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. 2021 Apr 7;7(15):eabe2261. doi: 10.1126/sciadv.abe2261

Fig. 5. BUB3 recruits USP7 to methylated FOXA1 for cell cycle gene regulation.

Fig. 5

(A) Co-IP in 293T cells transfected with indicated plasmids was performed using anti-FLAG M2 beads as shown in Fig. 4E, followed by WB. (B and C) Co-IP was performed in LNCaP cells using FOXA1, USP7, or IgG antibody (B) or FOXA1, EZH2, or IgG antibody (C). (D) LNCaP cells were infected with indicated lentivirus and treated with 20 μM MG132 for 16 hours. Co-IP was performed with IgG or FOXA1 antibody. (E) LNCaP cells expressing indicated plasmids were treated with 20 μM MG132 before co-IP with a FOXA1 antibody. (F) A working model showing that PRC2 methylates (me) FOXA1, which recruits BUB3 and USP7 to remove ubiquitination (Ub), thus preventing FOXA1 degradation by the proteasome system (barrel shape structure). (G) Venn diagram showing overlap between USP7-, BUB3-, and FOXA1-induced gene sets derived from RNA-seq (adjusted P < 0.05 and fold change > 3). (H) Gene ontology (GO) analysis of USP7/BUB3/FOXA1-coinduced genes. (I) Heatmap of USP7/BUB3/FOXA1-coinduced genes in LNCaP cells with indicated gene KD. (J and K) Genome browser view of two cell cycle genes showing FOXA1 ChIP-seq (top tracks), RNA-seq in siCtrl or EZH2-KD (green tracks), and RNA-seq in shCtrl, USP7-KD, BUB3-KD, and FOXA1-KD (bottom 4) LNCaP cells.