SP1 antagonizes H3K27me3 to shape chromatin landscapes for RNA polymerase II recruitment during gastrulation.

Following implantation, the epiblast undergoes gastrulation to form the three germ layers, a process requiring precise temporal control of developmental gene expression. However, the mechanisms governing RNA polymerase II (Pol II) engagement at developmental gene promoters during this critical stage remain poorly understood. Here, we present a genome-wide analysis of Pol II occupancy in mouse post-implantation embryos, revealing that nearly half of bivalent promoters are bound by Pol II in a lineage-specific and temporally ordered manner. This recruitment follows a stepwise chromatin remodeling cascade, with initial deposition of H3K27me3, followed by H3K4me3 acquisition and Pol II engagement. Through genetic perturbation, we show that KMT2B promotes Pol II loading via H3K4me3 deposition, whereas the Polycomb component EED restricts this process by maintaining H3K27me3. Notably, we identify the transcription factor SP1 as a critical facilitator of Pol II recruitment at bivalent loci. SP1 binding coincides with reduced H3K27me3 levels and enhanced Pol II occupancy, and its loss leads to chromatin re-silencing and transcriptional failure. Together, our findings establish a chromatin-based regulatory framework in which SP1 and histone modifications cooperatively license the transcriptional activation of developmental genes during germ layer formation.