Eukaryotic genomes vary greatly in terms of size, chromosome number and genetic complexity. Their temporal organization in the nucleus is complex, reflecting coordination between folding and functions of the DNA molecule. Here we used engineered karyotypes of budding yeast - in which 16 native chromosomes were fused into gigantic DNA molecules - to characterize effects of chromosome length on nuclear architecture and function. We observed that these megachromosomes occupy a larger fraction of nuclear volume than native chromosomes. Hi-C contact maps reveal marked changes in 3D structure corresponding to delocalization of deleted centromeres and telomeres. De-clustering of inactive centromeres results in their loss of early replication firing, highlighting a functional link between 3D organization and replication timing. The repositioning of former telomere-proximal regions further reveals a novel set of gene contacts among the FLO (flocculin) genes. Finally, the expected increase of cis contacts along fused chromosomes did not affect chromatin compaction throughout the cell cycle, however it revealed that the centromere-rDNA contact in anaphase, which extends only 300 kb in the wild type, cannot exceed 1.7 Mb. Together, these results highlight the relevance of engineered karyotypes to unveiling relationships between nuclear 3D organization and genome function.