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.