Fig. 3

Eaf1 directly suppresses expression of zebrafish foxo3b, an orthologue of human FOXO3a. (A) Alignment of zebrafish foxo3b and human FOXO3a amino acid sequences. Identical amino acids are indicated by dots; the phosphorylation sites by human AKT and putative phosphorylation sites by zebrafish akt are underlined and labeled by red color; the dominant negative form of either human FOXO3a or zebrafish Foxo3b is marked by blue arrows and numbered. (B) Phylogenetic tree of FOXO proteins was constructed by Neighbor Joining method. The bootstrap values are marked at each branch. Foxo3a mouse (NM_019740), Foxo3a Rat (NM_001106395), Foxo3a human (NM_001455), foxo3b zebrafish (NM_131085), Foxo3a zebrafish (NM_001009988), Foxo4 human (NM_5938), Foxo4 mouse (NM_018789), Foxo1 human (NM_002015), Foxo1 mouse (NM_019739), foxo1a zebrafish (NM_001077257), foxo1b zebrafish (NM_001082857). (C) Expression of foxo3b was increased dramatically by Eaf1–MO3-mediated eaf1 knockdown (b versus a; e versus d), but was decreased when mismatch mRNA of eaf1 was co-injected (c versus b; f versus e) at 10s and 30 hpf; semi-quantitative RT-PCR confirmed enhancement of foxo3b expression in eaf1 depleted-embryos, and reduction of foxo3b expression when mismatch mRNA of eaf1 was co-injected at 10s and 30 hpf (g). (D) Schematic depiction of different deletion constructs of foxo3b promoter luciferase reporter. (E) Expression of HA-tagged Eaf1 in 293 T cells was confirmed by Western blot using anti-HA monoclonal antibody. (F) Luciferase reporter assays for different deletion constructs of the foxo3b promoter in the presence or absence of HA-eaf1 in 293 T cells. The eaf1 response region on the foxo3b promoter was mapped to the 625 to 310 region (“+1” is designated as the transcription initial site; the translation starting site (ATG code) is located in “+1564”). (G) The suppressive role of eaf1 on foxo3b promoter constructs was re-evaluated in embryos. Ectopic expression of eaf1 in embryos dramatically inhibited transactivation function of the foxo3b promoter construct 625 to +1573, but not the promoter constructs 310 to +1573 or 143 to +1573. (H) The suppressive role of eaf1 on foxo3b promoter construct 625 to +1573 was further evaluated by eaf1 knockdown assays in embryos. Compared to standard morpholino (STD-MO) injection (control), the activity of foxo3b promoter 625 to +1537 was up-regulated dramatically by eaf1–MO1 injection, but the activity of the foxo3b promoter 310 to +1537 was not. (I) Further mapping for eaf1 response region in foxo3b promoter narrowed down to 385 to 310. (J) When the region of 385 to 310 in foxo3b promoter was deleted in both 2133 to +1573 promoter reporter and 625 to +1573 promoter reporter, ectopic expression of eaf1 could not suppress these two reporters′ activity. The dosage of promoter constructs was 1.25 pg/embryo; HA-empty vector and HA-eaf1, 62.5 pg/embryo; pRL-SV40 (internal control), 25.0 pg/embryo. (K) The expressions of HA-Eaf1, HA-Gata1, HA-Spi1 and HA-Foxo3b in embryos after plasmid injections were confirmed by Western blot using anti-HA monoclonal antibody. (L) Whole-embryo chromatin immunoprecipitation (E-ChIP) assays showed that Eaf1 bound to the foxo3b promoter at the region between 625 and 291. The foxo3b promoter fragment covering 625 to 291 was immunoprecipitated by anti-HA antibody (c, the fourth lane from left to right), but not by mouse IgG (control) (c, the third lane from left to right). Zebrafish β-actin promoter and the region of 2072 to 1770 in foxo3b promoter were used as negative controls (a and b). 25% of input DNA (total DNA) served as a positive control (a–c).