal counterpart to the adult β-globin. Research has shown that affected patients with higher than normal γ-globin show less severe symptoms. Therefore, reversing or preventing the hemoglobin switch from γ- to β- globin is a promising avenue of research for treating these diseases. KLF1 is an erythroid transcription factor involved in hemoglobin switching. Herein, we show that KLF1 directly regulates the γ-globin repressor gene LRF in both the mouse and human systems. KLF1 may also directly activate γ-globin expression by binding the promoter. In human HUDEP-2 cells, an increase in γ-globin expression is seen upon modest knockdown (~50%) of KLF1, whereas normal amounts of KLF1 are observed upon robust knockdown (>75%) of KLF1. The data suggest that KLF1 plays both a positive and negative role in γ-globin expression.

Krüppel-like factors KLF1 and KLF2 are closely related transcription factors with three zinc finger domains in their carboxy-termini. KLF1 (erythroid Krüppel-like factor, or EKLF) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β- 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 ϵ- (embryonic) and γ-globin (fetal) genes during embryonic erythropoiesis. Chromatin immunoprecipitation assays (ChIP) show that KLF1 and KLF2 bind to the promoters of the human ϵ- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-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 β-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 γ-globin gene in this fetal erythroid model. KLF1 knockdown in these cells affects mainly adult β- globin gene expression. However, the decrease in β- globin gene expression in KLF1 knockdown also affects the ratio of γ- to β- globin in these cells. H3K9Ac and H3K4me3 were decreased only at the β- 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 β- globin genes by direct binding to the promoters and LCR in the β- 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 γ- globin gene expression.

Hemoglobinopathies are some of the most common monogenic disorders in the world, affecting millions of people and representing a growing burden on health systems worldwide. Although the pathophysiology of sickle cell anemia and beta-thalassemia, two of the most common hemoglobinopathies, have been the focus of much research over the last century, patients affected by these diseases still lack a widely applicable and easily available cure. Sickle cell anemia and beta-thalassemia are caused by defects in the structure and production of the beta-globin chains that, along with the alpha-globin chains make up the heterotetrameric hemoglobin molecule. Studies geared towards re-expression of the silenced fetal gamma-globin gene in adult erythroid cells as a therapeutic strategy to alleviate the symptoms of beta-globin deficiencies have met with some success for the treatment of sickle cell anemia but not for beta-thalassemia. A better understanding of normal gamma-globin gene regulation will undoubtedly advance the development of more effective therapeutic strategies. Because many of the potential targets that may be modulated to achieve gamma-globin re-expression also have functions in erythroid cells other than regulating the gamma-globin gene, it is imperative to understand their role in all aspects of erythropoiesis before they are used for therapy. The current study focuses on the role of two Krüppel-like transcription factors, KLF1 and KLF2, which have known roles in the processes of primitive and definitive erythropoiesis as well as globin gene regulation. The regulation of primitive erythropoiesis by KLF1 and KLF2 is studied using the mouse as a model system because it is not possible to study primitive erythropoiesis in humans. Previous studies have shown that KLF1 and KLF2 are essential for and have overlapping roles in primitive erythropoiesis. Simultaneous ablation of KLF1 and KLF2 results in a severely anemic embryonic phenotype that is not evident in KLF1 or KLF2 single knockout embryos. In this study, we show that this anemia is caused by a paucity of blood cells, and exacerbated by diminished beta-like globin gene expression. The anemia phenotype is dose-dependent, and interestingly, can be ameliorated by a single copy of the KLF2, but not the KLF1 gene. The roles of KLF1 and KLF2 in maintaining both normal peripheral blood cell numbers and globin mRNA amounts are erythroid cell-specific. It was discovered that KLF2 has an essential function in erythroid precursor maintenance. KLF1 can partially compensate for KLF2 in this role, but is uniquely crucial for erythroid precursor proliferation, through its regulation of G1- to S-phase cell cycle transition. A more drastic impairment of primitive erythroid colony formation from embryonic progenitor cells occurs with simultaneous deficiency of KLF1 and KLF2, than with loss of a single factor. The regulation of human beta-like globin gene expression is studied using a recently developed in vitro system for the production of erythroid cells from umbilical cord blood hematopoietic precursor cells, representing a more “fetal” model of globin gene expression. Previous studies have shown that KLF1 binds to the promoters of the gamma- and beta-globin genes, while KLF2 binds to the promoter of the gamma-globin gene in cord blood-derived erythroid cells. Studies using transgenic mice carrying the entire human beta-globin locus had indicated that KLF1 and KLF2 positively regulate gamma-globin expression in mouse embryonic erythroid cells. We demonstrate in this study that KLF1 appears to have dual roles in the regulation of gamma-globin expression in human cord blood-derived definitive erythroid cells. Partial depletion of KLF1 causes elevated gamma-globin expression, while nearly complete depletion of KLF1 results in a down-regulation of gamma-globin expression. Of particular interest was the observation that KLF2 positively regulates gamma-globin expression in cord blood-derived erythroid cells. Surprisingly, KLF2 also positively regulates beta-globin expression in these cells. If regulation of gamma-globin by KLF2 proves to be a direct effect, KLF2 will join a very small group of factors known to directly activate gamma-globin expression.

Krüppel like factors (KLFs) are a family of 17 proteins whose main function is gene regulation by binding to DNA elements in the promoters of various genes. KLF transcription factors recognize CACCC-elements and act as activators or repressors of the gene expression. Among the 17 family members, KLF1, KLF2, and KLF4 share high homology to each other. KLF1 is the founding member of the family and is an erythroid-specific protein. KLF2 is expressed in erythroid, endothelial, and other cells. KLF4 is expressed in endothelial, smooth muscle, and other cells. In this thesis, the functions of these KLFs were reviewed in the context of subjects related to erythropoiesis and cardiovascular development. A mouse model lacking KLF1, KLF2, and KLF4 was used to investigate whether these genes have overlapping functions in regulating the embryonic β-globin genes during early embryogenesis. Quantitative RT-PCR assays were used to measure the expression level of Ey- and βh1- globin mRNA at embryonic day 9.5 (E9.5). It was found that KLF1-/-KLF2-/- and KLF1-/-KLF2-/-KLF4-/- embryos express significantly decreased amounts of Ey- and βh1-globin genes when compared to WT and KLF4-/- embryos. There were no significant changes in the levels of Ey- and βh1-globin mRNA between KLF1-/-KLF2-/- and KLF1-/-KLF2-/-KLF4-/- embryos. It was demonstrated here that KLF1 does not regulate KLF2 in mouse erythroid cells at E10.5.

The most widely transfused blood component is red blood cells (RBCs), and voluntary donation is the main resource for RBC transfusion. In the UK, 7,000 units of RBCs are transfused daily but this life-saving cell therapy is completely dependent on donors and there are persistent problems associated with transfusion transmitted infections and in blood group compatibility. Furthermore, the quality, safety and efficiency of donated RBCs gradually decrease with storage time. A number of novel sources of RBCs are being explored including the production of RBCs from adult haematopoietic progenitor cells, erythroid progenitor cell lines and induced pluripotent stem cells (iPSCs). The iPSC source could essentially provide a limitless supply and a route to producing cells that are matched to the recipient. A number of protocols have been described to produce mature RBCs from human pluripotent stem cells but they are relatively inefficient and would be difficult to scale up to the levels required for clinical translation. We tested and evaluated a defined feeder- and serum-free differentiation protocol for deriving erythroid cells from hiPSCs. RBC production was not efficient, the cells that were produced did not enucleate efficiently and they expressed embryonic rather than adult globin. We hypothesised that the production of RBCs from iPSCs could be enhanced by enforced expression of erythroid-specific transcription factors (TFs). Previous studies had demonstrated that Krüppel-like factor 1 (KLF1) plays an important role in RBC development and maturation so we generated iPSC lines expressing a tamoxifen-inducible KLF1-ERT2 fusion protein. Using zinc finger nuclease technology, we targeted the expression cassette to the AAVS1 locus to ensure consistent expression levels and to avoid integration site specific effects and/or silencing. These iKLF1 iPSCs were applied to our defined RBC differentiation protocol and the activity of KLF1 was induced by adding tamoxifen. Activation of KLF1 from day 10 accelerated erythroid differentiation and maturation with an increase in the proportion of erythroblasts, a higher level of expression of erythroid genes associated with maturation and an apparently more robust morphology. However, KLF1 activation had an anti-proliferation effect resulting in significantly less cell generated overall and HPLC analysis demonstrated that KLF1-activated cells expressed higher levels of embryonic globin compared to control iPSCs-derived cells. Many of the effects that were observed when KLF1 was activated from day 10 were not observed when activated from day 18. We therefore concluded that activation of exogenous KLF1 is able to promote erythroid cell production and maturation in progenitors (day 10) but not at the later stage of erythropoiesis (day 18). We hypothesised that KLF1 might require a co-factor to regulate RBC maturation and adult globin expression at the later stage of erythropoiesis. The TF, B-cell lymphoma/leukaemia 11a (BCL11A), plays a key role in the suppression of foetal globin expression, thereby completing globin switching to adult globin. Preliminary data showed that iPSC-derived erythroid cells were able to express adult globin when transduced with a BCL11A-expressing lentiviral-vector. Based on that finding we then generated an iPSC line expressing tamoxifen-inducible BCL11AERT2 and KLF1-ERT2 fusion proteins, applied this iBK iPSC line to our differentiation protocol. Activation of both TFs from day 18 slightly increased the expression of genes associated with RBC maturation and the inclusion of BCL11A appeared to eliminate the anti-proliferation effect of KLF1. Most importantly, activation of both BCL11A and KLF1 from day 18 of the differentiation protocol increased the production of α- globin (foetal / adult globin) indicating that some definitive-like erythroid cells might be generated by activation of both TFs at the later stage of erythroid differentiation. Collectively, these findings demonstrate that enforced expression of erythroid TFs could be a useful strategy to enhance RBC maturation from iPSCs.