Tumor Suppressor p16INK4A Regulates Polycomb-mediated DNA Hypermethylation in Human Mammary Epithelial Cells

Geoffrey M. Benton,Chibo Hong,Matthew B. Wilson,Giuseppe Zardo,Mahvash Sigaroudinia,Joseph F. Costello,Caroline J. Miller,Jane Fridlyand,Thea D. Tlsty,Paul A. Reynolds

doi:10.1074/jbc.m604175200

Geoffrey M. Benton, Chibo Hong + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m604175200

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Abstract

Alterations in DNA methylation are important in cancer, but the acquisition of these alterations is poorly understood. Using an unbiased global screen for CpG island methylation events, we have identified a non-random pattern of DNA hypermethylation acquired in p16-repressed cells. Interestingly, this pattern included loci located upstream of a number of homeobox genes. Upon removal of p16(INK4A) activity in primary human mammary epithelial cells, polycomb repressors, EZH2 and SUZ12, are up-regulated and recruited to HOXA9, a locus expressed during normal breast development and epigenetically silenced in breast cancer. We demonstrate that at this targeted locus, the up-regulation of polycomb repressors is accompanied by the recruitment of DNA methyltransferases and the hypermethylation of DNA, an endpoint, which we show to be dependent on SUZ12 expression. These results demonstrate a causal role of p16(INK4A) disruption in modulating DNA hypermethylation, and identify a dynamic and active process whereby epigenetic modulation of gene expression is activated as an early event in breast tumor progression.

Highlights

Loss of p16INK4A Activity Up-regulates Selected PcG Proteins— Because cells with loss of p16INK4A activity exhibit increased E2F activity, a known regulator of PcG expression, we wished to use microarray analysis to determine if factors involved in epigenetic silencing show altered expression in vHMEC compared with HMEC
To verify the gene expression profiling data, we assessed the mRNA levels of several PRC2 genes (SUZ12, EZH2, and EED) in HMEC and vHMEC populations using quantitative real-time PCR
Both SUZ12 and EZH2 mRNA is increased in vHMEC compared with HMEC, 4-fold and 9-fold, respectively (Fig. 1A)

Summary

Introduction

To determine if there is a causal relationship between loss of p16INK4A activity and increased expression of PcG genes in these primary human cells, we assessed lysates from HMEC after infection with retrovirus containing a small hairpin RNA (shRNA) to either GFP or p16INK4A by Western blot analysis. We by Targeted DNA Hypermethylation—To determine whether find vHMEC infected with retrovirus containing wild-type p16INK4A-mediated up-regulation of PcG proteins is associated p16INK4A have decreased expression of both SUZ12 and EZH2 with DNA hypermethylation, we compared the methylation when compared with vHMEC infected with retrovirus contain- profiles of populations of primary human mammary cells using ing empty vector (Fig. 1C).

Results

Conclusion