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. 2023 Feb 2;51(6):2655–2670. doi: 10.1093/nar/gkad043

Figure 4.

Figure 4.

SMAD3 binds to intron 3 of AR gene to promote AR mRNA expression. (A) Heatmap showing two replicates of SMAD3 ChIP-seq peaks relative to IgG negative control antibody. (B) SMAD3 ChIP-seq peak annotation. (C) The Homer motif analysis showing the significant enrichment of SBE motifs on SMAD3 peaks. (D) Image showing the SMAD3 ChIP-seq peaks at the AR gene. The red lines and numbers indicate the nine regions analyzed by ChIP-PCR as described in (E). (E) ChIP-PCR of SMAD3 showing the enrichment of SMAD3 at regions 6 and 7 that are located at the center of the major SMAD3 ChIP-seq peak. ChIP assays were performed on Rv1 cells with control and SMAD3 antibodies. Precipitated chromatin was analyzed by qPCR for the nine regions of AR gene as indicated in D. % of input was calculated and presented as mean ± SD (n = 3), and t test was used for statistical analysis (***P < 0.001). The comparison at other regions (1–5,8,9) was not significant. (F) ChIP-PCR of SMAD4 showing no enrichment of SMAD4 at regions 6 and 7. ChIP assays were performed with control and SMAD4 antibodies as described in E. % of input was calculated and presented as mean ± SD (n = 3), and t test was used for statistical analysis. The comparison at each of the 9 regions was not significant. (G) ChIP-PCR of SMAD3 showing the similar SMAD3 enrichment at regions 6 and 7 of AR gene between control and SMAD4-KD Rv1 cells. % of input was calculated and presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (ns, not significant). Region 9 serves as a negative control region for SMAD3 binding. (H) Real-time RT-PCR results showing that CRISPRi constructs targeting region 6 or 7 reduced the mRNA levels of AR, AR-V7 and AR targets in Rv1 cells. 3 sgRNAs (sg1,sg2,sg3) were simultaneously used to target the three SBEs at or near region 6. Three sgRNAs (sg4,sg5,sg6) were simultaneously used to target the 4 SBEs at region 7. Rv1 cells expressing the indicated CRISPRi constructs were analyzed by the real-time RT-PCR analysis of indicated genes. Quantification was presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001). (I) ChIP-PCR of Cas9 showing the enrichment of Cas9 at AR exon 2, region 6 or 7 in the CRISPRi-expressing Rv1 cells as described in H. % of input was calculated and presented as mean ± SD (n = 3), and t test was used for statistical analysis (ns, not significant; ***P < 0.001).