Project Summary With the completion of the Human Genome via the Telomere-to-Telomere Consortium, a complete characterization of the centromeric, pericentromeric, and telomeric regions of every chromosome, including the repeats (satellite remnants, transposable elements (TEs), retroviral insertions, etc.) is conceivable. Although it is widely accepted that centromeres are essential and necessary for proper chromosome segregation during cell division, there is a significant lack in fully understanding centromere dynamics. The limitations of previous technology are at the core of this knowledge gap in centromere entology. Current advances in sequencing and bioinformatic techniques provide an effective means to characterize and identify heterochromatic regions of the genome. Previous studies uncovered centromeric regions as more than long tandem arrays of higher order satellite repeats (HORs) that are comprised of transcriptionally active retroelement insertions displaying that repeat and retroelement transcription is necessary to identify topologically associating domains (TADs), understand the influences on 3D genome structure, and the modulation of activity of genes. We propose to determine how centromere transposable element (cenTEs) sequences influence centromeric function, and thus genome integrity in humans. This project provides a comprehensive analysis of the centromere landscape by studying three objectives. One, determine whether cenTEs are required transcription initiators in centromere assembly by delineating sites of transcription, and testing the impact of transcription initiation on assembly. Two, explain whether cenTEs are epigenetic drivers of centromere chromatin assembly by determining post-transcriptional localization of cenTE RNAs, test the impact of transcripts on centromere assembly, and describe cenTE mobile activity. And three, this study explains whether cenTEs are required for pre-and post- centromere stabilization by utilizing human artificial chromosome assays and long read sequencing. These findings will provide a novel understanding of centromere function and fitness. A comprehensive study such as this is paramount to understanding the complexities of human diseases such as cancer. This current technological golden age enables researchers to ask new questions to old problems utilizing creative techniques.