Cell density–dependent nuclear‐cytoplasmic shuttling of SETDB 1 integrates with Hippo signaling to regulate YAP 1‐mediated transcription

SETDB1 is a histone methyltransferase best known for its nuclear role in transcriptional silencing, yet it is also present in the cytoplasm, and the mechanism underlying its dynamic subcellular localization remains unclear. In this study, we identify cell density as a key regulator of SETDB1 subcellular localization and demonstrate that SETDB1 modulates the Hippo signaling pathway by promoting YAP1 phosphorylation and repressing YAP1–TEAD1 target gene (YTG) expression. Under low-density culture (LDC), SETDB1 is distributed across both the nucleus and cytoplasm, whereas in high-density culture (HDC), it is excluded from the nucleus and undergoes proteasomal degradation. SETDB1 depletion resulted in reduced YAP1 phosphorylation and increased nuclear YAP1 accumulation, particularly under HDC conditions. Transcriptomic analysis of SETDB1 knockout (SKO) cells revealed upregulation of numerous YTGs, indicating that SETDB1 functions as a transcriptional repressor of YTGs. Immunoprecipitation experiments showed that SETDB1 is recruited to YTG promoters via TEAD1 and competes with YAP1 for TEAD1 binding, thereby limiting YAP1-driven transcription. Together, our findings reveal that SETDB1 regulates Hippo pathway output through coordinated control of YAP1 phosphorylation and competitive transcriptional repression of YTGs, while dynamically trafficking between the nucleus and cytoplasm.