Saccharomyces cerevisiae Centromere RNA Is Negatively Regulated by Cbf1 and Its Unscheduled Synthesis Impacts CenH3 Binding

Genetics. 2019 Oct;213(2):465-479. doi: 10.1534/genetics.119.302528. Epub 2019 Aug 7.

Abstract

Two common features of centromeres are their transcription into noncoding centromere RNAs (cen-RNAs) 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 (WT) cells, and that 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 WT 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 the 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.

Keywords: Cbf1; Cse4; centromere; centromere RNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / genetics*
  • Centromere / genetics*
  • Chromatin / genetics
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosome Segregation / genetics
  • DNA Helicases / genetics*
  • Histones / genetics*
  • Kinetochores
  • Nucleosomes / genetics
  • RNA, Fungal / genetics
  • RNA, Untranslated / genetics
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / genetics*

Substances

  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
  • CBF1 protein, S cerevisiae
  • Chromatin
  • Chromosomal Proteins, Non-Histone
  • Histones
  • Nucleosomes
  • RNA, Fungal
  • RNA, Untranslated
  • Saccharomyces cerevisiae Proteins
  • PIF1 protein, S cerevisiae
  • DNA Helicases