EKLF and KLF2 have compensatory roles in embryonic β-globin gene expression and primitive erythropoiesis

Abstract

The Krü ppel-like C2/H2 zinc finger transcription factors (KLFs) control development and differentiation. Erythroid Krü ppel-like factor (EKLF or KLF1) regulates adult ␤-globin gene expression and is necessary for normal definitive erythropoiesis. KLF2 is required for normal embryonic Ey-and ␤h1-, but not adult ␤-globin, gene expression in mice. Both EKLF and KLF2 play roles in primitive erythroid cell development. To investigate potential interactions between these genes, EKLF/KLF2 double-mutant embryos were analyzed. EKLF ؊/؊ KLF2 ؊/؊ mice appear anemic at embryonic day 10.5 (E10.5) and die before E11.5, whereas single-knockout EKLF ؊/؊ or KLF2 ؊/؊ embryos are grossly normal at E10.5 and die later than EKLF ؊/؊ KLF2 ؊/؊ embryos. At E10.5, Ey-and ␤h1-globin mRNA is greatly reduced in EKLF ؊/؊ KLF2 ؊/؊ , compared with EKLF ؊/؊ or KLF2 ؊/؊ embryos, consistent with the observed anemia. Light and electron microscopic analyses of E9.5 EKLF ؊/؊ KLF2 ؊/؊ yolk sacs, and cytospins, indicate that erythroid and endothelial cells are morphologically more abnormal than in either single knockout. EKLF IntroductionKrüppel-Like factors (KLFs) are a family of DNA-binding proteins with sequence homology to the Drosophila transcription factor, Krüppel. KLFs have 3 C2/H2 zinc finger domains and share conserved residues located primarily within these domains. 1,2 Erythroid Krüppel-Like factor (EKLF or KLF1) was the first of 17 KLFs to be identified in mouse and man. 3 It is expressed specifically in erythroid cells and positively regulates the adult ␤-globin gene. 3,4 EKLF Ϫ/Ϫ mice develop fatal anemia during definitive (fetal liver) erythropoiesis, due to a defect in the maturation of red blood cells, and die by embryonic day 16 (E16). [5][6][7] Several other members of the KLF family, including KLF2 (lung Krüppel-like factor, LKLF), also are expressed in erythroid cells. [8][9][10][11] Based on phylogenetic analyses, the zinc finger domains of KLF2 and EKLF are very similar. 1,2,12,13 KLF2 Ϫ/Ϫ mice die between E12.5 and E14.5 due to heart failure and severe hemorrhaging, caused by defects in vascular endothelial cells and in stabilization of immature vessels by recruited smooth muscle cells. 14,15 Prior to E12.5, KLF2 Ϫ/Ϫ embryos have normal vasculogenesis and angiogenesis. 14,15 KLF2 also plays an important role in hematopoietic cell biology. We reported that KLF2 is essential for primitive (embryonic yolk sac) erythropoiesis and positively regulates the embryonic ␤-like globin genes in vivo. E10.5 KLF2 Ϫ/Ϫ primitive erythroid cells have abnormal morphology. 9 KLF2 also regulates T-cell activation. Deficiency of KLF2 leads to a decrease in the peripheral T-cell pool 16 due to defective thymocyte emigration. 17 Overexpression of KLF2 in mice inhibits proinflammatory activation of peripheral blood monocytes. 18 It was initially reported that EKLF does not affect embryonic/ fetal globin gene expression. Interestingly, however, EKLF is expressed very early in mouse and chicken development, as early as the primitive streak stage, fo...

Transcription Factors KLF1 and KLF2 Positively Regulate Embryonic and Fetal β-Globin Genes through Direct Promoter Binding

“…Because KLF1 and KLF2 are located in close proximity on mouse chromosome 8, recombination between the two genes is rare. Therefore, a mouse model that has the KLF1 and KLF2 KO alleles on the same DNA homolog was generated (12). Transgenic mice that carry the entire human ␤-globin locus (Tg-HBB) were described previously (19,20).…”

“…Mouse embryonic yolk sacs and blood cells were collected as described (12). For the conditional KO studies, mice with a floxed KLF2 allele were obtained from Dr. Jerry Lingrel (22).…”

“…The developmental expression pattern of KLF1 and KLF2 mRNA correlates with globin gene expression. KLF1 and KLF2 regulate the mouse Ey-and ␤h1-globin genes that are expressed at E9.5, whereas KLF1, but not KLF2, regulates the adult ␤-globin genes expressed at E12.5 (12,17).…”

“…KLF1 and KLF2 Regulate Human ⑀-and ␥-Globin Gene Expression-KLF1 and KLF2 regulate the mouse embryonic ␤-globin genes, Ey and ␤h1, during primitive erythropoiesis (12). When KLF1 and KLF2 are simultaneously ablated, Ey-and ␤h1-globin mRNA amounts are reduced by severalfold to less than amounts measured in either single KO (12).…”

“…KLF1 and KLF2 can partially functionally compensate for each other in regulating the mouse embryonic ␤-globin genes. When both KLF1 and KLF2 are simultaneously ablated in mice, the amounts of Ey-and ␤h1-globin mRNA are reduced more than in KLF1 or KLF2 single knockouts (12). Like KLF1, KLF2 recruits proteins with histone acetyltransferase activity such as CBP, p300 and PCAF (18).…”