Disruption of BRD4 at H3K27Ac-enriched enhancer region correlates with decreased c-Myc expression in Merkel cell carcinoma

Abstract

Pathologic c-Myc expression is frequently detected in human cancers, including Merkel cell carcinoma (MCC), an aggressive skin cancer with no cure for metastatic disease. Bromodomain protein 4 (BRD4) regulates gene transcription by binding to acetylated histone H3 lysine 27 (H3K27Ac) on the chromatin. Super-enhancers of transcription are identified by enrichment of H3K27Ac. BET inhibitor JQ1 disrupts BRD4 association with super-enhancers, downregulates proto-oncogenes, such as c-Myc, and displays antitumor activity in preclinical animal models of human cancers. Here we show that an enhancer proximal to the c-Myc promoter is enriched in H3K27Ac and associated with high occupancy of BRD4, and coincides with a putative c-Myc super-enhancer in MCC cells. This observation is mirrored in tumors from MCC patients. Importantly, depleted BRD4 occupancy at the putative c-Myc super-enhancer region by JQ1 correlates with decreased c-Myc expression. Thus, our study provides initial evidence that super-enhancers regulate c-Myc expression in MCC.

Keywords: BET inhibitor; BET, bromodomain and extra-terminal domain family; BRD, bromodomain; BRD4; ChIP, Chromatin immunoprecipitation; H3K27Ac; JQ1; MCC, Merkel cell carcinoma; Merkel cell carcinoma; qPCR, quantitative PCR; qRT-PCR, quantitative reverse transcription PCR; super-enhancer.

Figure 1.

Myc over-expressing cell lines exhibit enrichment of H3K27Ac and BRD4 at the c-Myc putative super-enhancer region. (A) Immunoblotting demonstrates c-Myc overexpression in MCC-3 and MCC-5 cells, and similar BRD4 levels in all MCC cells. Tubulin and GAPDH have been used as loading control for immunoblotting. In the c-Myc immunoblot, the black dividing line separates images grouped from different parts of the same blot. (B) qRT-PCR analysis shows elevated levels of c-Myc transcription in MCC-3 and MCC-5 cells. The mRNA expression of c-Myc has been normalized to that of MRPS2. (*** P < 0.001) indicates significantly higher levels of c-Myc transcripts. (C-D) Chromatin immunoprecipitation performed in MCC cell lines followed by qPCR analysis using primers of putative c-Myc super-enhancer regions demonstrates a higher co-occupancy of H3K27Ac and BRD4 at the c-Myc putative super-enhancer region. In the graphs, relative enrichment represents average fold enrichment of the target promoter in immunoprecipitation (IP) vs. input, normalized to β-actin. The total histone H3 ChIP signal has been used to normalize H3K27Ac ChIP signal. Regions of 5000 base pairs (bp) upstream and downstream to the target sequence were used as controls to ensure that the enrichment is specific to the target sequence. (***P < 0.001) indicates significant enrichment in MCC-3 and MCC-5 cells. In all the graphs, data is presented as mean ± SEM of 3 independent experiments, where SEM is standard error mean represented by the error bar.

Figure 2.

Human MCC tumors with c-Myc overexpression are associated with high levels of H3K27Ac and BRD4 occupancy at the c-Myc putative super-enhancer region. (A-B) ChIP-qPCR reveals that MCC tumors with c-Myc overexpression (Tumor #11, #12 and #16) show higher co-occupancy of H3K27Ac and BRD4 at the putative c-Myc super-enhancer region as compared to tumors with basal c-Myc expression (Tumor #10 and #15). Regions of 5000 base pairs (bp) upstream and downstream to the target sequence were used as controls to ensure that the enrichment is specific to the target sequence. Relative enrichment is the average fold enrichment of the target promoter in IP vs. input, normalized to β-actin. ChIP signal for H3K27Ac is normalized to total H3. Data is presented as mean ± SEM, where SEM is standard error mean of triplicate analysis represented by the error bar.

Figure 3.

BET inhibitor JQ1 depletes BRD4 enrichment at the c-Myc putative super-enhancer region in MCC-3 xenograft tumors. (A) Significant suppression of tumor growth are detected in both MCC-3 (**P < 0.01) and MKL-1 (*P < 0.1) xenograft tumors. A greater reduction of tumor growth is observed in MCC-3 xenograft tumors with c-Myc overexpression (75.6% vs. 48.9%). Data is presented as final tumor volume ± SEM from tumor bearing NSG mice treated with vehicle or JQ1. (B-C) JQ1 significantly reduced c-Myc expression in MCC-3 xenograft tumors as demonstrated by representative immunoblotting (B) and qRT-PCR analysis (C) (**P < 0.01 vs. vehicle treatment) of 3 biological replicates. In the c-Myc immunoblot, the black dividing line separates images grouped from different parts of the same blot. The c-Myc mRNA expression has been normalized to that of MRPS2. (D) JQ1 depletes BRD4 occupancy at the putative c-Myc super-enhancer regions in MCC-3 xenograft tumors by ChIP-qPCR analysis (***P < 0.001 vs. vehicle treatment). Regions of 5000 base pairs (bp) upstream and downstream to the target sequence were used as controls to ensure that the enrichment is specific to the target region. Relative enrichment represents average fold enrichment of the target promoter in IP vs. input, normalized to β-actin. ChIP signal for H3K27Ac is normalized to total H3. Data is presented as mean ± SEM. Error bars in the graph represent SEM from independent analysis of 3 biological replicates. (+) and (−) represents drug (JQ1) and vehicle treatment, respectively.