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Updated: Oct 26, 2025
A Method to Study de novo Formation of Chromatin Domains
Published on: August 23, 2019
SUMOylated non-canonical polycomb PRC1.6 complex as a prerequisite for recruitment of transcription factor RBPJ
Małgorzata Sotomska1, Robert Liefke2,3, Francesca Ferrante1
1Institute of Biochemistry, Justus-Liebig University of Giessen, Friedrichstrasse 24, 35392, Giessen, Germany.
Background:
Notch signaling controls cell fate decisions in many contexts during development and adult stem cell homeostasis and, when dysregulated, leads to carcinogenesis. The central transcription factor RBPJ assembles the Notch coactivator complex in the presence of Notch signaling, and represses Notch target gene expression in its absence.
Results:
We identified L3MBTL2 and additional members of the non-canonical polycomb repressive PRC1.6 complex in DNA-bound RBPJ associated complexes and demonstrate that L3MBTL2 directly interacts with RBPJ. Depletion of RBPJ does not affect occupancy of PRC1.6 components at Notch target genes. Conversely, absence of L3MBTL2 reduces RBPJ occupancy at enhancers of Notch target genes. Since L3MBTL2 and additional members of the PRC1.6 are known to be SUMOylated, we investigated whether RBPJ uses SUMO-moieties as contact points. Indeed, we found that RBPJ binds to SUMO2/3 and that this interaction depends on a defined SUMO-interaction motif. Furthermore, we show that pharmacological inhibition of SUMOylation reduces RBPJ occupancy at Notch target genes.
Conclusions:
We propose that the PRC1.6 complex and its conjugated SUMO-modifications provide a favorable environment for binding of RBPJ to Notch target genes.
Insights
The PRC1.6 complex and SUMOylation help RBPJ bind to Notch target genes, influencing cell fate and development. This interaction is crucial for regulating gene expression in biological processes.
Area of Science:
- Molecular Biology
- Developmental Biology
- Epigenetics
Background:
- Notch signaling is vital for cell fate determination during development and stem cell maintenance.
- Dysregulation of Notch signaling is implicated in cancer development.
- RBPJ (Recombination signal binding protein for J-k) is a key transcription factor in Notch signaling, activating or repressing target genes.
Purpose of the Study:
- To investigate the role of the non-canonical polycomb repressive PRC1.6 complex in RBPJ-mediated Notch target gene regulation.
- To determine the interaction between L3MBTL2, a component of PRC1.6, and RBPJ.
- To explore the involvement of SUMOylation in RBPJ binding to target genes.
Main Methods:
- Identification of PRC1.6 components in RBPJ-associated complexes.
- Co-immunoprecipitation assays to confirm L3MBTL2-RBPJ interaction.
- Chromatin immunoprecipitation to assess RBPJ occupancy at target genes upon L3MBTL2 depletion.
- SUMOylation assays and investigation of RBPJ binding to SUMO2/3.
- Assessment of RBPJ occupancy following pharmacological inhibition of SUMOylation.
Main Results:
- L3MBTL2 and other PRC1.6 members were found in DNA-bound RBPJ complexes.
- L3MBTL2 directly interacts with RBPJ.
- Absence of L3MBTL2 reduced RBPJ occupancy at Notch target gene enhancers.
- RBPJ binds to SUMO2/3 via a SUMO-interaction motif.
- Inhibition of SUMOylation decreased RBPJ occupancy at Notch target genes.
Conclusions:
- The PRC1.6 complex, through its interaction with RBPJ, plays a significant role in regulating Notch target gene expression.
- SUMOylation of RBPJ or associated proteins facilitates RBPJ binding to enhancers.
- PRC1.6 and SUMO-modifications create a conducive environment for RBPJ's function in Notch signaling pathways.
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