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Inducible and Reversible Dominant-negative DN Protein Inhibition
Published on: January 7, 2019
Tudor-domain protein PHF20L1 reads lysine methylated retinoblastoma tumour suppressor protein
Simon M Carr1, Shonagh Munro1, Cari A Sagum2
1Department of Oncology, University of Oxford, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Headington, Oxford OX3 7DQ, UK.
Abstract:
The retinoblastoma tumour suppressor protein (pRb) classically functions to regulate early cell cycle progression where it acts to enforce a number of checkpoints in response to cellular stress and DNA damage. Methylation at lysine (K) 810, which occurs within a critical CDK phosphorylation site and antagonises a CDK-dependent phosphorylation event at the neighbouring S807 residue, acts to hold pRb in the hypo-phosphorylated growth-suppressing state. This is mediated in part by the recruitment of the reader protein 53BP1 to di-methylated K810, which allows pRb activity to be effectively integrated with the DNA damage response. Here, we report the surprising observation that an additional methylation-dependent interaction occurs at K810, but rather than the di-methyl mark, it is selective for the mono-methyl K810 mark. Binding of the mono-methyl PHF20L1 reader to methylated pRb occurs on E2F target genes, where it acts to mediate an additional level of control by recruiting the MOF acetyltransferase complex to E2F target genes. Significantly, we find that the interplay between PHF20L1 and mono-methyl pRb is important for maintaining the integrity of a pRb-dependent G1-S-phase checkpoint. Our results highlight the distinct roles that methyl-lysine readers have in regulating the biological activity of pRb.
Insights
Methylation of retinoblastoma protein (pRb) at lysine 810 has two distinct roles. Mono-methylation recruits PHF20L1 to regulate cell cycle checkpoints, revealing new insights into pRb regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Epigenetics
Background:
- The retinoblastoma tumor suppressor protein (pRb) is crucial for cell cycle control.
- pRb activity is regulated by post-translational modifications, including phosphorylation and methylation.
- Lysine 810 (K810) methylation influences pRb's interaction with other proteins and its function.
Purpose of the Study:
- To investigate the role of K810 methylation in pRb function.
- To identify novel methylation-dependent interactions of pRb.
- To understand how K810 methylation regulates cell cycle checkpoints.
Main Methods:
- Mass spectrometry to identify methylated sites and interacting proteins.
- Chromatin immunoprecipitation (ChIP) to analyze protein binding on target genes.
- Cell-based assays to assess cell cycle progression and checkpoint integrity.
Main Results:
- K810 methylation of pRb is associated with distinct reader proteins.
- Di-methylation at K810 recruits 53BP1, integrating pRb with DNA damage response.
- Mono-methylation at K810 recruits PHF20L1, which recruits the MOF complex to E2F target genes.
- The PHF20L1-pRb interaction is essential for maintaining the G1-S phase checkpoint.
Conclusions:
- pRb methylation at K810 has dual roles depending on the methylation state (mono- vs. di-).
- PHF20L1 acts as a novel reader for mono-methyl K810 pRb, regulating gene expression and cell cycle.
- These findings reveal distinct functions of methyl-lysine readers in pRb regulation and cell cycle control.
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