BRCA1 prevents R-loop-associated centromeric instability

Carine Racca,Claire Francastel,Patrick Calsou,Sébastien Britton,Sabrine Hédouin,Florence Larminat

doi:10.1038/s41419-021-04189-3

Abstract

Centromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive α-satellite sequences, which are actively transcribed throughout the cell cycle. Centromeres play an essential role in chromosome inheritance and genome stability through coordinating kinetochores assembly during mitosis. Structural and functional alterations of the centromeres cause aneuploidy and chromosome aberrations which can induce cell death. In human cells, the tumor suppressor BRCA1 associates with centromeric chromatin in the absence of exogenous damage. While we previously reported that BRCA1 contributes to proper centromere homeostasis, the mechanism underlying its centromeric function and recruitment was not fully understood. Here, we show that BRCA1 association with centromeric chromatin depends on the presence of R-loops, which are non-canonical three-stranded structures harboring a DNA:RNA hybrid and are frequently formed during transcription. Subsequently, BRCA1 counteracts the accumulation of R-loops at centromeric α-satellite repeats. Strikingly, BRCA1-deficient cells show impaired localization of CENP-A, higher transcription of centromeric RNA, increased breakage at centromeres and formation of acentric micronuclei, all these features being R-loop-dependent. Finally, BRCA1 depletion reveals a Rad52-dependent hyper-recombination process between centromeric satellite repeats, associated with centromere instability and missegregation. Altogether, our findings provide molecular insights into the key function of BRCA1 in maintaining centromere stability and identity.

Highlights

Centromeres are the essential chromatin domains which coordinate the assembly of kinetochores, the proteinaceous complexes required for the attachment of the spindle microtubules to chromosomes during mitosis
Using DRIP and immunofluorescence, we showed that human centromeric repeats are prone to R-loop formation during interphase, a behavior which was previously observed in yeast [56, 57], for other repeats throughout the human genome [32] and during mitosis [17]
We revealed that centromeric chromatin association of BRCA1 is promoted by R-loops in undamaged cells

Summary

INTRODUCTION

Centromeres are the essential chromatin domains which coordinate the assembly of kinetochores, the proteinaceous complexes required for the attachment of the spindle microtubules to chromosomes during mitosis. DNA was resuspended in 200 μl of elution buffer (5 mM Tris-HCl pH 8) and fragmented on ice by sonication using a microtip for 2 × 10 s Despite these links between BRCA1 and centromere, it is still unknown what drives the localization of BRCA1 at centromeres and how BRCA1 contributes to centromere stability in undamaged cells. At the end of the incubation, 25 μl of protein A magnetic beads (Diagenode) prewashed 2−3 times with binding buffer were added to the DNA/antibody complex and incubated for at least 4 h at

MATERIALS AND METHODS

Racca et al 4

Findings

DISCUSSION