CtBP1/2 oligomerization promotes G9a-Mediated transcriptional repression.

Corepressors CtBP1 and CtBP2 (CtBP1/2) are evolutionarily conserved transcriptional regulators that repress gene expression by recruiting chromatin modifiers, yet the structural basis of this process remains elusive. Here, we identify a direct interaction between CtBP1/2 and the histone H3 lysine 9 (H3K9) methyltransferase G9a. Crystallographic and biochemical analyses reveal that a CtBP1/2 tetramer simultaneously engages two G9a molecules through a motif within the pre-SET domain of G9a, which is absent in its paralog GLP. This interaction enhances G9a catalytic activity in a manner strictly dependent on the oligomeric state of CtBP1/2. Disruption of CtBP2 tetramerization diminishes its association with G9a and abolishes enzymatic activation, underscoring the functional importance of CtBP1/2 oligomerization. In colorectal cancer (CRC) cells, CtBP2 and G9a co-occupy the PTEN promoter, where disruption of their interface reduces H3K9me2 deposition, derepresses PTEN expression, attenuates PI3K-AKT signaling, and impairs CRC cell proliferation. Together, these findings establish a structural framework for CtBP-mediated regulation of G9a activity and highlight the CtBP1/2-G9a complex as a potential therapeutic target in colorectal cancer.