Ubiquitylation of MYC couples transcription elongation with double-strand break repair at active promoters (RNAseq)

MYC family proteins are oncogenic transcription factors that can globally affect the function of RNA Polymerase II (RNAPII). The ability of MYC proteins to promote transcription elongation depends on their ubiquitination, but the underlying mechanism and its biological relevance are unknown. Here we show that MYC and the Polymerase II associated factor, PAF1c, interact directly and their function is mutually dependent, since the specific binding of MYC to active promoters depends on PAF1c and, conversely, PAF1c is required for MYC-dependent pause release. Upon binding, PAF1c is rapidly transferred from MYC onto RNAPII and this transfer is driven by the HUWE1 ubiquitin ligase. Both MYC and HUWE1 globally control histone H2B ubiquitylation, which promotes transcriptional elongation and alters chromatin structure for double-strand break repair. Consistently, MYC suppresses the accumulation of double-strand breaks at promoters in response to topoisomerase II inhibition. While depletion of PAF1c has only minor effects on MYC-dependent gene expression, MYC induces rampant transcription-dependent DNA damage in PAF1c-depleted cells. We propose that the HUWE1-dependent transfer of PAF1c from MYC onto RNAPII is critical for absorbing the topological stress accompanied with deregulated and oncogenic transcription. Overall design: U2OS cells which stably express MYC TetOn system (Walz et al, 2014) were infected with pGIPZ shCTR9 or shCDC73 for 24 h, the infection medium was diluted 1:2 and incubated for 24 h. Cells were split 1:3 and selected with puromycin (0.2 ug/ml) for 24 h. Medium was changed and cells were either treated with doxycicline (1ug/ml) or ethanol for 24 h.

MYC家族蛋白属于致癌性转录因子，可全局性影响RNA聚合酶II（RNAPII）的功能。MYC蛋白促进转录延伸的能力依赖于其自身的泛素化修饰，但其背后的分子机制与生物学相关性至今尚未阐明。本研究证实，MYC与聚合酶II相关因子复合物（Polymerase II Associated Factor complex，PAF1c）存在直接相互作用，且二者的功能相互依赖：MYC与活性启动子的特异性结合依赖于PAF1c；反之，PAF1c亦是MYC依赖的转录暂停释放过程所必需的。二者结合后，PAF1c会快速从MYC转移至RNAPII，这一转移过程由HUWE1泛素连接酶所介导。MYC与HUWE1均可全局性调控组蛋白H2B的泛素化修饰，该修饰可促进转录延伸并改变染色质结构以适配双链断裂修复。与此一致的是，MYC可抑制拓扑异构酶II抑制剂处理后启动子区域双链断裂的积累。尽管PAF1c的敲低仅对MYC依赖的基因表达产生轻微影响，但在PAF1c敲低的细胞中，MYC会引发大量依赖于转录的DNA损伤。我们提出，HUWE1介导的PAF1c从MYC向RNAPII的转移，对于缓解失调性致癌转录所伴随的拓扑压力至关重要。整体实验设计：将稳定表达MYC TetOn系统（Walz等，2014）的U2OS细胞用pGIPZ shCTR9或shCDC73感染24小时，随后将感染培养基按1:2比例稀释并继续培养24小时。将细胞以1:3比例传代，并用嘌呤霉素（0.2 μg/ml）筛选24小时。更换培养基后，分别用多西环素（1 μg/ml）或乙醇处理细胞24小时。