Notch signaling drives many aspects of neoplastic phenotype. Here, we report that the Integrator complex (INT) is a new component of the Notch transcriptional supercomplex. Together with Notch Activation Complex Kinase (NACK), INT activates Notch1 target genes by driving RNA polymerase II (RNAPII)-dependent transcription, leading to tumorigenesis. Size exclusion chromatography and CBF-1/RBPJ/Suppressor of Hairless/Lag-1 (CSL)-DNA affinity fast protein liquid chromatography (FPLC) was used to purify Notch/CSL-dependent complexes for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) were performed to investigate transcriptional regulation of Notch target genes. Transfection of Notch Ternary Complex components into HEK293T cells was used as a recapitulation assay to study Notch-mediated transcriptional mechanisms. Gene knockdown was achieved via RNA interference and the effects of protein depletion on esophageal adenocarcinoma (EAC) proliferation were determined via a colony formation assay and murine xenografts. Western blotting was used to examine expression of INT subunits in EAC cells and evaluate apoptotic proteins upon INT subunit 11 knockdown (INTS11 KD). Gene KD effects were further explored via flow cytometry. We identified the INT complex as part of the Notch transcriptional supercomplex. INT, together with NACK, activates Notch-mediated transcription. While NACK is required for the recruitment of RNAPII to a Notch-dependent promoter, the INT complex is essential for RNAPII phosphorylated at serine 5 (RNAPII-S5P), leading to transcriptional activation. Furthermore, INT subunits are overexpressed in EAC cells and INTS11 KD results in G2/M cell cycle arrest, apoptosis, and cell growth arrest in EAC. This study identifies the INT complex as a novel co-factor in Notch-mediated transcription that together with NACK activates Notch target genes and leads to cancer cell proliferation.

中文翻译：

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NACK 和 INTEGRATOR 协同作用以激活 Notch 介导的肿瘤发生转录

Notch 信号传导驱动肿瘤表型的许多方面。在这里，我们报告整合器复合体 (INT) 是 Notch 转录超复合体的一个新组成部分。INT 与 Notch 激活复合物激酶 (NACK) 一起通过驱动 RNA 聚合酶 II (RNAPII) 依赖性转录激活 Notch1 靶基因，从而导致肿瘤发生。尺寸排阻色谱法和 CBF-1/RBPJ/无毛/Lag-1 (CSL)-DNA 亲和快速蛋白质液相色谱法 (FPLC) 抑制器用于纯化 Notch/CSL 依赖性复合物，用于液相色谱-串联质谱法 (LC- MS/MS) 分析。进行染色质免疫沉淀 (ChIP) 和定量聚合酶链反应 (qPCR) 以研究 Notch 靶基因的转录调控。将 Notch 三元复合物成分转染到 HEK293T 细胞中被用作重演试验来研究 Notch 介导的转录机制。通过 RNA 干扰实现基因敲低，并通过集落形成测定和小鼠异种移植物确定蛋白质消耗对食管腺癌 (EAC) 增殖的影响。Western印迹用于检查EAC细胞中INT亚基的表达并评估INT亚基11敲低（INTS11 KD）后的凋亡蛋白。通过流式细胞术进一步探索基因KD效应。我们将 INT 复合体鉴定为 Notch 转录超复合体的一部分。INT 与 NACK 一起激活 Notch 介导的转录。虽然将 RNAPII 募集到 Notch 依赖性启动子需要 NACK，INT 复合物对于在丝氨酸 5 (RNAPII-S5P) 处被磷酸化的 RNAPII 至关重要，从而导致转录激活。此外，INT 亚基在 EAC 细胞中过表达，INTS11 KD 导致 EAC 中 G2/M 细胞周期停滞、细胞凋亡和细胞生长停滞。本研究将 INT 复合物确定为 Notch 介导的转录中的一种新型辅助因子，它与 NACK 一起激活 Notch 靶基因并导致癌细胞增殖。