<scp>GATA1</scp> Drives Human Erythropoiesis via Liquid–Liquid Phase Separation

The spatial organization and transcriptional activity of transcription factors are increasingly recognized to be regulated by liquid-liquid phase separation (LLPS). Whether GATA1, the master regulator of erythropoiesis, undergoes LLPS and how this process influences erythroid development have remained unknown. Here, we show that GATA1 forms dynamic nuclear condensates in HEK293T, HUDEP2 progenitors, and erythroleukemia cells, as well as concentration-dependent droplets in vitro. These condensates exhibit hallmark properties of LLPS, including fusion behavior, sensitivity to 1,6-hexanediol, and rapid fluorescence recovery after photobleaching (FRAP). Domain deletion and mutational analyses revealed intrinsically disordered region 2 (IDR2) as the primary driver of GATA1 LLPS. Importantly, the congenital anemia-associated R307H mutation and dephosphorylation-mimicking S310A substitution within IDR2 disrupted droplet formation and abolished FRAP recovery. Mechanistically, loss of LLPS reduced GATA1 chromatin occupancy, impaired its assembly with cofactors such as FOG1 and LMO2, and abrogated activation of erythroid gene promoters in luciferase assays, leading to defective terminal differentiation. Together, these findings uncover a previously unrecognized mechanism by which GATA1 regulates erythropoiesis through LLPS and highlight modulation of its IDR2 domain and post-translational modifications as potential therapeutic strategies for erythroid-related disorders.