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

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.

Polo kinase recruitment via the constitutive centromere-associated network at the kinetochore elevates centromeric RNA

“…Recent studies found that a deletion of the gene encoding the CDEI-binding protein, Cbf1, increased centromeric, non-coding transcripts and chromosome missegregation [92,93]. Although our CDC5ΔC-AME1 suppressor screen identified mutants of transcription elongation and mRNA processing genes, we felt that Cdc5 was unlikely to be directly affecting centromeric (CEN) transcription for various reasons.…”

“…Although our CDC5ΔC-AME1 suppressor screen identified mutants of transcription elongation and mRNA processing genes, we felt that Cdc5 was unlikely to be directly affecting centromeric (CEN) transcription for various reasons. First, although deletion of Cbf1 increases CEN transcription, this is not lethal [92,93]. Second, a deletion of Cbf1 is synthetic lethal with a number of kinetochore mutants [94], which suggests that increased CEN transcription could be a consequence of kinetochore disruption.…”

“…Cse4 CENP-A deposition in budding yeast occurs in S-phase after replication of the CEN DNA [6]. Recently, it was shown that Cbf1 is involved in restraining CEN transcription to late S-phase after CEN replication [92,93]. We asked whether Cse4 localization was affected by the Cdc5 association with the COMA complex and examined Cse4 foci in cells expressing CDC5ΔC-AME1.…”

“…In addition, we found that the increased CEN1 and CEN3 RNA was dependent on SPT4, whereas CEN8 was not ( Fig 6A). It has been proposed that centromeres are differentially transcriptionally regulated [93], which may explain the difference between CEN1/3 and CEN8. These results indicate that association of Cdc5 to the COMA complex can upregulate CEN RNA levels, which largely depends on the elongation and mRNA-processing factor Spt4.…”

“…Since Cbf1 was recently identified as a repressor of CEN transcription [92,93], we hypothesized if the CDC5ΔC-AME1 fusion results in hyperactivation of CEN transcription then overexpression of CBF1 may suppress the growth phenotype. However, CBF1 overexpression driven by either the CUP1 (in the presence of copper to increase expression) or GAL1 promoters were not sufficient to suppress the CDC5ΔC-AME1 growth defect (S15A and S15B Fig).…”