Klfd binds to miR-144 promoter to activate its transcription. (A,B) Markedly decreased expression of miR-144 in Klfd-deficient 22 hpf embryos (panel A arrows), whereas increased expression of miR-144 was observed in 22 hpf embryos injected with 20 pg of capped klfd mRNAs (panel B arrows). (C) Schematic representation of 5.0-kb miR-144 promoter upstream of the transcriptional start sites (+1). The positions of CACCC boxes were indicated by the vertical arrows and Arabic numbers. Horizontal arrows indicate the primers located to the promoter regions (I-V). EH453145 is the GenBank accession number of cDNA encoding zebrafish miR-144/miR-451 precursor sequence. (D) The Klfd selectively interacts with the proximal CACCC boxes (region V) of miR-144 promoter. The Klfd only binds to the proximal miR-144 promoter at 22 hpf but not at 16 hpf. (E) A proposed model of miR-144--Klfd-mediated trans-acting mechanism in the regulation of embryonic α-E1 globin.