SETD2 methyltransferase activity promotes correct transcription initiation and termination

SETD2 is a methyltransferase responsible for depositing histone H3 lysine 36 trimethylation (H3K36me3). Loss of its enzymatic activity occurs in some cancers, including renal cell carcinoma. SETD2 mutations have been linked to delayed transcription termination, but have not been explored in depth. Here, using nascent transcriptomics in SETD2 knockout and patient-derived cells, we reveal a dichotomy in SETD2 functions depending on the affected protein-coding gene. The majority of genes, named class I, are dependent on SETD2 function for transcription initiation, yet terminate transcription in the usual locations. In contrast, for class II genes, corresponding to 15-25% of active protein-coding genes, transcription initiation is robust in absence of SETD2 activity; however, widespread transcriptional readthrough occurs. Defective termination following SETD2 loss/mutation is associated with increased cryptic transcription initiation and impaired 3' pre-mRNA cleavage. Additionally, alternative polyadenylation upon SETD2 activity loss is highly cell type specific, and no relationship with transcription readthrough was observed. We demonstrate that methyltransferase activity of SETD2 stimulates proper initiation, prevents cryptic initiation and promotes efficient 3' end processing, however it does so indirectly. Overall design: POINTseq profiling U2OS parental cells and SETD2 KO cells.

SETD2是负责催化组蛋白H3赖氨酸36三甲基化（H3K36me3）的甲基转移酶。其酶活性缺失可见于包括肾细胞癌在内的多种癌症。SETD2突变已被证实与转录终止延迟相关，但相关机制尚未得到深入探究。本研究通过在SETD2敲除细胞及患者来源细胞中开展新生转录组学分析，揭示了SETD2的功能呈现二分性，具体取决于其所作用的蛋白编码基因类别：占多数的I类基因的转录起始依赖SETD2功能，但其转录终止位点仍保持正常；与之相对的II类基因，约占活跃蛋白编码基因的15%~25%，即使在SETD2活性缺失的情况下，转录起始仍可高效进行，但会出现广泛的转录通读现象。SETD2缺失或突变引发的转录终止缺陷，与隐秘转录起始增加及3'前体mRNA剪切受损密切相关。此外，SETD2活性缺失所导致的可变多聚腺苷酸化具有高度的细胞类型特异性，且未观察到其与转录通读存在关联。本研究证实，SETD2的甲基转移酶活性可间接促进正常转录起始、阻断隐秘转录起始，并提升3'端加工效率。实验总体设计：采用POINTseq技术对U2OS亲本细胞及SETD2敲除细胞进行测序分析。