The SWI/SNF-related protein SMARCA3 is a histone H3K23 ubiquitin ligase that regulates H3K9me3 in cancer.

Histone ubiquitination is a crucial post-translational modification (PTM) regulating chromatin function, yet many histone ubiquitination sites and the enzymes that control them remain poorly understood. Here, we identify SMARCA3, a SWI/SNF-related protein frequently downregulated in colorectal cancer (CRC), as an E3 ubiquitin ligase that targets histone H3 at lysine 23 (H3K23). We demonstrate that SMARCA3 histone ubiquitination activity is stimulated by the repressive H3K9me3 mark. Loss of SMARCA3 reduces both H3K23Ub and H3K9me3, increasing chromatin accessibility at promoters and enhancers enriched for pioneer transcription factor motifs. This chromatin "rewiring" alters the transcriptional landscape, driving upregulation of cancer-promoting genes. We validate this mechanism in CRC cell lines and patient-derived organoids, where SMARCA3 loss reduces H3K23Ub and H3K9me3. In xenograft mouse models, overexpression of wild-type SMARCA3, but not a RING domain mutant, suppresses tumor growth. Together, our findings define SMARCA3 as a key chromatin regulator contributing to CRC pathogenesis through epigenetic mechanisms.