Distinct promoters, subjected to epigenetic regulation, drive the expression of two clusterin mRNAs in prostate cancer cells

• CLU 2 is a low abundance mRNA of the CLU gene.
• Epigenetic drugs up-regulate CLU 2 expression in prostate cancer cells.
• We identify a new promoter of CLU, P2, that drives CLU 2 expression.
• Histone-3 tail modifications at P2 promote chromatin relaxation and CLU 2 transcription.

The human clusterin (CLU) gene codes for several mRNAs characterized by different sequences at their 5’ end. We investigated the expression of two CLU mRNAs, called CLU 1 and CLU 2, in immortalized (PNT1a) and tumorigenic (PC3 and DU145) prostate epithelial cells, as well as in normal fetal fibroblasts (WI38) following the administration of the epigenetic drugs 5-aza-2'-deoxycytidine (AZDC) and trichostatin A (TSA) given either as single or combined treatment (AZDC-TSA).Our experimental evidences show that:a) CLU 1 is the most abundant transcript variant.b) CLU 2 is expressed at a low level in normal fibroblasts and virtually absent in prostate cancer cells.c) CLU 1, and to a greater extent CLU 2 expression, increased by AZDC-TSA treatment in prostate cancer cells.d) Both CLU 1 and CLU 2 encode for secreted CLU.e) P2, a novel promoter that overlaps the CLU 2 Transcription Start Site (TSS), drives CLU 2 expression.f) A CpG island, methylated in prostate cancer cells and not in normal fibroblasts, is responsible for long-term heritable regulation of CLU 1 expression.g) ChIP assay of histone tail modifications at CLU promoters (P1 and P2) shows that treatment of prostate cancer cells with AZDC-TSA causes enrichment of Histone3(Lys9)acetylated (H3K9ac) and reduction of Histone3(Lys27)trimethylated (H3K27me3), inducing active transcription of both CLU variants.In conclusion, we show for the first time that the expression of CLU 2 mRNA is driven by a novel promoter, P2, whose activity responds to epigenetic drugs treatment through changes in histone modifications.