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Supplemental Material for Chen et al., 2019
figure
posted on 2019-08-07, 16:25 authored by Chi-Fu Chen, Thomas J. Pohl, Angela Chan, Joshua S. Slocum, Virginia A. Zakiantwo
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.