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.
Highlights
PRb is the archetypal tumour suppressor that is directly mutated or its protein product functionally inactivated in the vast majority of human tumours.[1]
PRb activity is modulated by the activity of cyclin-CDK complexes, which phosphorylate pRb to induce the release of E2F transcription factors. pRb can undergo additional post-translational modifications (PTMs), including acetylation and lysine methylation, which further impact on pRb activity.[5,6,7,8]
We used biotinylated pRb peptides to screen the chromatin-associated domain array (CADOR), a platform developed to identify protein domains that bind modified peptides, which includes tudor, MBT, plant homeodomain (PHD) and chromodomains,[25] and previously used to identify 53BP1.9 When this screen was performed with a mono-methylated K810 pRb peptide, we identified the tudor domain protein PHF20L1 (Figure 1b)
Summary
One of the most important functions for the pRb protein in cells is to regulate the transition from G1 into S phase, and this activity is mediated in part by modulating the activity of the E2F transcription factors.[2,3] Direct binding of pRb to E2F coincides with an inhibition of transcription and cell cycle arrest, though the recruitment of histone-modifying enzymes by pRb contributes to this effect.[2]. Under conditions of cellular stress, CDK-dependent phosphorylation of pRb is inhibited by the induction of CDK inhibitor proteins such as p21, and via direct methylation of K810 in pRb by the methyltransferase SET7/9.8 The recruitment of the tudor domain protein 53BP1 to di-methylated K810 subsequently occurs, enabling pRb activity to be integrated with the DNA damage response.[9]
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