TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution

Abstract

R-loops, which consist of a DNA/RNA hybrid and a displaced single-stranded DNA (ssDNA), are increasingly recognized as critical regulators of chromatin biology. R-loops are particularly enriched at gene promoters, where they play important roles in regulating gene expression. However, the molecular mechanisms that control promoter-associated R-loops remain unclear. The epigenetic ‘reader’ Tudor domain-containing protein 3 (TDRD3), which recognizes methylarginine marks on histones and on the C-terminal domain of RNA polymerase II, was previously shown to recruit DNA topoisomerase 3B (TOP3B) to relax negatively supercoiled DNA and prevent R-loop formation. Here, we further characterize the function of TDRD3 in R-loop metabolism and introduce the DExH-box helicase 9 (DHX9) as a novel interaction partner of the TDRD3/TOP3B complex. TDRD3 directly interacts with DHX9 via its Tudor domain. This interaction is important for recruiting DHX9 to target gene promoters, where it resolves R-loops in a helicase activity-dependent manner to facilitate gene expression. Additionally, TDRD3 also stimulates the helicase activity of DHX9. This stimulation relies on the OB-fold of TDRD3, which likely binds the ssDNA in the R-loop structure. Thus, DHX9 functions together with TOP3B to suppress promoter-associated R-loops. Collectively, these findings reveal new functions of TDRD3 and provide important mechanistic insights into the regulation of R-loop metabolism.

Resolution of R-loops by topoisomerase III-β (TOP3B) in coordination with the DEAD-box helicase DDX5

“…We also found that the helicase DHX9 interacts with TOP3B, which is consistent with a recent study reporting a role for DHX9 in R-loop resolution (Yuan et al, 2021). However, by contrast to our results showing that DHX9 interacts with TOP3B independently of TDRD3, that study found that the TOP3B interaction was TDRD3 dependent (in the MCF7 cell line) and that the TDRD3-DHX9 complex worked independently of TOP3B to resolve promoter-associated R-loops (Yuan et al, 2021). Further studies are therefore warranted to decipher how DHX9 and other TOP3B interacting helicases couple with TOP3B for the resolution of different R-loops.…”

“…Consistent with the role of TOP3B in suppressing R-loops, many of these helicases have known roles in R-loop metabolism, namely, AQR, DDX21, DHX9, DHX19A (Chakraborty et al, 2018;Cristini et al, 2018;Hodroj et al, 2017;Paulsen et al, 2009;Sollier et al, 2014;Song et al, 2017). We also found that the helicase DHX9 interacts with TOP3B, which is consistent with a recent study reporting a role for DHX9 in R-loop resolution (Yuan et al, 2021). However, by contrast to our results showing that DHX9 interacts with TOP3B independently of TDRD3, that study found that the TOP3B interaction was TDRD3 dependent (in the MCF7 cell line) and that the TDRD3-DHX9 complex worked independently of TOP3B to resolve promoter-associated R-loops (Yuan et al, 2021).…”

“…Whereas DDX5 was detected in the interactome of TOP3B both in WT and TDRD3KO HCT116 cells, as expected, TDRD3 was detected only in WT cells (Figure 5A; Table S4). Consistent with a recent publication (Yuan et al, 2021), an additional DNA-RNA helicase, DHX9, was detected as a TOP3B interaction partner in both WT and TDRD3KO HCT116 cells (Figure 5A).…”

FMRP Directly Interacts With R-Loop and Shows Complex Interplay With the Dhx9 Helicase

“…On one hand, DHX9 knockdown HEK293T cells showed increased R-loop formation, suggesting that DHX9 prevents R-loop accumulation (Cristini et al, 2018). Consistent with this observation it was recently shown that DHX9 is recruited by the TDRD3/ Top3β complex to remove R-loops at specific target genes (Yuan et al, 2021). On the other hand, in cells depleted of the SFPQ RNA splicing protein the loss of DHX9 led to reduced R-loop levels, suggesting that DHX9 in fact promotes R-loop formation by unwinding dsRNA when RNA splicing is impaired (Chakraborty et al, 2018).…”

Structural and biochemical basis for DNA and RNA catalysis by human Topoisomerase 3β

“…Together these experiments show that TDRD3 binding stabilizes the TOP3B-substrate complex and enhances the catalytic activity of TOP3B. These effects could result from two complementary and non-exclusive mechanisms: 1) increase of the DNA/RNA gate flexibility and opening-closing dynamics of TOP3B, multiplying the catalytic rate of TOP3B; 2) as TDRD3 binds both TOP3B and single-strand DNA/RNA 40 , 56 , it could serve as a bridge between TOP3B and the nucleic acid substrate, stabilizing the TOP3B-substrate complex for cleavage (Fig. 6j, k ).…”