Department of Urology, The First Affiliated Hospital of Nanchang University, Yongwai Street 17, Nanchang, 330006, China.
Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, China.
J Transl Med. 2023 Oct 12;21(1):716. doi: 10.1186/s12967-023-04429-4.
Androgen receptor (AR) activation and repression dual-functionality only became known recently and still remains intriguing in prostate cancer (PCa). MYC is a prominent oncogene that functionally entangles with AR signaling in PCa. Further exploration of AR regulatory mechanisms on MYC gene transcription bears clinical and translation significance.
Bioinformatics analysis of PCa cell line and clinical RNA-Seq and ChIP-Seq (chromatin immunoprecipitation-sequencing) datasets to anchor interactions of AR and MYC transcriptional networks. ChIP-qPCR and 3C (chromosome conformation capture) analyses to probe MYC distal regulation by AR binding sites (ABSs). CRISPR/Cas9-mediated genome-editing to specify functions of ABS within the 8q24-MYC locus on androgen-mediated MYC transcription. Global FoxA1 and HoxB13 distribution profiling to advance AR transcriptional mechanisms.
Here we recognize AR bi-directional transcription mechanisms by exploiting the prominent 8q24-MYC locus conferring androgen hyper-sensitivity. At ~ 25 Kb downstream of the MYC gene, we identified an undefined ABS, P10. By chromatin analyses, we validated androgen-dependent spatial interaction between P10 and MYC-Promoter (MYC-Pro) and temporal epigenetic repression of these MYC-proximal elements. We next designed a CRISPR/Cas9-mediated double genomic knock-out (KO) strategy to show that P10-KO slightly lessened androgen-elicited MYC transrepression in LNCaP-AR cells. In similar genomic editing assays, androgen-mediated MYC repression became slightly deepened upon KO of P11, an ABS in the PVT1 gene locus highly enriched in AR-binding motifs and peaks. We also investigated multiple ABSs in the established PCAT1 super-enhancer that distally interacts with MYC-Pro for transactivation, with each KO pool consistently shown to relieve androgen-elicited MYC repression. In the end, we systemically assessed androgen effects in the 8q24-MYC locus and along PCa genome to generalize H3K27ac and BRD4 re-distribution from pioneer factors (FoxA1 and HoxB13) to AR sites.
Together, we reconciled these observations by unifying AR dual-functions that are mechanistically coupled to and equilibrated by co-factor redistribution.
雄激素受体 (AR) 的激活和抑制双重功能最近才被发现，但其在前列腺癌 (PCa) 中的作用仍令人着迷。MYC 是一个突出的癌基因，它在 PCa 中与 AR 信号功能上纠缠在一起。进一步探索 AR 对 MYC 基因转录的调节机制具有临床和转化意义。
对 PCa 细胞系和临床 RNA-Seq 和 ChIP-Seq（染色质免疫沉淀测序）数据集进行生物信息学分析，以锚定 AR 和 MYC 转录网络的相互作用。ChIP-qPCR 和 3C（染色体构象捕获）分析探测 AR 结合位点 (ABS) 对 MYC 远端的调节。使用 CRISPR/Cas9 介导的基因组编辑来指定 8q24-MYC 基因座上 AR 结合位点内的功能，以调节雄激素介导的 MYC 转录。对全局 FoxA1 和 HoxB13 分布进行分析，以推进 AR 转录机制。
在这里，我们通过利用赋予雄激素超敏性的突出的 8q24-MYC 基因座来识别 AR 的双向转录机制。在 MYC 基因的下游约 25kb 处，我们鉴定了一个未定义的 ABS，即 P10。通过染色质分析，我们验证了 P10 和 MYC-启动子 (MYC-Pro) 之间雄激素依赖性的空间相互作用，以及这些 MYC 近端元件的时空表观遗传抑制。我们接下来设计了一种 CRISPR/Cas9 介导的双基因组敲除 (KO) 策略，以表明 P10-KO 略微减轻了 LNCaP-AR 细胞中雄激素诱导的 MYC 转录抑制。在类似的基因组编辑实验中，P11-KO 略微加深了雄激素介导的 MYC 抑制，P11 是 PVT1 基因座中富含 AR 结合基序和峰的 ABS。我们还研究了在已建立的 PCAT1 超级增强子中多个 ABS，这些 ABS 与 MYC-Pro 远端相互作用以进行转录激活，每个 KO 池都一致显示出减轻雄激素诱导的 MYC 抑制。最后，我们系统地评估了雄激素在 8q24-MYC 基因座和整个 PCa 基因组中的作用，以概括从先驱因子 (FoxA1 和 HoxB13) 到 AR 位点的 H3K27ac 和 BRD4 再分布。
总的来说，我们通过统一 AR 的双重功能来解释这些观察结果，这些功能在机制上与共因子的再分布相关联并达到平衡。
