R-loops shape the H2A.Z landscape and promote balanced lineage allocation during differentiation

expression in mouse embryonic stem cells (mESCs) to achieve short-term, global R-loop depletion and to systematically interrogate their impact on chromatin structure and lineage specification. We find that R-loop loss has a minimal effect on steady-state gene expression or self-renewal. Instead, it leads to a striking reduction in H2A.Z occupancy at both active and bivalent promoters, accompanied by increased nucleosome density, revealing a previously unrecognized role for R-loops in maintaining promoter architecture. During gastruloid differentiation, R-loop-depleted mESCs exhibit accelerated ectodermal differentiation, along with dysregulation of lineage-specific transcription factors and impaired cell-cell signaling. Consistent with these alterations, R-loop-depleted cells show widespread perturbations in gene regulatory networks across several early cell types. These findings uncover a critical role for R-loops in shaping the H2A.Z chromatin landscape and preserving balanced lineage trajectories during early development, offering new insights into the epigenomic regulation of stem cell fate.