Integrator endonuclease drives promoter-proximal termination at all RNAPII transcribed loci (ChIP-Seq 2)

Pausing of RNA polymerase II (RNAPII) in early elongation is critical for gene regulation. Paused RNAPII can be released into productive elongation by the kinase P-TEFb or targeted for premature termination by the Integrator complex. Integrator comprises endonuclease and phosphatase activities, driving termination through cleavage of nascent RNA and removal of stimulatory phosphorylation. To probe the direct consequences of Integrator activity, we generated a degron system to rapidly deplete the Integrator endonuclease INTS11. Degradation of INTS11 elicits a nearly universal increase in RNAPII escape from promoter regions. However, these RNAPII complexes fail to achieve optimal elongation rates and reveal continued Integrator phosphatase activity. Short transcripts are thus selectively upregulated by INTS11 loss, including many non-coding RNAs, transcription factors and signaling regulators. Together, our data indicate a common function for INTS11 at all RNAPII loci, with differential effects on particular genes, pathways or RNA biotypes reflecting transcript lengths rather than Integrator specificity. Overall design: ChIP-seq examination of 4 samples (2 biological replicate sets each of INTS11Halo cells: DMSO- and PROTAC-treated for RNAPII Ser5P)

RNA聚合酶II（RNA polymerase II, RNAPII）在早期延伸阶段的暂停对基因调控至关重要。处于暂停状态的RNAPII可被激酶P-TEFb激活进入有效延伸，或被Integrator复合物（Integrator complex）介导进入过早终止过程。Integrator复合物兼具核酸内切酶与磷酸酶活性，通过切割新生RNA并去除刺激性磷酸化来驱动转录终止。为探究Integrator活性的直接效应，我们构建了降解子（degron）系统以快速降解Integrator的核酸内切酶INTS11。INTS11的降解几乎全局性地提升了RNAPII从启动子区域的逃逸效率，但此类RNAPII复合物无法达到最优延伸速率，同时揭示了Integrator磷酸酶活性的持续存在。因此，短转录本会因INTS11缺失被选择性上调，其中包含大量非编码RNA（non-coding RNAs）、转录因子及信号调控因子。综合来看，我们的数据表明INTS11在所有RNAPII基因座上均发挥共性功能，其对特定基因、通路或RNA亚型的差异化影响实则由转录本长度决定，而非Integrator的特异性。整体实验设计：对4组样本开展染色质免疫沉淀测序（ChIP-seq）检测，具体为两组生物学重复的INTS11Halo细胞，分别经二甲基亚砜（DMSO）与蛋白水解靶向嵌合体（PROTAC）处理，用于检测RNA聚合酶II Ser5磷酸化形式（RNAPII Ser5P）。