R-loops at centromeric chromatin contribute to defects in kinetochore integrity and chromosomal instability in budding yeast

Abstract

R-loops, the by-product of DNA-RNA hybridization and the displaced single stranded DNA, have been identified in bacteria, yeasts, and other eukaryotic organisms. The persistent presence of R-loops contributes to defects in DNA replication and repair, gene expression, and genomic integrity. R-loops have not been detected at centromeric ( CEN) chromatin in wild-type budding yeast. Here we used an hpr1∆ strain that accumulates R-loops to investigate the consequences of R-loops at CEN chromatin and chromosome segregation. We show that Hpr1 interacts with CEN-histone H3 variant, Cse4, and prevents the accumulation of R-loops at CEN chromatin for chromosomal stability. DNA-RNA immunoprecipitation (DRIP) analysis showed an accumulation of R-loops at CEN chromatin which was reduced by overexpression of RNH1 in hpr1∆ strains. Increased levels of ssDNA, reduced levels of Cse4 and its assembly factor Scm3, and mislocalization of histone H3 at CEN chromatin were observed in hpr1∆ strains. We determined that accumulation of R-loops at CEN chromatin contributes to defects in kinetochore biorientation and chromosomal instability (CIN), and these phenotypes are suppressed by RNH1 overexpression in hpr1∆ strains. In summary, our studies provide mechanistic insights into how accumulation of R-loops at CEN contributes to defects in kinetochore integrity and CIN.

The Role of Human Centromeric RNA in Chromosome Stability

“…R-loops, the byproduct of DNA-RNA hybridization, have important physiological functions but are also contributing to defects in genome integrity and chromosome fragility (Aguilera and García-Muse, 2012). In budding yeast, the accumulation of R-loops at the centromere is associated with a defect in kinetochore integrity (Mishra et al, 2021). In human, DHX9 is one element generating centromeric R-loops (Chakraborty et al, 2018), and while R-loops causes genomic instability in S phase (Buisson et al, 2015), they are necessary for faithful mitosis through a mitotic specific ATR pathway, independent of DNA damage or replication, but dependent of Aurora B and R-loops, ensuring accurate chromosome segregation (Aze et al, 2016;Kabeche et al, 2018).…”

Oncogenic lncRNAs alter epigenetic memory at a fragile chromosomal site in human cancer cells

“…R-loops that are formed during transcription in cis occupy 5% of the mammalian genomes ( 70 ). R-loops play an essential role in centromeric CENP-A maintenance and kinetochore integrity, ultimately accounting for chromosome stability ( 51 , 71 – 74 ). We speculate that a three-pronged battle between transcription, R-loop occupancy, and CENP-A invasion could challenge the mechanical integrity of the 8q24 locus DNA backbone, placing it under physical strain ( 75 , 76 ).…”

Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores.

“…When both RNases were removed and Rio1 depleted (rnh1∆ rnh2∆ RIO1-AID strain + 500µM auxin), CEN and periCEN RNA levels dropped with 50-80% (Fig. 4b), attesting that R-loops curtail RNAPII activity at centromeres and pericentromeres 20,31,37 .…”