show Abstracthide AbstractGenome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in Genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ-cell genes during differentiation and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we find that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ-cell genes into a silenced state and activating a group of oocyte genes and Nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, crosstalk between genome architecture and NPCs is essential for successful cell fate transitions. Overall design: Sequencing of duplicate samples after performing CUT&RUN on Bam GKD ovaries (enriched for undifferentiated stages), WT (differentiated stages) and stwl GKD ovaries for the transcription factor Stwl, the boundary element BEAF as well as histone marks: H3K4me3, H3k27ac, H3k9me3, H3k27me3 and H3k4me1