A trimeric USP11-USP7-TCEAL1 complex stabilizes paused RNAPII to sustain oncogenic transcription [ChIP-seq]

During early transcription, RNA polymerase II (RNAPII) undergoes a series of structural transitions controlled by cyclin-dependent kinases. Whether protein ubiquitylation and proteasomal degradation affect the fate of RNAPII at core promoters is less well understood. Here we show that the deubiquitinating enzyme USP11 and its heterodimeric partner USP7 form a trimeric complex with TCEAL1, a member of the poorly understood TCEAL (TCEA/TFIIS-like) protein family. TCEAL1 shares sequence homology with the RNAPII interaction domain of the TCEA/TFIIS elongation factor, which controls the fate of backtracked RNAPII. TCEAL1 stabilizes complexes of USP11 with USP7 and with RNAPII. TCEAL1 is recruited to core promoters upon RNAPII pausing and globally enhances the chromatin association of paused RNAPII. Mechanistically, the USP11/USP7/TCEAL1 complex competes with TFIIS for binding to core promoters and protects RPB8, an essential subunit of RNAPII, from degradation, likely preventing TFIIS-mediated transcript cleavage and RNAPII disassembly. In neuroblastoma and other tumors, TCEAL1-dependent genes define a TGF-beta-dependent gene expression program that is characteristic of mesenchymal and invasive tumor cell types, suggesting that the USP11/USP7/TCEAL1 trimer stabilizes paused RNAPII to support a critical oncogenic gene expression program. Using ChIP-sequencing, we assessed changes in the binding of TCEAL1, TFIIS, total RNA Polymerase II and Ser2-phophorylated RNA Polymerase II to chromatin in SHEP cells under control conditions and after exogenous expression of an OHT-activated MYCN-ER construct. The SHEP cells used in the various different experiments further contained either a Dox-inducible shRNA targeting TCEAL1 or an constitutive exogenous overexpression of HA-tagged TCEAL1 (either wild-type or mutant). In some experiments, SHEP cells were also treated with the CDK7 or CDK9 inhibitors THZ1 or NVP-2, respectively. In addition, poly(A)-RNA-sequencing was used for differential gene expression analysis in SHEP cells with an OHT-activated MYCN transgene and a Dox-inducible shRNA targeting TCEAL1.

在转录早期，RNA聚合酶II（RNAPII）会经历一系列由细胞周期蛋白依赖性激酶调控的构象转变。目前对于蛋白质泛素化与蛋白酶体降解是否会影响核心启动子处RNAPII的命运，尚不清楚。本研究发现，去泛素化酶USP11与其异二聚体伴侣USP7，可与TCEAL1形成三聚体复合物；TCEAL1属于研究尚不充分的TCEAL（TCEA/TFIIS样）蛋白家族成员。TCEAL1与调控回溯态RNAPII命运的TCEA/TFIIS延伸因子的RNAPII结合结构域存在序列同源性。TCEAL1可稳定USP11与USP7、以及USP11与RNAPII的复合物。当RNAPII发生暂停时，TCEAL1会被招募至核心启动子区域，并在全基因组水平增强暂停态RNAPII的染色质结合能力。从机制层面来看，USP11/USP7/TCEAL1复合物可与TFIIS竞争结合核心启动子区域，并保护RNAPII的必需亚基RPB8免于降解，从而可能阻断TFIIS介导的转录本切割与RNAPII解离过程。在神经母细胞瘤及其他肿瘤中，受TCEAL1调控的基因可构成一套依赖于转化生长因子β（TGF-β）的基因表达程序，该程序是间质样与侵袭性肿瘤细胞表型的特征；这提示USP11/USP7/TCEAL1三聚体可通过稳定暂停态RNAPII，来维持一套关键的致癌基因表达程序。本研究通过染色质免疫共沉淀测序（ChIP-sequencing），分析了对照组与转染他莫昔芬激活型MYCN-ER融合蛋白表达载体的SHEP细胞中，TCEAL1、TFIIS、总RNA聚合酶II以及丝氨酸2磷酸化RNA聚合酶II与染色质的结合情况变化。本研究中使用的各类SHEP细胞，分别携带靶向TCEAL1的多西环素诱导型短发夹RNA（Dox-inducible shRNA），或组成型过表达HA标签标记的TCEAL1（包括野生型与突变型两种）。部分实验中，SHEP细胞还分别接受了细胞周期蛋白依赖性激酶7（CDK7）抑制剂THZ1与细胞周期蛋白依赖性激酶9（CDK9）抑制剂NVP-2的处理。此外，本研究还通过聚腺苷酸RNA测序（poly(A)-RNA-sequencing），对携带他莫昔芬激活型MYCN转基因与靶向TCEAL1的多西环素诱导型shRNA的SHEP细胞进行了差异基因表达分析。