[KLF2 and EKLF have compensatory and critical roles in primitive erythropoiesis.] We hypothesize that KLF2 acts in an erythroid cell autonomous manner, directly binding to globin genes.
The first aim i s to further elucidate the mechanistic role of KLF2 in erythropoiesis. [The stage of maturation of KLF2-/- embryonic erythroid cells will be determined with a flow cytometry assay, and the ability of these cells to differentiate in vitro with exogenous factors will be assessed.] Mice with a conditional knockout (KO) the KLF2 gene (KLF2flox allele) have been bred with Cre recombinase mice with an erythroid-specific promoter (LCR-beta pr-Cre), and >90% of the KLF2flox alleles can be deleted. This system will be used to determine whether the cell morphology, globin gene expression, differentiation and apoptotic effects of KLF2 on primitive erythroid cells are cell autonomous, as expected. Using this system, we will also test the role of KLF2 in definitive (adult) erythropoiesis, measuring the same parameters. ChIP (chromatin immunoprecipitation) assays will be used to determine if KLF2 interacts with CACCC elements in the beta-globin locus in vivo, using K562, HEL and MEL cell lines expressing an inducible, tagged construct encoding KLF2. [If possible, ChIP assays will be performed on native mouse erythroid precursors].
The second aim i s to mechanistically study the compensatory roles of KLF2 and EKLF in erythropoiesis using [our established] double KO mouse model. EKLF is expressed in primitive erythropoiesis, and [EKLF-/-KLF2-/- embryos are severely anemic at embryonic day 10.5, when single KOs appear unaffected. To assess the maturation and differentiation of EKLF-/-KLF2-/- embryonic erythroid cells, flow cytometry and progenitor assays will be used.] To study the effects of gene dosage on red cell morphology and globin gene expression, EKLF-/-KLF2 and EKLF KLF2-/- will be compared to wildtype, EKLF-/-, KLF2-/- and EKLF-/-KLF2-/- embryos. We will determine whether EKLF-/-KLF2-/- embryos have reduced gamma-globin mRNA compared to wildtype, using mice with the human beta-globin locus. [EKLF-/-KLF2-/- yolk sacs have abnormal endothelial cells, which is not the case in single KO yolk sacs. We will generate embryos lacking EKLF and KLF2 in erythroid cells only, to determine whether the endothelial defects are directed by erythroid cells.] Our long-term goal is to use KLF2 in strategies to increase embryonic/fetal beta-globin, to treat patients with beta-hemoglobinopathies.
|Vinjamur, Divya S; Alhashem, Yousef N; Mohamad, Safa F et al. (2016) Krüppel-Like Transcription Factor KLF1 Is Required for Optimal ?- and ?-Globin Expression in Human Fetal Erythroblasts. PLoS One 11:e0146802|
|Vinjamur, Divya S; Wade, Kristen J; Mohamad, Safa F et al. (2014) Krüppel-like transcription factors KLF1 and KLF2 have unique and coordinate roles in regulating embryonic erythroid precursor maturation. Haematologica 99:1565-73|
|Chiplunkar, Aditi R; Lung, Tina K; Alhashem, Yousef et al. (2013) Krüppel-like factor 2 is required for normal mouse cardiac development. PLoS One 8:e54891|
|Alhashem, Yousef N; Vinjamur, Divya S; Basu, Mohua et al. (2011) Transcription factors KLF1 and KLF2 positively regulate embryonic and fetal beta-globin genes through direct promoter binding. J Biol Chem 286:24819-27|