AbstractWe herein employ in situ Hi-C with an auxin-inducible degron (AID) system to examine the effect of chromatin remodeling on 3D genome organization in yeast. Eight selected ATP-dependent chromatin remodelers representing various subfamilies contribute to 3D genome organization differently. Among the studied remodelers, the temporary depletions of Chd1p, Swr1p, and Sth1p (a catalytic subunit of the Remodeling the Structure of Chromatin [RSC] complex) cause the most significant defects in intra-chromosomal contacts, and the regulatory roles of these three remodelers in 3D genome organization differ depending on the chromosomal context and cell cycle stage. Furthermore, even though Chd1p and Isw1p are known to share functional similarities/redundancies, their depletions lead to distinct effects on 3D structures. The RSC and cohesin complexes also differentially modulate 3D genome organization within chromosome arm regions, whereas RSC appears to support the function of cohesin in centromeric clustering at G₂ phase. Our work suggests that the ATP-dependent chromatin remodelers control the 3D genome organization of yeast through their chromatin-remodeling activities.