Supplemental Material for Chen et al., 2019
figureposted on 07.08.2019 by Chi-Fu Chen, Thomas J. Pohl, Angela Chan, Joshua S. Slocum, Virginia A. Zakian
Figures are generally photos, graphs and static images that would be represented in traditional pdf publications.
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.