Project Summary The human centromere contains non-coding repetitive alpha-satellite DNA sequences that is under active RNA polymerase (RNAP) II-catalyzed transcription. The centromeric transcription has been proposed to play an important role in the deposition of centromere proteins to centromeric chromatin and centromeric cohesion. However, these conclusions are facing a challenge because the approaches to suppress centromeric transcription in these studies were not specific to centromeres. Therefore, it is critically important to develop novel approaches to specifically inactivate centromeric transcription without significantly altering global gene transcription and then determine the functions of centromeric transcription. In addition, how centromeric transcription is regulated is also poorly understood. The overall objective of this application is to determine the functioning mechanisms underlying centromeric transcription. Based on the published and our preliminary data, we hypothesize that centromeric transcription orchestrated by specific factors and histone modifications during the cell cycle promotes centromeric cohesion that is essential for chromosome segregation. This hypothesis will be tested by pursuing three specific aims: 1) determining the functions of centromeric transcription. We will examine centromeric cohesion when centromeric transcription is specifically suppressed using our newly developed approach. 2) elucidating the epigenetic regulation of centromeric transcription. We will test the role of the histone mark H3K4 di-methylation (me2) in centromeric transcription and its regulation. 3) identifying the key regulators for centromeric transcription. We will test the functioning mechanism of Cyclin-dependent kinase (Cdk)11 in centromeric transcription. At the completion of the proposed research, we will expect to have determined the functioning mechanisms of centromeric transcription. These results will have an important positive impact because they will contribute to a conceptual framework for the future identification and development for potential cancer therapeutic targets.