Polycomb-mediated regulation of the INK4/ARF tumour suppressor locus


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Emma Anderton1,Marc Rodriguez-Niedenfuhr1,Goedele Maertens1,Selma El Messaoudi-Aubert1,Tomas Racek1,Julie Stock1,Gordon Peters1
1Cancer Research UK London Research Institute, London, United Kingdom

Background

The Polycomb group (PcG) of transcriptional repressors participate in two classes of multi-component complex, termed PRC2 and PRC1, that respectively establish and bind to histone H3 trimethylated on lysine 27 (H3K27me3). One of their key targets is the INK4a/ARF tumour suppressor locus whose products are important for the implementation of senescence, a cell intrinsic defence against oncogenic mutations. We aim to understand how PcG proteins regulate INK4a/ARF and how oncogenic signalling overrides epigenetic silencing by PcG complexes.

Method

Primary human fibroblasts were infected with retroviral vectors encoding cDNAs or shRNAs against PcG proteins and histone modifying enzymes. The effects on the INK4a/ARF locus were analysed by chromatin immunoprecipitation, and the expression of INK4a or other genes of interest was assessed by qRT-PCR and western blotting.

Results

Activation of the RAS-RAF-MEK pathway causes removal of the H3K27me3 mark from the INK4a locus, accompanied by up-regulation of the H3K27 demethylase JMJD3 (KDM6B) and down-regulation of the H3K27 methyltransferase EZH2 (KMT6). In human fibroblasts, ectopic expression of JMJD3 activates INK4a, but not ARF, and causes a p16INK4a-dependent senescence-like arrest, whereas shRNA-mediated knockdown of JMJD3 reduces the expression of p16INK4a and alleviates oncogene-induced senescence.

We have also shown that multiple distinct PRC1 complexes bind simultaneously to a limited region of the INK4a locus and that knockdown of any one of these PRC1 complex components results in de-repression of p16INK4a.

Conclusion

Oncogenic RAS overturns PcG-mediated silencing of INK4a via activation of histone demethylase JMJD3 and down-regulation of methyltransferase EZH2. Our findings directly implicate JMJD3 in the regulation of INK4a during oncogene-induced senescence and suggest that JMDJ3 has the capacity to act as a tumour suppressor.

The discovery that the INK4a gene can be regulated by several distinct PRC1 complexes raises important questions about the mechanisms of PcG-mediated repression and whether this is a general feature of PcG-regulated genes.