Abstract
BackgroundNotch 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.MethodsSize 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.ResultsWe 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.ConclusionsThis 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.DmMSvvPJYVoBWTrbTitSG7Video abstract
Highlights
Notch signaling drives many aspects of neoplastic phenotype
Integrator is part of the Notch transcriptional supercomplex To investigate the mechanism of Notch transcriptional regulation, we sought to identify key downstream components that link the Notch Ternary Complex (NTC) to activation of transcription
In contrast to Notch Activation Com‐ plex Kinase (NACK) depletion, Integrator subunit 11 (INTS11) KD does not affect the occupancy of unmodified RNA polymerase II (RNAPII) indicating that INTS11 is required for the activation of RNAPII while NACK is important for its recruitment to Notch target genes (Fig. 2e, g) [7]
Summary
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. Many diseases including cancer are driven by abnormal cell signaling and deregulation of transcription It is Shersher et al Cell Commun Signal (2021) 19:96 generally accepted that Notch functions as a transcriptional activator, and that aberrantly activated Notch signaling drives many aspects of the neoplastic phenotype [1,2,3]. NACK is an atypical kinase and likely serves as a scaffold for the formation of a higher-order transcriptional complex and is required for the recruitment of RNAPII to target promoters [7,8,9]. These events in turn lead to the activation of a Notch transcriptional cascade that drives tumorigenesis
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