Fig. 5

Fig. 5

(A-F) ChIP experiments with the indicated antibodies on cdh5 (A, B), esama (C, D) and Fli1b (E, F) regulatory sequences. P, promoter; E, enhancer. Samples were analyzed in duplicates from 2 experiments. Fold enrichment was calculated relative to the input and to a negative region on the genome and then relative to the mock ChIP (No antibody). (G) Schematic representation of the transgene TgBAC(cdh5:GAL4FF) containing two putative cis-regulatory elements, a promoter region (P) and an enhancer (E), separated by ~20kb with the GAL4FF reporter gene inserted at the TSS of cdh5. (H-K) Confocal projections of transgenic TgBAC(cdh5:GAL4FF);Tg(UAS:GFP) embryos at 28 hpf. Control morphant is on the top left panel (H), prmt5 morphant embryos were not injected (I) or injected by either prmt5WT mRNA (J) or the catalytic mutant form prmt5MUT (K) mRNA. The fluorescent intensity is colored-coded, from the Low intensity (L) in black to High intensity (H) in white. Scale bar 100 μm. (L) Average GFP fluorescence intensity per confocal projection for control, prmt5 morphant embryos not injected or injected by prmt5WT mRNA or prmt5 MUT, from 3 independent experiments with at least 3 animals per condition. One-way ANOVA was performed. *P<0.05, **P<0.01. (M) Schematic representation of the transgene Tg(cdh5:GAL4VP16) containing the two putative cis-regulatory elements next to each other (E and P), upstream of GAL4VP16 reporter gene. (N, O) Confocal projection of transgenic Tg(cdh5:GAL4VP16);Tg(UAS:KAEDE) embryos at 26 hpf injected with either a control morpholino (N) or a prmt5 morpholino (O). The fluorescence intensity is color- coded, from the Low intensity (L) in black to High intensity (H) in white as in panels (H-F). (P)- Average KAEDE fluorescence intensity for control and for prmt5 morphant embryos, from 3 independent experiments with at least 5 animals per condition. T-test was performed. (Q) Proposed model to depict the function of Prmt5 in zebrafish endothelial cells. The transcription factor Etv2 bound on promoters and selective enhancers of endothelial specific genes, could recruit a complex including Prmt5, Brg1 and the mediator complex which favors the formation of a chromatin loop thereby facilitating the transcription of these genes. Dashed lines indicate potential interactions for the recruitments of Prmt5, Brg1 and/or the mediator complex by Etv2.