Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers.

Transcription factors (TFs) bind to enhancers and recruit H3K4me1 methyltransferase KMT2D, chromatin remodeler BAF, and H3K27 acetyltransferase p300 to activate transcription. However, the role of chromatin modifiers in regulating de novo binding of TFs on enhancers remains unclear. Using a robust nuclear translocation system, we show that the muscle lineage-determining TF MyoD binds to chromatin pervasively within one hour, with half of induced MyoD binding sites co-occupied by KMT2D, BAF, and p300. On the majority of these MyoD(+) enhancers, acute depletion of KMT2D or short-term inhibition of BAF or p300 enzymatic activity markedly reduces de novo binding of MyoD as well as that of KMT2D, BAF, and p300. On enhancers with intact MyoD binding despite these perturbations, we observe a cooperative recruitment among chromatin modifiers. Similar interdependent relationships are observed between the signal-dependent TF Glucocorticoid Receptor and KMT2D, BAF, and p300. Together, our findings show that chromatin modifiers are not only downstream effectors but also required for de novo binding of TFs on enhancers, refining a model of enhancer establishment as a process governed by functional cooperation rather than a strict hierarchy.