The goal of this proposal is to study the opposing and complementary roles of Kr ppel-like factor 1 (EKLF or KLF1) and KLF2 in embryonic and fetal erythropoiesis. This is biologically important because it could facilitate effective therapeutic strategies for the hemoglobinopathies. KLF1 and KLF2 are closely related transcription factors that activate the mouse embryonic -like globin genes.
Aim 1 is to define the roles of KLF1 and KLF2 in -globin gene expression. KLF1 binds the -globin promoter in in vitro differentiated CD34+ human cord blood cells, but in contrast to its role in embryonic globin gene expression, it negatively regulates the -globin gene in fetal cells. KLF2 binds robustly to the -globin promoter in fetal cells, at the same -globin CACCC consensus site. We will determine whether KLF2 positively regulates -globin expression, using KLF2 knockdown (KD) and overexpression in in vitro differentiated CD34+ human cord blood cells. The hypothesis that KLF1 has a net negative effect on -globin gene expression by precluding KLF2 binding will be tested, by knocking down KLF1 in in vitro differentiated CD34+ cells.
Aim 2 is to define the mechanistic roles of KLF1 and KLF2 in organizing the global chromatin structure of the -globin locus in embryonic and fetal erythroid cells. KD in in vitro differentiated CD34+ human cord blood cells and knockout (KO) mouse models will be used to determine if KLF1 and KLF2 are required for recruitment of BRG1 and CBP/p300 chromatin remodeling complexes to the -globin LCR and promoters. We will test whether the factors are required for formation of the 5'HS2 and 5'HS3 DNase I hypersensitive sites. We will also determine whether KLF1 and KLF2 are required for recruitment of RNA polymerase II to, and chromatin looping between, the LCR and -globin genes.
In Aim 3, the effects of KLF1 and KLF2 on mouse embryonic erythropoiesis will be investigated. At E10.5, KLF1-/-KLF2-/- (double KO) embryos are pale and anemic, but KLF1-/- and KLF2-/- are grossly normal. KLF1-/-KLF2-/- embryos have less peripheral blood cells, and form primitive erythroid progenitor colonies that mature more slowly and are less hemoglobinized than normal controls. KLF1 and KLF2 appear to coordinately control the formation and maturation of mouse primitive erythroid progenitors. To test this, the roles of KLF1 and KLF2 in determining the number and nature of erythroid progenitor colonies formed, cell proliferation, maturation and apoptosis will be tested using single and double KO mouse embryos. Based on the gross phenotypes of embryos, we expect a larger number and more mature colonies from progenitor assays with KLF1-/-KLF2+/- than with KLF1+/-KLF2-/-. Preliminary data suggests that KLF1 and KLF2 regulate genes involved in proliferation. We will determine the amounts of mRNA for KLF1 and KLF2 target genes, such as FoxM1, Myc, CD24a antigen, sphingosine kinase 1, Pura and parathyroid hormone 1 receptor, in KLF1+/-KLF2-/-, KLF1-/-KLF2+/-, KLF2-/-, KLF1-/- and KLF1-/-KLF2-/- embryos, and in colonies derived from erythroid progenitor cells.
It is important to understand the regulation of the globin genes by the DNA-binding proteins called transcription factors, and the normal processes of embryonic and fetal red blood cell development to facilitate effective therapeutic strategies for the hemoglobinopathies. Treatment of sickle cell anemia and the thalassemias can involve turning the fetal globin gene back on in adult red blood cells. This work will increase the understanding of how transcription factors switch the globin genes on and off in the embryo and fetus.
|Perkins, Andrew; Xu, Xiangmin; Higgs, Douglas R et al. (2016) Krüppeling erythropoiesis: an unexpected broad spectrum of human red blood cell disorders due to KLF1 variants. Blood 127:1856-62|
|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|