RAD51AP1 regulates ALT-HDR through chromatin-directed homeostasis of TERRA

Abstract

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA.

Keywords: ALT; RAD51AP1; TERRA; cancer; chromatin; homology-directed repair; telomere; transcription.

Figure 1.. RAD51AP1 binds to TERRA and TERRA R-loops.

(A) Northern dot-blot of TERRA RNA immunoprecipitation (RIP) of endogenous RAD51AP1 and RAD51 in U2OS (n=4) and LM216J (n=2) ALT cells and HeLa LT (n=2) telomerase expressing (TEL+) cells. (B) Quantification of % TERRA detected in RAD51AP1 and RAD51 RIPs. Data represent mean ± SEM. (C) Electromobility Shift Assay (EMSA) of RAD51AP1 binding TERRA RNA (left) and ssDNA oligonucleotides (right) and quantification of shifted nucleic acid substrates. Data represent mean ± S.D, n=3. (D) EMSA of RAD51AP1 WT (left) and K6K7 (nucleic acid binding mutant, right) binding synthetic TERRA R-loops and quantification of shifted R-loop. * indicates a DNA contaminant in the substrate. Data represent mean ± S.D, n=3. (E) Top: Western blots showing RAD51AP1 and FLAG-TRF1-FokI protein levels after RAD51AP1 knockdown in (WT, left)- and (DA, right)- TRF1-FokI induced U2OS cells. Bottom: Southern dot-blot of DNA-RNA immunoprecipitation (DRIP) with S9.6 antibodies after RAD51AP1 knockdown in (WT, left)- and (DA, right)- TRF1-FokI induced U2OS cells. (F) Quantification of telomeric R-loops. Data represent mean ± SEM, n=3. p values are indicated and generated by One way ANOVA. See also Figure S1.

Figure 2.. The RAD51AP1 proximal interactome contains RNA processing and transcription-associated complexes.

(A) Right. Schematic of TRF1 and RAD51AP1 BioID. Left. Western blots showing the expression of BirA-TRF1 and BirA-RAD51AP1 in U2OS cells. * indicates the band corresponding to the expected protein. (B) GO-term annotation and ranking of TRF1RAD51AP1 enriched proteins by biological processes and molecular functions using DAVID. (C) Clustering of distinct functional protein groups identified by TRF1-RAD51AP1 BioID. (D) Spectral counts of selected TRF1 (left bars) and RAD51AP1 (right bars) BioID hits from the indicated functional groups. (E) Representative images and (F) quantification of APBs (PML and TRF2 localization) after knockdown of the indicated proteins in U2OS cells. All data represent mean ± SEM, n=3. All scale bars, 5μm. (G) Quantification of telomeres staining positive for EdU incorporation after knockdown of the indicated proteins in U2OS cells synchronized in G2. All data represent mean ± SEM, n=4 (H) Box plot showing the quantification of telomere DRIP assays (n=3). Horizontal lines and boxes represent the mean ± SEM and min-max range of values from individual experiments. (I-K) Quantification of (I) micronuclei with TTAGGG FISH signals (n=2), (J) fragile telomeres (n=2) and (K) signal-free chromosome ends (i.e., telomere loss) (n=2) after knockdown of the indicated proteins in U2OS cells. Each circle in J and K represents a single data point, mean ± SEM are represented by black lines. p values are indicated and generated by One way ANOVA. See also Figures S2, S3 and Table S1.

Figure 3.. RAD51AP1 co-regulates TERRA suppression during ALT.

(A) Representative images and (B) quantification of TERRA (RNA FISH) and Ubiquitin-K119 of histone H2A (Ub-H2A) (IF) at t-DSBs after ATMi or EZH2i (10μM, 4hrs each). (C) Representative images and (D) quantification of TERRA (RNA-FISH) and ubiquitinated histone H2A-K119 (Ub-H2A) (IF) at telomeric double-strand breaks (t-DSBs generated by FLAG tagged WT-TRF1-FokI) after siRNA-mediated knockdown of indicated proteins. (E) Representative images and (F) quantification of TERRA (left) and Ub-H2A (right) at t-DSBs in scrambled (SCRM) and TERRA ASO depleted U2OS cells that were co-transfected with either control (CTRL) or RAD51AP1 siRNAs. (G) Representative images and (H) quantification of TERRA (left) and Ub-H2A (right) at t-DSBs in U2OS cells that were transfected with either control (CTRL), SETX or/and RAD51AP1 siRNAs. All box and whiskers plots show the interquartile and min-max ranges. The median is represented by the horizontal black line. Data from triplicate independent experiments are shown. p values are indicated and generated by One way ANOVA. All scale bars, 5μm. See also Figure S4.

Figure 4.. RAD51AP1 prevents transcription-replication collisions (TRCs) during ALT.

(A) Schematic of proximity ligation assay (PLA) of RNAPII (phospho-Serine 2) and PCNA at sites of t-DSB induction (mCherry-TRF1-FokI). (B) Representative IF images of PLA signals (green) and t-DSBs (red) in PDS5a, ZMYND8, BRG1 and RAD51AP1 depleted U2OS cells expressing WT-TRF1-FokI. Controls include PLA reactions where no primary antibody was added and expression of DA-TRF1-FokI. (C) Quantification of the % PLA signals (in PLA positive cells) that co-localize with t-DSBs from B. Each circle in C represents a single data point i.e., % PLA-TRF1-FokI co-localizations per cell. Mean ± SEM are represented by black lines. Data from at least 3 independent experiments per condition are shown. p values are indicated and generated by One way ANOVA. All scale bars, 5μm.

Figure 5.. The RAD51AP1 SUMO-SIM regulatory axis is required for TERRA suppression.

(A) Schematic showing the location of RAD51AP1 functional domains, SUMO modifications and known amino acid substitutions that alter DNA/RNA binding. (B) Western blot of RAD51AP1 knockdown and complementation with wildtype and mutant FLAG-tagged RAD51AP1 proteins. (C) Representative images of TERRA (RNA-FISH) at telomeres (TRF2) after knockdown of RAD51AP1 and complementation with FLAG-tagged RAD51AP1 mutants in U2OS cells synchronized in G2-phase. (D) Representative image of Ub-H2A at telomeres (TRF2) after knockdown of RAD51AP1 and complementation with FLAG-tagged RAD51AP1 mutants in U2OS cells synchronized in G2. (E) Quantification of full-length (FL) and mutant FLAG-tagged RAD51AP1 localization at telomeres in U2OS cells depleted of endogenous RAD51AP1. (F) Quantification of TERRA localization and (G) Ub-H2A accumulation at telomeres. Box and whiskers plots in F and G show the interquartile and min-max ranges. The median is represented by the horizontal black line. Data from triplicate independent experiments are shown. (H) Western blot of U2OS cells stably expressing myc-tagged RAD51AP1. (I) Quantification of TERRA recovered from U2OS cells expressing myc-tagged wildtype, K269R and ΔSIM RAD51AP1. Data represent mean ± SEM, n=3 biological replicates. p values are indicated and generated by One way ANOVA. All scale bars, 5μm.

Figure 6.. Proposed model of RAD51AP1 dependent TERRA regulation during ALT-HDR.

Top. TERRA (in yellow) transcription destabilizes telomeric chromatin, causing telomere replicative stress and DNA breaks. Middle. RNAPII and TERRA transcription are paused at the telomere DSB (t-DSB) by chromatin modulation involving BRG1, PDS5a and ZMYND8, and ubiquitination of chromatin (yellow circle; Ub). In addition, by sliding nucleosomes aside, chromatin remodelers like BRG1 make the break site accessible for repair. RAD51-RAD51AP1 binding to TERRA promotes the capture of homologous telomeric DNA sequences and strand invasion. Bottom. Together, TERRA and RAD51AP1 promote D-loop formation by RAD51. It is possible that TERRA located proximal to or annealed with the displaced strand of the D-loop allows RAD51AP1 binding to the D-loop. This D/R loop structure might then be stabilized by SUMO-SIM mediated interactions between RAD51AP1 and the UAF1 complex that further reinforces TERRA suppression by maintaining repressive chromatin at the t-DSB and possibly shielding these DNA: RNA hybrids from their premature resolution by factors that include SETX. Once telomere DNA synthesis and extension (blue dashed arrow) has successfully initiated and completed, these structures can be dismantled and RNAPII mediated transcription across telomeres reinstated.