Fig. 4

ccr9a Is a Direct Target of Irf4a

(A) Co-expression of irf4a and ccr9a in the CHT of 2-dpf embryos revealed by double color FISH. Scale bar, 100 µm.

(B) qPCR of irf4a and ccr9a in GFP⁺ cells relative to negative cells sorted from coro1a:EGFP transgenic embryos at 2 dpf. p < 0.05; p < 0.01.

(C) Schematic diagram of the ChIP and reporter assay. The ccr9a-CF and CR are the control forward and reverse primers, which are used to amplify the ccr9a promoter region without the conserved Irf4 binding site. The ccr9a-1F and 1R represent primers used to amplify the promoter region with two conserved Irf4 binding sites within 500 bp upstream of 5′-ATG-3′. AICE, AP-1-IRF composite elements; EICE, Ets-IRF composite elements.

(D) Myc was detected in embryos injected with irf4a-myc mRNA by western blot.

(E) Direct binding of ccr9a promoter by Irf4a examined by ChIP assay.

(F) qPCR of DNA fragments obtained from ChIP assay with primers (ccr9a-1F, 1R or CF, CR). p < 0.001.

(G) Reporter assay showed the regulation of ccr9a promoter by Irf4a full-length CDS. M1 indicates that 5′-TGAT-3′ within AICE was mutated to 5′-GTCG-3′, and M2 indicates that 5′-GGAA-3′ within EICE was mutated to 5′-TTCC-3′. p < 0.01; p < 0.05.

(H) Expression of rag1 in the thymus (red arrow) of the control and ccr9a morphants at 4 dpf. All data are mean ± SD.

See also Figure S4 and Table S2.