Fig. 7: DOT1L and AR co-regulate MYC expression through a distant enhancer.
From: Histone methyltransferase DOT1L coordinates AR and MYC stability in prostate cancer
a ChIP-seq tracks of AR in LNCaP cells treated with R1881 (GSM353644), H3K27ac in LNCaP cells (GSM686937), H3K79me2 in Vehicle and EPZ-treated LNCaP cells (EPZ 1μM, 8 days), (top—bottom). b ChIP-seq tracks of AR and H3K27ac in 3 patient samples (GSE120738) at the MYC enhancer. c Enrichment of AR, DOT1L, H3K79me2, H3K27ac, H3K4me2 and RNA Pol II at MYC enhancer in PDX tumors. d AR and H3K79me2 enrichment at MYC enhancer in LNCaP cells treated with Vehicle or DHT for 3 h. e Enrichment of AR, DOT1L, H3K79me2, H3K27ac, H3K4me2 and RNA Pol II at the MYC enhancer in LNCaP cells treated with Vehicle or 1 μM EPZ for 8 days. f Enrichment of AR, DOT1L, H3K79me2, H3K27ac, H3K4me2 and RNA Pol II at the MYC enhancer in LNCaP cells transduced with EV or AR and treated with Vehicle or 1 μM EPZ for 8 days. g MYC expression in LNCaP EV or AR cells treated with Vehicle or EPZ for 8 days. h Quantitation of colony formation assays in LNCaP EV or AR cells treated with Vehicle or 1 μM EPZ for 12 days. i Results of genomic DNA PCR following CRISPR-Cas9-mediated deletion of AR-binding region in LNCaP, PC3, and LNCaP-MYC cells leading to a wild type full length band product and a deletion product. Representative image shown from 3 independent experiments. j Cells per FOV plotted for LNCaP, PC3 and LNCaP MYC (n = 5) transfected with gRNAs for CRISPR-Cas9-mediated deletion of the AR-binding site at the MYC enhancer after 5 days relative to Cas9 alone cells. k Relative MYC mRNA expression in LNCaP, PC3 (n = 5), and LNCaP-MYC (n = 5) transfected with or without gRNAs for CRISPR-Cas9 deletion of MYC enhancer after 3 days. l Graphical summary of DOT1L dependent regulation of MYC expression and its association with AR and MYC protein stability. Dashed lines represent decreased abundance. Gray lines represent inactive pathways. Statistical tests: P value determined by two-tailed t test (c–h, j, k). Error bars represent S.E.M. n = 3 biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
