CDYL1-dependent decrease in lysine crotonylation at DNA double-strand break sites functionally uncouples transcriptional silencing and repair

Previously, we showed that CDYL1 is recruited to DNA double-strand breaks (DSBs) to promote homologous recombination (HR) repair and foster transcriptional silencing. Yet, how CDYL1 elicits DSB-induced silencing is not fully understood. Here, we identify a CDYL1-dependent local decrease in the transcriptionally active marks lysine crotonylation (PanKcr) and crotonylated histone residue H3K9cr at AsiSI-induced DSBs, which correlates with transcriptional silencing. Mechanistically, we reveal that CDYL1 crotonyl-CoA hydratase activity counteracts PanKcr and H3K9cr at DSB sites, which triggers the eviction of the transcription elongation factor ENL and foster transcriptional silencing. Furthermore, genetic inhibition of CDYL1 hydratase activity blocks the reduction in H3K9cr and alleviates DSB-induced silencing, while HR efficiency unexpectedly remains intact. Therefore, our results functionally uncouple the repair and silencing activity of CDYL1 at DSBs. In a broader context, we address a long-standing question concerning the functional relationship between HR repair and DSB-induced transcriptional silencing, suggesting that they may occur independently.