Abstract
CLU (clusterin) is a tumor suppressor gene that we have previously shown to be negatively modulated by the MYCN proto-oncogene, but the mechanism of repression was unclear. Here, we show that MYCN inhibits the expression of CLU by direct interaction with the non-canonical E box sequence CACGCG in the 5'-flanking region. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. MYCN physically binds in vitro and in vivo to EZH2, a component of the Polycomb repressive complex 2, required to repress CLU. Notably, EZH2 interacts with the Myc box domain 3, a segment of MYC known to be essential for its transforming effects. The expression of CLU can be restored in MYCN-amplified cells by epigenetic drugs with therapeutic results. Importantly, the anticancer effects of the drugs are ablated if CLU expression is blunted by RNA interference. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU.
Highlights
The neuroblastoma oncogene MYCN and the PRC2 members EZH2 and SUZ12 are regulators of gene transcription
We firstly investigated whether MYCN could interact with the putative E boxes in vitro
We quickly ruled out BS1 as a genuine E box sequence, but a probe containing BS2 could be shifted by cell lysates containing exogenously expressed MYCN protein
Summary
The neuroblastoma oncogene MYCN and the PRC2 members EZH2 and SUZ12 are regulators of gene transcription. Results: MYCN and PRC2 form a repressive complex on the promoter of the tumor suppressor gene CLU. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU. It was observed that MYC proteins can suppress gene expression indirectly, by interacting with sequence-specific transcription factors such as SP1 and MIZ1 and bringing transcriptional co-repressors near the transcription initiation site of the growth suppressor gene p21 (10 –12) Using this mechanism, MYCN could induce transcriptional silencing of genes involved in negative regulation of cell proliferation and transformation. A sensible hypothesis is that the aberrant expression of MYCN could modify gene expression both via direct and indirect mechanisms
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