HIRA protects telomeres against R-loop-induced instability in ALT cancer cells

Abstract

Inactivating mutations in chromatin modifiers, like the α-thalassemia/mental retardation, X-linked (ATRX)-death domain-associated protein (DAXX) chromatin remodeling/histone H3.3 deposition complex, drive the cancer-specific alternative lengthening of telomeres (ALT) pathway. Prior studies revealed that HIRA, another histone H3.3 chaperone, compensates for ATRX-DAXX loss at telomeres to sustain ALT cancer cell survival. How HIRA rescues telomeres from the consequences of ATRX-DAXX deficiency remains unclear. Here, using an assay for transposase-accessible chromatin using sequencing (ATAC-seq) and cleavage under targets and release using nuclease (CUT&RUN), we establish that HIRA-mediated deposition of new H3.3 maintains telomeric chromatin accessibility to prevent the detrimental accumulation of nucleosome-free single-stranded DNA (ssDNA) in ATRX-DAXX-deficient ALT cells. We show that the HIRA-UBN1/UBN2 complex deposits new H3.3 to prevent TERRA R-loop buildup and transcription-replication conflicts (TRCs) at telomeres. Furthermore, HIRA-mediated H3.3 incorporation into telomeric chromatin links productive ALT to the phosphorylation of serine 31, an H3.3-specific amino acid, by Chk1. Therefore, we identify a critical role for HIRA-mediated H3.3 deposition that ensures the survival of ATRX-DAXX-deficient ALT cancer cells.

Keywords: ALT; CP: Cancer; CP: Molecular biology; HIRA; R-loop; cancer; histone; telomere.

Figure 1.. HIRA maintains chromatin accessibility in ATRX-deficient ALT cells

(A) Schematic of the ATAC-seq methodology. Tn5 transposase is active at accessible chromatin regions, enabling the isolation and detection of DNA sequences at open chromatin through next-generation sequencing (NGS) and downstream analysis. The graph shows the normalized count of total telomeric motifs (TTAGGG) that contain ≥3 tandem telomeric repeats in CTRL and IMR90 ATRX-KO ALT cells expressed as means ± standard error of the mean (SEM) from three biological replicates (n = 3). p values were calculated using a paired t test (one tailed). (B) Schematic of the CUT&RUN methodology. Protein A/G-MNase is recruited to the H3.3-bound primary antibody and cleaves around the binding site upon activation with Ca²⁺, followed by downstream solubilization and sequencing of isolated DNA fragments underlying H3.3. The graph shows normalized telomere repeat counts from H3.3-associated reads in CTRL and IMR90 ATRX-KO ALT cells expressed as means ± SEM from three biological replicates (n = 3). p values were calculated using a paired t test (one tailed). (C) Left: representative images of ssTelo detected by non-denaturing (native) FISH after siRNA knockdown of HIRA or H3.3 in IMR90 ATRX-KO ALT cells. Right: the graph displays the number of ssTelo signals detected per cell in each condition. Individual data points of ≥600 cells and the mean ± SEM derived from three biological replicates (n = 3) are shown. (D) Left: telomeric western dot blot of ssDNA, double-stranded DNA (dsDNA), and Southern blot detection of telomeric and Alu repeat DNA in CTRL and IMR90 ATRX-KO ALT cells following knockdown of HIRA. Right: the fold change and SEM of detected telomeric ssDNA calculated relative to telomeric dsDNA derived from three biological replicates (n = 3). (E) Representative images of TERRA RNA-FISH and TRF2 IF in IMR90 ATRX-KO ALT cells following HIRA and H3.3 knockdown. The graph displays the percentage of TERRA-positive telomeres as the percentage of co-localization of TERRA and TRF2. Individual data points of ≥200 cells and the mean ± SEM derived from three biological replicates (n = 3) are shown. (F) Top: relative levels of R-loops at telomeres in CTRL and IMR90 ATRX-KO ALT cells after knockdown of HIRA or H3.3. Bottom: relative levels of R-loops at telomeres of samples pre-treated with RNaseH1 before pull-down. All data in (C)–(E) represent the mean ± SEM from three biological replicates (n = 3) except the data in (F), which represent the mean ± SEM from four biological replicates (n = 4). p values in (C) and (E) were calculated using a one-way ANOVA, in (D) by unpaired t test, and in (F) by a two-way ANOVA. Scale bar: 5 μM. See also Figure S1.

Figure 2.. HIRA-mediated H3.3 deposition suppresses TERRA R-loops and transcription-replication conflicts

(A) Western blot analysis of ATRX protein expression with doxycycline for 10 days in iATRX U2OS cells expressing wild-type (WT) or the catalytically inactive D450A (DA) TRF1-FokI after siRNA knockdown of HIRA or histone H3.3. γTUB is the loading control. (B) Relative levels of telomeric R-loops after ATRX re-expression in iATRX U2OS cells expressing WT or DA TRF1-FokI after siRNA knockdown of HIRA or H3.3. Data represent mean ± SEM from four biological replicates (n = 4). p values were calculated using a two-way ANOVA. (C) Top: western blot analysis of the re-expression of ATRX protein with doxycycline for 10 days in iATRX U2OS cells after siRNA knockdown of FANCM. γTUB is the loading control. Bottom: relative levels of R-loop signals at telomeres after FANCM depletion with or without ATRX re-expression in iATRX U2OS cells. All data represent mean ± SEM from three biological replicates (n = 3). p values were calculated by a two-way ANOVA. (D) Left: representative images of SNAP-H3.3 at telomere DNA breaks in control and HIRA or FANCM-depleted U2OS cells expressing WT or inactive (DA) TRF1-FokI. (E) The graph displays the percentage of SNAP-histone H3.3-positive TRF1-FokI telomeres. Individual data points of ≥200 cells and the mean ± SEM derived from three biological replicates (n = 3) are shown. p values were calculated using a one-way ANOVA. Scale bar: 5 μM. (F) Representative images of PCNA and RNAPII proximity ligation assay (PLA) foci at TRF1-FokI telomere DNA breaks after HIRA or histone H3.3 knockdown in iATRX U2OS cells with (+dox) and without (—dox) ATRX expression. Arrows indicate co-localized PLA and TRF1-FokI foci. (G) The graph displays the percentage of PCNA-RNAPII PLA-positive TRF1-FokI telomeres in iATRX cells after siRNA knockdown of HIRA or H3.3. Individual data points of ≥200 cells and the mean ± SEM derived from three biological replicates (n = 3) are shown. p values were calculated using a two-way ANOVA. Scale bar: 5 μM. See also Figure S2.

Figure 3.. Deposition of new histone H3.3 by HIRA-UBN is required for productive ALT-HDR

(A) Cartoon of HIRA-mediated recycling of old H3.3 via HIRA-ASF1a and the deposition of newly synthesized histone H3.3 via HIRA-UBN. (B) Western blot analysis of HIRA, UBN1, H3.3, and ASF1a in U2OS cells after siRNA knockdown of the indicated corresponding proteins. γTUB is the loading control. (C) Representative images of SNAP-H3.3 at telomere DNA breaks in control and HIRA-, UBN-, and ASF1a-depleted U2OS cells expressing WT or inactive (DA) TRF1-FokI. (D) The graph displays the percentage of SNAP-H3.3-positive TRF1-FokI telomeres in each condition. Individual data points of ≥200 cells and the mean ± SEM derived from three biological replicates (n = 3) are shown. p values were calculated using a one-way ANOVA. Scale bar: 5 μM. (E) Left: representative images of ssTelo detected by non-denaturing (native) FISH after siRNA knockdown of HIRA, histone H3.3, UBN, and ASF1a in U2OS cells. Right: the graph displays the number of ssTelo signals detected per cell in each condition. Individual data points of ≥1,200 cells and the mean ± SEM derived from three biological replicates (n = 3) are shown. p values were calculated using a one-way ANOVA. Scale bar: 5 μM. (F) Western dot blot of ssDNA and Southern blot detection of telomeric DNA in U2OS cells following knockdown of HIRA, histone H3.3, UBN, and ASF1a. The graph shows the fold change and the SEM of detected ssDNA calculated relative to telomeric dsDNA derived from three biological replicates (n = 3). p values were calculated using a one-way ANOVA. (G) Relative levels of R-loop signal at telomeres after HIRA and UBN depletion in U2OS cells expressing either WT or inactive (DA) TRF1-FokI. All data represent mean ± SEM from four biological replicates (n = 4). p values were calculated using a one-way ANOVA. (H) Representative images of telomeric FISH on metaphase chromosomes from HIRA-, H3.3-, or UBN-depleted U2OS cells. Purple and orange sections display examples of telomere loss (also termed signal-free ends) and telomere fragility, respectively. (I) Graphs on the left and right display the percentages of telomere loss (signal-free ends) and fragility (multiple FISH signals per chromatid) observed per metaphase, respectively. Individual data points of ≥55 metaphases counted and the mean ± SEM derived from three biological replicates (n = 3) are shown. p values were calculated using a one-way ANOVA. Scale bar: 5 μM. (J) Representative images of focal EdU accumulation at telomere DNA breaks in control and HIRA-, histone H3.3-, and UBN-depleted U2OS cells expressing WT or inactive (DA) TRF1-FokI. White arrows indicate co-localizing signals. (K) The graph displays the percentage of EdU-positive TRF1-FokI telomeres in the indicated conditions. Individual data points of ≥150 cells and the mean ± SEM derived from three biological replicates (n = 3) are shown. p values were calculated using a one-way ANOVA. Scale bar: 5 μM. See also Figure S3.

Figure 4.. Histone H3.3 serine 31 is essential to suppress TERRA-associated telomere dysfunction

(A) Western blot analysis of chromatin-associated HIRA, H3.3, phospho-H3.3 S31 (H3.3S31ph), acetyl-H3K27 (H3K27ac), H3S10ph, and cyclin B1 after siRNA knockdown of HIRA in U2OS cells. Ponceau-stained histones are shown as loading. (B) Representative images of metaphase chromosomes stained with DAPI and anti-H3.3S31ph antibodies. (C) The graph displays the frequency of metaphase spreads displaying H3.3S31ph on chromosome arms observed in the indicated conditions. ≥500 metaphases were analyzed in each condition, and the mean ± SEM derived from three biological replicates (n = 3) is shown. p values were calculated using a Student’s t test. Scale bar: 5 μm. (D) Left: representative images of ssTelo detected by non-denaturing (native) FISH after siRNA knockdown of endogenous histone H3.3 and complementation with exogenous WT and mutant histone H3.3. Scale bar: 5 μM. (E) The graph displays the number of ssTelo signals detected per cell in each condition. Individual data points of ≥300 cells and the mean ± SEM derived from three biological replicates (n = 3) are shown. p values were calculated using a two-way ANOVA. Scale bar: 5 μm. (F) Top: representative TERRA RNA-FISH and TRF2 immunofluorescence images in DMSO- and CHK1i-treated U2OS cells. Bottom: the graph displays the percentage of TERRA-positive TRF2 foci detected per cell in each condition. Individual data points of ≥500 cells and the mean ± SEM derived from two biological replicates (n = 2) are shown. p values were calculated using an unpaired t test. Scale bar: 5 μM. (G) Top: relative levels of R-loops at telomeres in DMSO- and Chk1i-treated U2OS cells. Bottom: relative levels of R-loops at telomeres of samples pre-treated with RNaseH1 before pull-down. p values were calculated using an (top) unpaired t test and (bottom) a two-way ANOVA. (H) Summary model of the contribution of HIRA to telomeric chromatin in ATRX-DAXX-deficient ALT cells. See discussion and Figure S4.