Background <p>Chromosome conformation within the nucleus is essential for genome function. These have primarily been studied at the scale of loops and compartments, or at lower spatial resolution using traditional in situ hybridization in chemically fixed samples. However, the mesoscale organization of single chromosomes in vivo, shaped by the interplay between chromatin architectural proteins and histone modifications, remains partially understood. In this study, we interrogated the mesoscale conformations of interphase chromosomes in live human osteoblasts and transformed osteosarcoma cells, focusing on chromosome 19.</p> Results <p>Chromosome conformations were quantified by the aspect ratio of the principal axes of gyration tensors. In osteoblasts, approximately 81% of chromosome 19 are observed to consist of regions characterized by highly extended organizations, with aspect ratios approximately four times greater than those of spheres. In contrast, in osteosarcoma cells, the chromosome displays an extensively collapsed conformation, with aspect ratios more closely approximately that of a sphere. In both cell types, the chromosome’s conformation is bimodal and the balance between these two modes differs very significantly between the two cell types. While the mesoscopic conformation is considerably stable, it is superimposed on dynamic, smaller scale regions. Additional results reveal that this significant conformational shift is independent of the cell cycle but co-regulated by CTCF, cohesion, and H3K27 modifications.</p> Conclusions <p>Our findings provide new insights into the coordinated complex regulatory mechanisms governing mesoscale chromosome organization in normal and transformed osteogenic tissues.</p>