DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations

Fonctions de BRCA1 dans le maintien de la stabilité chromosomique lors de la progression mitotique

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Etude des réponses de cellules de mammifères aux agents pontant l'ADN (identification de gènes impliqués dans le sensibilité à la mitomycine C)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Fonctions et régulation de la protéine suppresseur de tumeurs BRCA1 dans la réponse cellulaire aux poisons du fuseau mitotique

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells.

Zeocin is a member of the bleomycin/phleomycin family of antibiotics, known to bind and cleave DNA. We established human SK-OV-3 cells that stably express the Zeocin resistance gene (Sh ble) using an ecdysone-inducible mammalian expression system. Surprisingly, our results demonstrated that Zeocin, added in the culture medium to maintain the expression of the ecdysone receptor, was responsible for the formation of DNA strand breaks in the recombinant cells. This suggests that the Zeocin is not completely detoxified and is still able to cleave DNA, despite the stable expression of the Sh ble gene in the recombinant clones. Our study indicates that one needs to be very cautious in the interpretation of data involving stable cell lines selected with Zeocin

OPA1 cleavage depends on decreased mitochondrial ATP level and bivalent metals.

International audienceOPA1, an intra-mitochondrial dynamin GTPase, is a key actor of outer and inner mitochondrial membrane dynamic. OPA1 amino-terminal cleavage by PARL and m-AAA proteases was recently proposed to participate to the mitochondrial network dynamic in a DeltaPsi(m)-dependent way, and to apoptosis. Here, by an in vitro approach combining the use of purified mitochondrial fractions and mitochondrial targeting drugs, we intended to identify the central stimulus responsible for OPA1 cleavage. We confirm that apoptosis induction and PTPore opening, as well as DeltaPsi(m) dissipation induce OPA1 cleavage. Nevertheless, our experiments evidenced that decreased mitochondrial ATP levels, either generated by apoptosis induction, DeltaPsi(m) dissipation or inhibition of ATP synthase, is the common and crucial stimulus that controls OPA1 processing. In addition, we report that ectopic iron addition activates OPA1 cleavage, whereas zinc inhibits this process. These results suggest that the ATP-dependent OPA1 processing plays a central role in correlating the energetic metabolism to mitochondrial dynamic and might be involved in the pathophysiology of diseases associated to excess of iron or depletion of zinc and ATP