Fig. 7
CSRP2BP activates the N-cadherin promoter by acetylating histone H4. A-C Acetylation of H4, H4K5, H4K8, H4K12 and H4K16 was analysed by Western blotting. Total H4 was used as a loading control. The quantification of Western Blot was performed by ImageJ software. All data are presented as mean ± SD of 3 independent experiments (n = 3). D Histone H4 acetylation levels in the N-cadherin promoter region were determined by ChIP‒PCR assay. IgG was used as the negative control. E ChIP‒QPCR assays were performed using antibodies against Ac-H4 and CSRP2BP in Hela-CSRP2BP cells, Hela-shCSRP2BP cells and their respective controls. IgG was used as a negative control. F H4K5ac levels in the N-cadherin promoter region were analysed by ChIP‒QPCR assay. IgG was used as a negative control. G Immunoprecipitation assay was performed to evaluate that CSRP2BP promoted H4 acetylation through K5 site. Flag-HA-tagged H4 plasmid (H4-WT) and flag-HA-tagged H4K5 mutant plasmid (H4K5 MUT) was respectively transfected into Hela-CSRP2BP cells. IgG was used as the negative control. The level of H4Ac was detected by Western blotting. H A luciferase reporter assay was used to detect the effect of CSRP2BP HAT domain mutation (CSRP2BP MUT) on its binding to SMAD4 to promote N-cadherin promoter transcription in Hela cells (n = 3). I Graphic model of CSRP2BP as discussed in the text. In cervical cancer, CSRP2BP causes a significant increase in H4 acetylation at lysine (K) sites 5 and 12 (K5 and K12). Moreover, CSRP2BP promoted N-cadherin transcription by binding the transcription factor SMAD4 to the N-cadherin promoter and enhancing cervical cancer EMT and metastasis. DNA (grey line); acetylated histone sites (histone 4 lysine 5, brown ball; histone 4 lysine 12, red ball). All data are mean ± SD of 3 independent experiments performed in triplicate. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)