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| Status |
Public on Feb 07, 2022 |
| Title |
A transcriptional roadblock protects yeast centromeres |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Centromeres are the chromosomal loci essential for faithful chromosome segregation during cell division. Although centromeres are transcribed and produce non-coding RNAs (cenRNAs) that affect centromere function, we still lack a mechanistic understanding of how centromere transcription is regulated. Here, using a targeted RNA isoform sequencing approach, we identified the transcriptional landscape at and surrounding all centromeres in budding yeast. Overall, cenRNAs are derived from transcription readthrough of pericentromeric regions but rarely span the entire centromere and are a complex mixture of molecules that are heterogeneous in abundance, orientation, and sequence. While most pericentromeres are transcribed throughout the cell cycle, centromere accessibility to the transcription machinery is restricted to S-phase. This temporal restriction is dependent on Cbf1, a centromere-binding transcription factor, that we demonstrate acts locally as a transcriptional roadblock. Cbf1 deletion leads to an accumulation of cenRNAs at all phases of the cell cycle which correlates with increased chromosome mis-segregation that is partially rescued when the roadblock activity is restored. We propose that a Cbf1-mediated transcriptional roadblock protects yeast centromeres from untimely transcription to ensure genomic stability.
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| Overall design |
We performed long-read isoform sequencing (Iso-Seq) combined with transcriptional enrichment to identify the full reportoire of centromeric RNAs (cenRNAs) in S. cerevisiae. We found that transcription at and around centromeres is complex and heterogenous between chromosomes and highly regulated during the cell cycle. Transcription initiates in the pericentromeric domain and usually terminates before entering the centromere, making cenRNAs a small subset of the surrounding RNA repertoire. While most pericentromeres are transcribed throughout the cell cycle, centromeres only become accessible to the transcription machinery in S phase. We found that centromere accessibility is regulated via a transcription roadblock mechanism mediated by Cbf1. Restoring transcription roadblock activity at CDEI partially rescues chromosome segregation defects, suggesting that one activity of Cbf1 at centromeres is to safeguard this domain from surrounding transcription. Overall, our findings highlight the importance of shielding centromeres from transcription during most cell cycle phases, in order to protect kinetochore function and ensure genomic stability.
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| Contributor(s) |
Hedouin S, Logsdon GA, Underwood JG, Biggins S |
| Citation(s) |
35253883 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| F32 GM134558 |
Centromere Sequence, Variation, and Function |
UNIVERSITY OF WASHINGTON |
Glennis Amelia Logsdon |
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| Submission date |
Nov 17, 2021 |
| Last update date |
May 06, 2022 |
| Contact name |
Glennis Amelia Logsdon |
| E-mail(s) |
glogsdon@uw.edu
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| Organization name |
University of Washington
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| Street address |
3720 15th AVE NE, S413A
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platforms (1) |
| GPL30973 |
Sequel II (Saccharomyces cerevisiae) |
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| Samples (16)
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| GSM5694121 |
Wild-type, G1 phase (t=0 min), Input, Rep 1 |
| GSM5694122 |
Wild-type, G1 phase (t=0 min), Input, Rep 2 |
| GSM5694123 |
Wild-type, S phase (t=40 min), Input, Rep 1 |
| GSM5694124 |
Wild-type, S phase (t=40 min), Input, Rep 2 |
| GSM5694125 |
Wild-type, G1 phase (t=0 min), HybCap, Rep 1 |
| GSM5694126 |
Wild-type, G1 phase (t=0 min), HybCap, Rep 2 |
| GSM5694127 |
Wild-type, S phase (t=40 min), HybCap, Rep 1 |
| GSM5694128 |
Wild-type, S phase (t=40 min), HybCap, Rep 2 |
| GSM5694129 |
cbf1Δ, G1 phase (t=0 min), Input, Rep 1 |
| GSM5694130 |
cbf1Δ, G1 phase (t=0 min), Input, Rep 2 |
| GSM5694131 |
cbf1Δ, S phase (t=40 min), Input, Rep 1 |
| GSM5694132 |
cbf1Δ, S phase (t=40 min), Input, Rep 2 |
| GSM5694133 |
cbf1Δ, G1 phase (t=0 min), HybCap, Rep 1 |
| GSM5694134 |
cbf1Δ, G1 phase (t=0 min), HybCap, Rep 2 |
| GSM5694135 |
cbf1Δ, S phase (t=40 min), HybCap, Rep 1 |
| GSM5694136 |
cbf1Δ, S phase (t=40 min), HybCap, Rep 2 |
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| Relations |
| BioProject |
PRJNA781283 |
| SRA |
SRP346633 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE189063_220131-all_reads.1bpcounts.summary.tsv.gz |
702 b |
(ftp)(http) |
TSV |
| GSE189063_RAW.tar |
144.9 Mb |
(http)(custom) |
TAR (of BAM, BW) |
| GSE189063_all_reads.100bpcounts.divergent.left.summary.tsv.gz |
431 b |
(ftp)(http) |
TSV |
| GSE189063_all_reads.100bpcounts.divergent.right.summary.tsv.gz |
422 b |
(ftp)(http) |
TSV |
| GSE189063_all_reads.1bp_meanreadlength.allsummary.tsv.gz |
7.4 Kb |
(ftp)(http) |
TSV |
| GSE189063_all_reads.50bp_meanreadlength.left.allsummary.tsv.gz |
7.2 Kb |
(ftp)(http) |
TSV |
| GSE189063_all_reads.50bp_meanreadlength.right.allsummary.tsv.gz |
6.7 Kb |
(ftp)(http) |
TSV |
| GSE189063_all_reads.50bpcounts.left.summary.tsv.gz |
691 b |
(ftp)(http) |
TSV |
| GSE189063_all_reads.50bpcounts.right.summary.tsv.gz |
697 b |
(ftp)(http) |
TSV |
| GSE189063_all_reads.entireCDEcounts.summary.tsv.gz |
535 b |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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