Human THO –Sin3A interaction reveals new mechanisms to prevent R‐loops that cause genome instability

Abstract

R-loops, formed by co-transcriptional DNA-RNA hybrids and a displaced DNA single strand (ssDNA), fulfill certain positive regulatory roles but are also a source of genomic instability. One key cellular mechanism to prevent R-loop accumulation centers on the conserved THO/TREX complex, an RNA-binding factor involved in transcription elongation and RNA export that contributes to messenger ribonucleoprotein (mRNP) assembly, but whose precise function is still unclear. To understand how THO restrains harmful R-loops, we searched for new THO-interacting factors. We found that human THO interacts with the Sin3A histone deacetylase complex to suppress co-transcriptional R-loops, DNA damage, and replication impairment. Functional analyses show that histone hypo-acetylation prevents accumulation of harmful R-loops and RNA-mediated genomic instability. Diminished histone deacetylase activity in THO- and Sin3A-depleted cell lines correlates with increased R-loop formation, genomic instability, and replication fork stalling. Our study thus uncovers physical and functional crosstalk between RNA-binding factors and chromatin modifiers with a major role in preventing R-loop formation and RNA-mediated genome instability.

The DNA damage response acts as a safeguard against harmful DNA–RNA hybrids of different origins

“…Although R loop-driven replication fork stalling is likely the reason behind ATR activation, the displaced ssDNA of R loops in principle could also promote local ATR activation as it has been reported at centromeres in mitotic cells [61]. Together with our recent data [11], our results support that DNA-RNA hybrids not necessarily impact replication fork velocity. Indeed, so far, both faster and slower forks have been detected in conditions of transcription-replication conflicts.…”

“…Importantly, this damage was partially dependent on transcription as it was reduced by cordycepin treatment ( Fig EV2E). Furthermore, although RNase H overexpression caused DNA damage by itself in siC-treated cells, in agreement with previous reports [11], it caused no further damage in ATR-depleted cells. By contrast, RNase H overexpression caused a slight decrease in the occurrence of DNA breaks in cells depleted of specific factors of the 9-1-1/ATR/CHK1 pathway, such as siRAD9A-, siRAD17-, and siCHK1-treated cells ( Fig 3B), even though they did not show statistical significance.…”

“…Indeed, so far, both faster and slower forks have been detected in conditions of transcription-replication conflicts. Thus, slower fork speed was detected in cells depleted for FACT [26], histone H1 [62,63], ASF/SF2, and TOP1 [23], whereas faster fork speed was reported in SIN3A-and THOC1-depleted cells [7,11]. Instead, obstacles in the DNA should cause replication fork stalling.…”

“…A number of reports indicate that replication impairment is a major mechanism of DNA-RNA hybrid-mediated genetic instability [11,[19][20][21][22][23][24]. Along this line, R loop and R loop-mediated DNA damage accumulation was reported in cells depleted of the doublestrand break (DSB) repair and tumor suppressor genes BRCA1 and BRCA2, the Fanconi anemia (FA) factors involved in inter-strand crosslink (ICL) repair or the FACT chromatin reorganizer complex, required for the progression of replication fork (RF) through chromatin [25][26][27][28][29].…”

Yra1-bound RNA–DNA hybrids cause orientation-independent transcription–replication collisions and telomere instability

“…Artificially stabilized RNA-DNA hybrids cause genome instability when encountered by the replication fork in both head-on and codirectional orientation; otherwise, only RNA-DNA hybrids involved in head-on transcription-replication collisions have a major effect on genome instability. These findings may help understand the role in preventing R-loop accumulation and R-loop-mediated genome instability of specific functions such as histone deacetylase Sin3A, the THO complex, or the Fanconi anemia pathway, which have been shown to impair replication fork progression through R loops when mutated (García-Rubio et al 2015;Schwab et al 2015;Madireddy et al 2016;Salas-Armenteros et al 2017). Similarly, these results raise the question of whether overabundance of the Yra1 ortholog ALY protein in a significant proportion of tumoral cells may be related to high levels of stabilized RNA-DNA hybrids that contribute to genome instability (Dominguez-Sanchez et al 2011).…”