ZFIN Image: Zhu et al., 2021, Figure 5

Image

Figure Caption

Figure 5

Ttf1 is accumulated on its binding sites in bms1l mutants. (A) A drawing to illustrate the head-on conflict between rDNA transcription and replication during the S-phase in higher eukaryotes. Binding of Ttf1 to the RFB-site prevents the conflict. Once the transcription is completed, Ttf1 is proposed to leave the RFB-site to allow progression of the replication fork; however, how Ttf1 is dissociated from the RFB-site remains unknown. (B) Western blotting showing an increase of Ttf1 protein level in bms1l^sq163/sq163 and bms1l^zju1/zju1 but not in rcl1^−/− compared with their corresponding siblings at 5dpf. Tubulin: loading control. ?: unknown protein. (C) Signal intensity of nucleolar Ttf1 in the gut epithelia was significantly higher in bms1l^sq163/sq163 and bms1l^zju1/zju1 than in WT and rcl1^−/−. Samples: three WT embryos, 364 cells counted; three bms1l^sq163/sq163 embryos, 183 cells counted; three bms1l^zju1/zju1 embryos, 98 cells counted; three rcl1^−/− embryos, 176 cells counted. (D) Ratio of the nucleoli displaying intervening signals of Ttf1 and Rpa2 in the gut epithelia was significantly higher in bms1l^zju1/zju1 than in WT. Samples: three WT embryos, 264 cells examined; three bms1l^zju1/zju1 embryos, 140 cells examined. (E) ChIP‒seq identification of Ttf1-binding sites (peak-1 to peak-4) downstream of the 3ʹ-end of rDNA gene on chromosome 5 (Chr.V). (F) ChIP‒qPCR analysis showing the strong binding of Flag-tagged Ttf1a to peak-1 and peak-3 but not to the 18S (left panel) or 28S (right panel) rDNA. (G) Endogenous Ttf1 showed significant enrichment on its binding sites (ChIP-peak: peak-1 to peak-4) in bms1l^sq163/sq163 (163 MU) and bms1l^zju1/zju1 (zju1 MU) than in WT at 3dpf. IgG and ChIP-18S: negative controls. (H and I) ChIP‒qPCR analysis showing that, in the presence of Mg²⁺, the binding of purified Ttf1a to peak-3 depends on GTP but not ATP (H) and is correlated to the dosage of purified Ttf1a (I). Lower panel in I: Ttf1a was examined by western blotting. In C, D, and F‒H, **P < 0.01, ***P < 0.001; NS, no significance.

Acknowledgments

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ J. Mol. Cell Biol.