Fig. 6: MTA1 expression is regulated by TRIM21-mediated ubiquitination, and ERα promotes TRIM21 transcriptional activation.

A TRIM21 hinders MTA1 expression in MCF-7 and MDA-MB-231 cells. The protein expression of MTA1 was detected after overexpression or knockdown of TRIM21 cells. B MTA1 interacts with TRIM21. Whole cell lysates of MDA-MB-231 cells overexpressing MTA1 were immunoprecipitated with control IgG or FLAG antibodies, followed by western blotting with the indicated antibodies. C HEK293T cells were transfected with FLAG-MTA1 alone or together with MYC-TRIM21 for 36 h, then immunoprecipitated with anti-FLAG and anti-MYC antibodies, followed by western blotting. D, E HEK293T (D) and MCF-7 (E) cells were transfected with FLAG-tagged MTA1, HA-tagged Ub, and MYC-tagged TRIM21 alone or together for 24 h and treated with 20 μM MG132 for 8 h. The cell lysate was immunoprecipitated with an anti-FLAG antibody and western blotting was performed with the corresponding antibody. F TRIM21 affects the protein stability of MTA1. MDA-MB-231 cells were transfected with control or TRIM21 siRNA for 40 h and then treated with MG132 (20 μM) for 8 h. Cell lysates were immunoprecipitated with MTA1 antibody, and western blotting was performed using the corresponding antibodies. G Western blotting revealed that TRIM21 affected the protein stability of MTA1. MDA-MB-231 cells transfected with control or TRIM21 siRNA were treated with CHX (50 μg/ml) for 0 h, 8 h, 16 h, and 24 h. H, I qRT-PCR and western blot results show that E2 treatment (H) or FLAG-ERα transfection (I) promotes the expression of MTA3 and TRIM21 and inhibits the expression of MTA1 in MCF-7 and T-47D cells. J ChIP assays showed that ERα binds in the promoter region of MTA3 and TRIM21. K, L Primer pairs #1–7 were synthesized to cover the promoter region of TRIM21, and a qChIP-based promoter walking experiment was performed using ERα antibody (K) or MTA1 (L) antibodies. M, N Luciferase activity of TRIM21 promoter (−2000 to +100) reporters in MDA-MB-231 cells with E2 treatment (M) or FLAG-ERα (N) transfection after transfection with vector and MTA1. O The proposed regulatory mechanisms of ERα, MTA3, MTA1, and TRIM21. Error bars represent the mean ± SD in H–N. *p < 0.05, **p < 0.01; two-tailed unpaired t-test.