The regulation and function of centromeric transcription in the budding yeast Saccharomyces cerevisiae

Abstract

Every cell must distribute its genetic materials equally to its daughter cells in cell divisions. Errors in chromosome segregation result in cells with an abnormal number of chromosomes, which is a hallmark of cancers and may cause spontaneous abortion or birth defects. Thus, understanding the mechanisms that ensure accurate chromosome segregation is fundamentally important and medically relevant. The centromere is a chromatin domain on the chromosome acting as a platform for kinetochore formation, which allows microtubule attachment and ensures accurate chromosome segregation during mitosis and meiosis. Centromere is proposed to be regulated mainly by epigenetics. Recently, transcriptional activity has been found in the centromere of many eukaryotes, and increasing evidence indicates that centromeric transcription and its corresponding non-coding centromeric RNAs (cenRNAs) may involve in controlling the activity of centromere epigenetically. Budding yeast S. cerevisiae possesses the simplest form of centromere in eukaryotes, and its centromere is originally thought to be DNA sequence-dependent without any epigenetic control. In this study, I have identified that budding yeast centromere is regulated epigenetically by centromeric transcription. To characterize budding yeast centromeric transcription, I have demonstrated that cenRNAs are long non-coding RNAs induced in S phase of the cell cycle, coincident with the CENP-A loading time. The induction of centromeric transcription is DNA replication-dependent. Centromeric transcription is repressed by kinetochore protein Cbf1 and histone H2A variant Htz1. Deletion of CBF1, HTZ1, or both, resulted in an increase of centromere-containing plasmid (CEN plasmid) loss. Double deletion of CBF1 and HTZ1 down-regulate the protein level of CENP-A chaperone HJURPScm3, and kinetochore proteins CENP-ACse4, CENP-CMif2 and SurvivinBir1. A novel RNAi system is developed to knockdown cenRNAs from all the centromeres, and resulted in centromere malfunction, suggesting that cenRNAs regulate centromere function in trans. cenRNA knockdown partially rescues the CEN plasmid loss phenotype in cbf1Δ and htz1Δ, indicating that disruption of centromere function in these deletion mutants are partially due to derepressed cenRNA level. This work demonstrates that centromeric transcription is tightly regulated, and a fine balance of cenRNAs level is important for normal centromere function.