The Role of Mediator Subunit MED30 in Licensing the c-Myc Oncogenic Program [ChIP-seq]

Despite extensive functional and structural investigation of the Mediator complex, the basis for selective functional requirements for specific Mediator subunits for the actions of specific DNA binding transcription factors remain incompletely understood. Here, we report the MED30 subunit of Mediator, one of the core subunits maintaining the complex, plays a critical role in regulating the MYC transcription program in cancer cells. This is consequent to MED30 being required for stabilizing Mediator complex and MYC binding on the genome, involving low-affinity hydrophobic interaction functions of the MYC N-terminal Intrinsic Disordered Region (IDR). A mutational screen reveals that mutations in critical residues in the MYC coiled-coil domain structurally adjacent to the IDR of MED30 strongly inhibit tumor growth, even in the presence of wild-type MED30, mimicking the effects of MED30 deletion. This study provides new insights into the principles governing the Mediator complex actions MYC-related biological processes. To map the binding sites of Mediator component MED17, MED30 and MYC, these factors ChIP-seq were performed in Mia PaCa-2 cells with antibodies MED17 (invitrogen PA5-30314), MED30 (from Dr. Thomas G. Boyer, UT health San Antonio, homemade) and Myc (CST #9402), respectively. To test the effect of MED30 knockdown in Myc's binding, MYC ChIP-seq was perform with or without three days 0.5ug/ml Dox treatment in Dox-induced shMED30 Mia PaCa-2 cells. To test the domain/motif contribution to binding for Myc, letivrus vector expressing HA-tagged Myc fragment and WT in Dox-induced manner were intergrated into Mia PaCa-2 cells, and 0.5ug/ml Dox was administrated for 1 day, another WT sample was not treated with Dox, serving as negative control.

尽管对中介体复合物（Mediator complex）已开展了广泛的功能与结构研究，但针对特定DNA结合转录因子发挥作用时，中介体复合物特定亚基所具备的选择性功能需求的分子基础，目前仍未完全阐明。本研究报道，作为维持中介体复合物稳定的核心亚基之一，MED30在癌细胞调控MYC转录程序的过程中发挥关键作用。该作用源于MED30可稳定中介体复合物及MYC在基因组上的结合，此过程依赖于MYC N端内在无序区域（IDR，Intrinsic Disordered Region）的低亲和力疏水相互作用功能。突变筛选实验显示，MYC卷曲螺旋结构域中与MED30的IDR在结构上相邻的关键残基发生突变后，即便存在野生型MED30，也可显著抑制肿瘤生长，其效应与MED30敲除的表型一致。本研究为解析调控中介体复合物参与MYC相关生物学过程的分子机制提供了全新视角。为绘制中介体组分MED17、MED30与MYC的结合位点，我们分别使用针对MED17的抗体（invitrogen PA5-30314）、MED30抗体（由德克萨斯大学圣安东尼奥健康科学中心Thomas G. Boyer博士惠赠，自制）以及MYC抗体（CST #9402），在Mia PaCa-2细胞中开展了染色质免疫共沉淀测序（ChIP-seq）实验。为验证MED30敲低对MYC结合的影响，我们在多西环素（Dox）诱导的shMED30 Mia PaCa-2细胞中，分别施加或不施加0.5μg/ml多西环素处理3天，随后开展MYC ChIP-seq实验。为探究MYC的结构域/基序对结合的贡献，我们将携带HA标签的MYC片段及野生型MYC的慢病毒（原文疑似笔误为letivrus，正确应为lentivirus）表达载体以多西环素诱导的方式整合至Mia PaCa-2细胞中，其中一组施加0.5μg/ml多西环素处理1天，另一组未施加多西环素处理作为阴性对照。