TY - JOUR
T1 - Saccharomyces cerevisiae centromere RNA is negatively regulated by Cbf1 and its unscheduled synthesis impacts CenH3 binding
AU - Chen, Chi Fu
AU - Pohl, Thomas J.
AU - Chan, Angela
AU - Slocum, Joshua S.
AU - Zakian, Virginia A.
N1 - Funding Information:
We thank A. Aguilera, K. Cimprich, D. Koshland, and B. Luke for answering questions about R-loops and providing protocols to detect them; D. Rubinstein for help with statistical analysis of the data in Figure 6; M. Basrai, S. Biggins, K. Bloom, and T. Kunkel for discussions on cen- RNAs and sharing data prior to publication; C. J. Decoste and the Princeton Molecular Biology FACS facility for help with flow cytometry; and Carly Geronimo for comments and suggestions on the manuscript. This work was funded by grant 1R35 GM-118279 to V.A.Z. from the National Institutes of Health. C.-F. C. was funded in part by a grant from the New Jersey Commission on Cancer Research (NJCCR). T.J.P. was funded in part by grants from the FORD Foundation, the Burroughs Wellcome Fund Postdoctoral Enrichment Program, and the NJCCR.
Funding Information:
We thank A. Aguilera, K. Cimprich, D. Koshland, and B. Luke for answering questions about R-loops and providing protocols to detect them; D. Rubinstein for help with statistical analysis of the data in Figure 6; M. Basrai, S. Biggins, K. Bloom, and T. Kunkel for discussions on cen-RNAs and sharing data prior to publication; C. J. Decoste and the Princeton Molecular Biology FACS facility for help with flow cytometry; and Carly Geronimo for comments and suggestions on the manuscript. This work was funded by grant 1R35 GM-118279 to V.A.Z. from the National Institutes of Health. C.-F. C. was funded in part by a grant from the New Jersey Commission on Cancer Research (NJCCR). T.J.P. was funded in part by grants from the FORD Foundation, the Burroughs Wellcome Fund Postdoctoral Enrichment Program, and the NJCCR.
Publisher Copyright:
© 2019 by the Genetics Society of America.
PY - 2019
Y1 - 2019
N2 - 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 rnh1D cells but only in mid-late S phase. However, fork pausing at centromeres was not elevated in rnh1D cells but rather was due to centromere-binding proteins, including Cbf1. Strains with increased cen-RNA lost centromere plasmids at elevated rates. In cbf1D 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.
AB - 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 rnh1D cells but only in mid-late S phase. However, fork pausing at centromeres was not elevated in rnh1D cells but rather was due to centromere-binding proteins, including Cbf1. Strains with increased cen-RNA lost centromere plasmids at elevated rates. In cbf1D 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.
KW - Cbf1
KW - Centromere
KW - Centromere RNA
KW - Cse4
UR - http://www.scopus.com/inward/record.url?scp=85073091039&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85073091039&partnerID=8YFLogxK
U2 - 10.1534/genetics.119.302528
DO - 10.1534/genetics.119.302528
M3 - Article
C2 - 31391265
AN - SCOPUS:85073091039
SN - 0016-6731
VL - 213
SP - 465
EP - 479
JO - Genetics
JF - Genetics
IS - 2
ER -