Klfd selectively transactivates α-E1 globin promoter. (A) WISH analysis of klfd mRNAs at the indicated stages. The klfd is first detected in the ICM of 18 hpf embryos. Embryos are lateral views with the head to the left. (B) Forced expression of klfd mRNAs (20 pg) increases α-E1 globin expression at 24 hpf (arrows). (C) Knockdown of Klfd protein by morpholino in vivo. Western blot analysis of embryos injected with 5-MM control (Ctrl) or klfd-specific morpholino oligonucleotides at the indicated doses. The proteins extract from 20 embryos at 22 hpf were loaded into each lane. WT indicates wild-type. Actin was used as a loading control. (D) Knockdown of Klfd reduces α-E1 globin expression at 22 hpf (arrows). (E) Decreased hemoglobin staining by O-dianisidine at 36 and 54 hpf (arrows). (F) Knockdown of Klfd is able to significantly rescue the overproduction and ectopic expression of α-E1 globin induced by miR-144 deficiency (arrow). The mpo expression was not affected. (G-I) The Klfd selectively binds to the distal CACCC elements of α-E1 gene promoter. E-ChiP analysis of chromatin extracted from 16 and 22 hpf embryos. PCR was performed using the primers located to the indicated promoter regions for α-E1 (G, regions I-III), β-E1 (G, regions IV and V), alas2 (H, regions I and II), and β-E3 (I, regions III and IV). The Klfd was significantly enriched at the CACCC boxes (region I) of α-E1 globin promoter at 22 hpf (g) but not at 16 hpf. The results were repeated 4 times with separate batches of chromatin preparations.