MLL methyltransferases regulate H3K4 methylation to ensure CENP-A assembly at human centromeres

The active state of centromeres is epigenetically defined by the presence of CENP-A interspersed with histone H3 nucleosomes. While the importance of dimethylation of H3K4 for centromeric transcription has been highlighted in various studies, the identity of the enzyme(s) depositing these marks on the centromere is still unknown. The MLL (KMT2) family plays a crucial role in RNA polymerase II (Pol II)-mediated gene regulation by methylating H3K4. Here, we report that MLL methyltransferases regulate transcription of human centromeres. CRISPR-mediated down-regulation of MLL causes loss of H3K4me2, resulting in an altered epigenetic chromatin state of the centromeres. Intriguingly, our results reveal that loss of MLL, but not SETD1A, increases co-transcriptional R-loop formation, and Pol II accumulation at the centromeres. Finally, we report that the presence of MLL and SETD1A is crucial for kinetochore maintenance. Altogether, our data reveal a novel molecular framework where both the H3K4 methylation mark and the methyltransferases regulate stability and identity of the centromere.