Summary/Abstract Mammalian cells initiate DNA replication at multiple sites along each chromosome at different times, following a temporal replication program. We have used a chromosome-engineering strategy to identify cis-acting loci that control this replication-timing program on individual human chromosomes. We found that Cre/loxP- mediated translocations, affecting eight different autosomes, display a delay in replication timing and structural instability of entire chromosomes. Subsequently, we found that Cre/loxP-mediated disruption of the lncRNA genes ASAR6 and ASAR15 result in delayed replication of human chromosomes 6 and 15, respectively. ASAR6 and ASAR15 share numerous characteristics, including: 1) random mono-allelic expression of lncRNAs that can physically “coat” entire chromosomes in cis; 2) asynchronous replication between alleles; 3) genetic disruption results in structural instability of their respective chromosomes; and 4) ectopic integration of transgenes causes delayed replication of entire chromosomes in cis. In earlier studies, we detected the delayed replication and structural instability phenotypes following rearrangement of numerous human and mouse chromosomes, suggesting that all mammalian chromosomes are regulated by similar mechanisms. Our work suggests that all mammalian chromosomes are regulated by similar loci. Therefore, we are proposing that all mammalian chromosomes contain “Inactivation/Stability Centers” (I/SCs), which normally function to promote proper replication timing, monoallelic gene expression and structural stability of individual chromosomes. We believe that I/SCs are as fundamentally important to mammalian chromosome biology as telomeres, centromeres, or origins of replication. Thus, under this scenario every mammalian chromosome contains four essential cis-acting elements, origins, centromeres, telomeres, and I/SCs all functioning to ensure proper replication, segregation and stability of each chromosome. This proposal is designed to elucidate the mechanisms by which ASAR6 and ASAR15 RNAs control chromosome replication timing, to identify chromatin interaction sites of ASAR6 and ASAR15 RNAs, and to determine the mechanisms by which ASAR6 and ASAR15 function to localize chromosomes within the 3D space of the nucleus, and if they control the genome interaction maps of chromosomes 6 and 15. If successful the experiments described in this proposal will help to establish a new paradigm for mammalian chromosome biology, and will serve as the foundation for a fourth type of essential chromosomal element, the “Inactivation/Stability Center”.