Systematic identification of a 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 homology-directed repair (HDR) and foster transcriptional silencing. Yet, how CDYL1 elicits DSB-induced silencing is not fully understood. Here, we systematically 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 AsiSI sites, which triggers the eviction of the transcriptional 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 HDR 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 HDR and DSB-induced transcriptional silencing, suggesting that they may occur independently. HighlightsO_LISystematic identification of a local decrease in lysine crotonylation PanKcr and H3K9cr at AsiSI-induced DSBs that correlates with transcriptional silencing. C_LIO_LICDYL1 crotonyl-CoA hydratase activity downregulates Kcr at DSBs. C_LIO_LIKcr reduction at DSBs promotes ENL eviction and fosters transcriptional silencing. C_LIO_LICDYL1 roles in DSB-induced transcriptional silencing and HDR are functionally uncoupled. C_LI