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Updated: Aug 18, 2025
Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands
Published on: January 2, 2018
The structure, binding and function of a Notch transcription complex involving RBPJ and the epigenetic reader protein
Daniel Hall1, Benedetto Daniele Giaimo2, Sung-Soo Park3
1University of Cincinnati College of Medicine, Department of Molecular Genetics, Biochemistry and Microbiology, Cincinnati, OH, USA.
Abstract:
The Notch pathway transmits signals between neighboring cells to elicit downstream transcriptional programs. Notch is a major regulator of cell fate specification, proliferation, and apoptosis, such that aberrant signaling leads to a pleiotropy of human diseases, including developmental disorders and cancers. The pathway signals through the transcription factor CSL (RBPJ in mammals), which forms an activation complex with the intracellular domain of the Notch receptor and the coactivator Mastermind. CSL can also function as a transcriptional repressor by forming complexes with one of several different corepressor proteins, such as FHL1 or SHARP in mammals and Hairless in Drosophila. Recently, we identified L3MBTL3 as a bona fide RBPJ-binding corepressor that recruits the repressive lysine demethylase LSD1/KDM1A to Notch target genes. Here, we define the RBPJ-interacting domain of L3MBTL3 and report the 2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA complex. The structure reveals that L3MBTL3 interacts with RBPJ via an unusual binding motif compared to other RBPJ binding partners, which we comprehensively analyze with a series of structure-based mutants. We also show that these disruptive mutations affect RBPJ and L3MBTL3 function in cells, providing further insights into Notch mediated transcriptional regulation.
Insights
Researchers identified L3MBTL3 as a Notch pathway corepressor, revealing its unique binding to RBPJ (CSL) via crystal structure analysis. This discovery offers new insights into Notch-mediated gene regulation and its role in diseases.
Area of Science:
- Molecular Biology
- Cell Signaling
- Structural Biology
Background:
- The Notch pathway is crucial for cell fate, proliferation, and apoptosis, with dysregulation linked to diseases like cancer.
- Notch signaling involves the transcription factor CSL (RBPJ), which can activate or repress gene expression.
- Corepressors like FHL1, SHARP, and Hairless modulate CSL's repressive function.
Purpose of the Study:
- To characterize L3MBTL3 as a novel RBPJ-binding corepressor in the Notch pathway.
- To define the RBPJ-interacting domain of L3MBTL3.
- To elucidate the structural basis of the RBPJ-L3MBTL3 interaction.
Main Methods:
- X-ray crystallography to determine the 2.06 Å structure of the RBPJ-L3MBTL3-DNA complex.
- Structure-based mutagenesis to analyze the L3MBTL3 binding motif.
- Cell-based assays to assess the functional impact of mutations.
Main Results:
- L3MBTL3 was confirmed as a bona fide RBPJ corepressor, recruiting LSD1/KDM1A to target genes.
- The crystal structure revealed an unusual binding motif between L3MBTL3 and RBPJ.
- Mutational analysis demonstrated the functional importance of this interaction in cellular processes.
Conclusions:
- The unique L3MBTL3-RBPJ interaction provides a new perspective on Notch-mediated transcriptional repression.
- Understanding this interaction may offer therapeutic targets for Notch-related disorders.
- Further research into L3MBTL3's role can illuminate complex gene regulatory networks.
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