The essential function of centromeres in chromosome segregation during cell division requires a complex cascade of epigenetic events involving changes to chromatin character and kinetochore assembly. Kinetochore formation during mitosis is the culmination of a cycle defined by the “loading”, or deposition, of newly synthesized CENP-A, a variant histone, into centromeric chromatin. CENP-Atis faithful assembly during late telophase/early G1 of the cell cycle is facilitated by its histone chaperone, HJURP. This assembly cascade is sensitive to perturbation by genetic, epigenetic and environmental insults, with catastrophic consequences for genome/cell stability, but the genomic elements that guide accurate CENP-A nucleosome assembly are not well understood. A central conundrum in understanding the genomic features that aQract CENP-A nucleosome assembly is the observation that established centromeres are replete with satellite DNA while de novo centromeres (e.g. neocentromeres) lack satellites, yet are defined by retroelements, such as LINE-1s. While it appears CENP-A nucleosome occupancy may not require specific DNA sequences, mounting evidence demonstrates that RNA is a critical component of the epigenetic cascade leading to faithful CENP-A nucleosome assembly. However, the sequence specificity, spatiotemporal requirements for and transcriptional regulation of these centromeric RNAs are currently unknown. In Preliminary Data, we show that centromeric retroelements (cenTEs) are sites of engaged RNA polymerase and are involved in the CENP-A assembly cascade at human centromeres, linking a common transcribed genomic feature to CENP-A assembly at both native and de novo centromeres in humans for the first time. Leveraging our expertise in centromere assembly, noncoding RNAs, chromosome engineering, and genomics, we have formulated three aims, each with an innovative approach that will allow us to provide an unbiased assessment of where within centromeres transcription initiates, when during the cell cycle transcript initiation and elongation occur, and how these transcripts mediate centromere nucleosome assembly. We will use chromosome engineering to directly test whether cenTEs and their transcriptional activity are sufficient to facilitate de novo centromere assembly on chromosomes. These engineered chromosomes provide a new model to study the processes guiding cenTE-mediated centromere assembly and stabilization, and to examine misregulated incorporation of centromeric histones. The outcomes of this study will fill a large gap in our current understanding of centromere assembly and maintenance in normal cells, and provide valuable insight into events underlying chromosome aberration presenting in human diseases, infertility, and cancers of high metastatic potential.