Supplemental Material for Chen et al., 2019

two common features of centromeres are their transcription into non-coding RNAs (cen-RNA) and their assembly into nucleosomes that contain a centromere-specific histone H3 (cenH3). Here we show that Saccharomyces cerevisiae cen-RNA was present in low amounts in wild type cells, and its appearance was tightly cell cycle regulated, appearing and disappearing in a narrow window in S phase after centromere replication. In cells lacking Cbf1, a centromere binding protein, cen-RNA was 5-12 times more abundant throughout the cell cycle. In wild type cells, cen-RNA appearance occurred at the same time as loss of Cbf1’s centromere binding, arguing that the physical presence of Cbf1 inhibits cen-RNA production. Binding of the Pif1 DNA helicase, which happens in mid-late S phase, occurred at about the same time as Cbf1 loss from the centromere, suggesting that Pif1 may facilitate this loss by its known ability to displace proteins from DNA. cen-RNAs were more abundant in rnh1Δ cells but only in mid-late S phase. However, fork pausing at centromeres was not elevated in rnh1Δ cells but rather was due to centromere binding proteins, including Cbf1. Strains with increased cen-RNA lost centromere plasmids at elevated rates. In cbf1Δ cells, where both the levels and cell cycle regulated appearance of cen-RNA were disrupted, the timing and levels of CenH3 centromere binding were perturbed. Thus, cen-RNAs are highly regulated and disruption of this regulation correlates with changes in centromere structure and function.