H3.3K36me3 Reader ZMYND11 Localizes to Gene Promoter and Regulates Transcription Initiation

ZMYND11 is the sole known reader of H3.3K36me3. It has been characterized as a transcriptional repressor that fine-tunes RNA polymerase II (Pol II) elongation through recognition of gene body-localized H3K36me3, based primarily on earlier ChIP-seq data showing predominant ZMYND11 occupancy at gene bodies. However, subsequent data suggest that ZMYND11 may also localize to promoter regions. Here, using CUT&Tag assay, we demonstrate that ZMYND11 robustly occupies promoter regions in mouse ESCs and MEFs, with its promoter enrichment positively correlating with gene expression levels and Pol II occupancy. We further identify formaldehyde crosslinking as a critical factor causing signal loss at transcription start sites in conventional ChIP-seq. Mechanistically, ZMYND11 deficiency reduces the pausing index of Pol II, H3.3, and H3K36me3, indicating impaired transcription initiation. ZMYND11 knockout in mouse ESCs induces transcriptomic changes, impairs cell proliferation, and aberrantly activates 2-cell-specific transcriptional programs via ROS accumulation. Our findings reveal a previously unappreciated role for ZMYND11 as a transcriptional initiator that stabilizes Pol II at promoters, establishing its essential function in maintaining embryonic stem cell homeostasis and advancing our understanding of its context-dependent transcriptional regulatory mechanisms.