Molecular Oncology Program, Division of Surgical Oncology, Dewitt Daughtry Family Department of Surgery, University of Miami, 1600 NW 10th Ave, Miami, FL, 33136, USA.
Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL, 33136, USA.
Cell Commun Signal. 2021 Sep 22;19(1):96. doi: 10.1186/s12964-021-00776-1.
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. Video abstract.
Notch 信号通路驱动肿瘤表型的多个方面。在这里，我们报告 Integrator 复合物（INT）是 Notch 转录超级复合物的一个新组件。与 Notch 激活复合物激酶（NACK）一起，INT 通过驱动 RNA 聚合酶 II（RNAPII）依赖性转录来激活 Notch1 靶基因，导致肿瘤发生。
使用排阻色谱和 CBF-1/RBPJ/无发突体/Lag-1（CSL）-DNA 亲和快速蛋白质液相色谱（FPLC）来分离 Notch/CSL 依赖性复合物，进行液相色谱-串联质谱（LC-MS/MS）分析。染色质免疫沉淀（ChIP）和定量聚合酶链反应（qPCR）用于研究 Notch 靶基因的转录调控。将 Notch 三元复合物组分转染到 HEK293T 细胞中，作为一种重编程实验来研究 Notch 介导的转录机制。通过 RNA 干扰进行基因敲低，并通过集落形成实验和小鼠异种移植来确定蛋白耗竭对食管腺癌（EAC）增殖的影响。Western blot 用于检测 EAC 细胞中 INT 亚基的表达，并评估 INT 亚基 11 敲低（INTS11 KD）后凋亡蛋白的表达。通过流式细胞术进一步探索基因 KD 效应。
我们鉴定了 INT 复合物是 Notch 转录超级复合物的一部分。INT 与 NACK 一起激活 Notch 介导的转录。虽然 NACK 是将 RNAPII 募集到 Notch 依赖性启动子所必需的，但 INT 复合物对于 RNAPII 在丝氨酸 5 位磷酸化（RNAPII-S5P）是必需的，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 中的 G2/M 细胞周期停滞、凋亡和细胞生长停滞。
本研究鉴定了 INT 复合物作为 Notch 介导的转录中的一个新的共因子，它与 NACK 一起激活 Notch 靶基因并导致癌细胞增殖。视频摘要。
