Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis

Merkel cell carcinoma (MCC) frequently contains integrated copies of Merkel cell polyomavirus DNA that express a truncated form of Large T antigen (LT) and an intact Small T antigen (ST). While LT binds RB and inactivates its tumor suppressor function, it is less clear how ST contributes to MCC tumorigenesis. Here we show that ST binds specifically to the MYC homolog MYCL (L-MYC) and recruits it to the 15-component EP400 histone acetyltransferase and chromatin remodeling complex. We performed a large-scale immunoprecipitation for ST and identified co-precipitating proteins by mass spectrometry. In addition to protein phosphatase 2A (PP2A) subunits, we identified MYCL and its heterodimeric partner MAX plus the EP400 complex. Immunoprecipitation for MAX and EP400 complex components confirmed their association with ST. We determined that the ST-MYCL-EP400 complex binds together to specific gene promoters and activates their expression by integrating chromatin immunoprecipitation with sequencing (ChIP-seq) and RNA-seq. MYCL and EP400 were required for maintenance of cell viability and cooperated with ST to promote gene expression in MCC cell lines. A genome-wide CRISPR-Cas9 screen confirmed the requirement for MYCL and EP400 in MCPyV-positive MCC cell lines. We demonstrate that ST can activate gene expression in a EP400 and MYCL dependent manner and this activity contributes to cellular transformation and generation of induced pluripotent stem cells.

默克尔细胞癌（Merkel cell carcinoma, MCC）常整合有默克尔细胞多瘤病毒DNA，该病毒可表达截短型大T抗原（Large T antigen, LT）与完整型小T抗原（Small T antigen, ST）。尽管LT可结合RB并使其肿瘤抑制功能失活，但ST在MCC肿瘤发生中的作用机制仍不明确。本研究发现，ST可特异性结合MYC家族同源蛋白MYCL（L-MYC），并将其招募至由15个亚基组成的EP400组蛋白乙酰转移酶及染色质重塑复合物。我们通过针对ST的大规模免疫沉淀实验结合质谱分析，鉴定出共沉淀的蛋白组分：除蛋白磷酸酶2A（Protein phosphatase 2A, PP2A）亚基外，还鉴定得到MYCL及其异二聚体伴侣MAX，以及EP400复合物。针对MAX与EP400复合物组分的免疫沉淀实验，进一步验证了它们与ST的相互作用。通过整合染色质免疫沉淀测序（Chromatin immunoprecipitation with sequencing, ChIP-seq）与RNA测序（RNA-seq）技术，我们证实ST-MYCL-EP400复合物可特异性结合特定基因启动子并激活其转录。在MCC细胞系中，MYCL与EP400是维持细胞活力所必需的，且可协同ST促进靶基因表达。全基因组CRISPR-Cas9筛选实验证实，在MCPyV阳性的MCC细胞系中，MYCL与EP400为细胞存活的必需基因。本研究表明，ST可通过EP400与MYCL依赖的方式激活基因表达，该活性可促进细胞转化及诱导多能干细胞的生成。