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. 2024 Jun 17;15(1):5151.
doi: 10.1038/s41467-024-49567-5.

Topoisomerase I is an evolutionarily conserved key regulator for satellite DNA transcription

Zhen Teng #  1 , Lu Yang #  1 , Qian Zhang #  1 , Yujue Chen  1 , Xianfeng Wang  1 , Yiran Zheng  1 , Aiguo Tian  1   2   3 , Di Tian  4 , Zhen Lin  2   4 , Wu-Min Deng  1   2 , Hong Liu  5   6   7
Affiliations

Topoisomerase I is an evolutionarily conserved key regulator for satellite DNA transcription

Zhen Teng et al. Nat Commun. .

Abstract

RNA Polymerase (RNAP) II transcription on non-coding repetitive satellite DNAs plays an important role in chromosome segregation, but a little is known about the regulation of satellite transcription. We here show that Topoisomerase I (TopI), not TopII, promotes the transcription of α-satellite DNAs, the main type of satellite DNAs on human centromeres. Mechanistically, TopI localizes to centromeres, binds RNAP II and facilitates RNAP II elongation. Interestingly, in response to DNA double-stranded breaks (DSBs), α-satellite transcription is dramatically stimulated in a DNA damage checkpoint-independent but TopI-dependent manner, and these DSB-induced α-satellite RNAs form into strong speckles in the nucleus. Remarkably, TopI-dependent satellite transcription also exists in mouse 3T3 and Drosophila S2 cells and in Drosophila larval imaginal wing discs and tumor tissues. Altogether, our findings herein reveal an evolutionally conserved mechanism with TopI as a key player for the regulation of satellite transcription at both cellular and animal levels.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. TopI is for α-satellite transcription in human cells.
a TopI inhibition decreases α-SatRNA levels. RNAs from HeLa cells treated with DMSO, CPT or TPT for 12 h were subjected for real-time PCR analysis. n = 4 biological replicates. b Effects of TopI inhibition on the transcription on different centromere 1 regions. RNAs from HeLa cells treated with DMSO or CPT (2.5 µM) and RNAs were subjected to real-time PCR analysis. n = 4 biological replicates. c TopI knockdown decreases α-SatRNA levels. HeLa cells were transfected with Luciferase or Top1 siRNAs (#1 and #2) and RNAs were extracted for real-time PCR analysis. n = 3 biological replicates. d IAA-induced TopI-mAID degradation decreases α-SatRNA levels. HeLa cells of TopI-mAID constructed by CRSPR/Cas9 were treated with IAA (1 µM) and RNAs were extracted for real-time PCR analysis. n = 3 biological replicates. e TopI inhibition globally decreases the levels of α-satellite high-order repeat (HOR) RNAs. HeLa cells treated with DMSO or CPT (2.5 µM) for 12 h and RNAs were extracted for RNA-Seq analysis. Average fold change for the transcript of each HOR upon CPT treatment is shown here. n = 2 biological replicates. f TopI inhibition does not decrease the transcription of telomeric and ribosomal repeats. RNAs from HeLa cells treated with DMSO or CPT (2.5 µM) for 12 h and were subjected to real-time PCR analysis. n = 3 biological replicates. g Comparison for the effects of TopI inhibition and RNAP II inhibition on α-satellite transcription. HeLa cells were treated with DMSO, α-amanitin (50 µg/ml) and CPT (2.5 µM) for 12 h, and RNAs were extracted for real-time PCR analysis. n = 4 biological replicates. h TopI inhibition represses transcriptional activity on α-satellite in G1 cells. Thymidine-arrested G1 HeLa cells were treated with DMSO or CPT (2.5 µM) and 5′-ethynyl uridine (EU) was added 1 h before harvest. Biological replicates (n = 3 for 1 h and n = 4 for others). All data here are presented as mean values +/− SEM. Two-sided Student’s T-test (a–d, f, h) and ANOVA followed by pairwise comparisons using Tukey’s test for (e). Quantification details for all figures are recorded in the Methods. ns, not significant (P > 0.1). Numeric values for P < 0.1 are shown. Source data are provided as Source Data file.
Fig. 2
Fig. 2. TopI binds RNAP II and promotes RNAP II elongation on α-satellite chromatin.
a TopI inhibition decreases RNAP II-pSer2 levels on α-satellite DNAs. HeLa cells treated with DMSO or CPT (2.5 µM) for 12 h were subjected to chromatin immunoprecipitation analysis with anti-RNAP II-pSer2 antibody. Biological replicates (n = 1 for beads, n = 4 for DMSO and CPT). b TopI inhibition reduces RNAP II-pSer2 levels on α-satellite DNAs in G1 cells. Stretched chromatin was prepared from thymidine-arrested HeLa cells treated with DMSO or CPT (2.5 µM) for 6 h and immunostaining was performed. n = 3 biological replicates. c Mitosis-specific inhibition of TopI reduces RNAP II-pSer2 levels on α-satellite DNAs. Mitotic HeLa cells were enriched by a brief treatment of nocodazole (5 µM). Collected mitotic cells were further treated with DMSO or CPT (2.5 µM) for 5 h and then subjected for chromosome spread followed by immunostaining. n = 3 biological replicates. d TopI physically interacts with RNAP II and phospho-RNAP II (pSer2). Lysates of HeLa cells transfected Luciferase or Top1 siRNAs (#1) were incubated with IgG or antibody against TopI. Pelleted proteins were blotted with the indicated antibodies. The physical interaction between TopI and non-phospho-RNAP II was recorded in Supplementary fig. 1e. “H” high exposure. e TopI and TopI-cleavage complex (cc) are present on the centromere in interphase. Stretched chromatin was prepared from log-phase HeLa cells and immunostaining was performed. Similar results were observed in at least two biological replicates. f TopI-cc is enriched on the centromere in mitosis. HeLa cells were briefly treated with nocodazole (5 µM) and mitotic cells were collected for chromosome spread followed by immunostaining. Similar results were observed in at least two biological replicates. Validation of TopI and Top-cc fluorescence signals were recorded in Supplementary Figs. 1e, f. All data here are presented as mean values +/− SEM. Two-sided Student’s T-test for (a–c). ns, not significant (P > 0.1). Numeric values for P < 0.1 are shown. The source data are provided as Source Data file.
Fig. 3
Fig. 3. Double-stranded breaks (DSBs) dramatically increase α-satellite transcription in a DNA damage checkpoint-independent manner in human cells.
a, b Etoposide, not ICRF, dramatically increases the levels of total and nascent α-satRNAs. Total and EU-labelled RNAs from HeLa cells treated with DMSO, etoposide (30 μM), or ICRF (20 or 40 μM), were subjected to real-time PCR analysis. Biological replicates (n = 4 for nascent in (a) and n = 3 for the others). c DSB analysis by comet assay. HeLa cells treated with DMSO, Etoposide (Etop, 30 μM), Phleomycin (Phleo, 80 μg/ml), ICRF (20 μM), MMC (5 μg/ml), Cisplatin (Cispl, 20 μg/ml), or CPT (2.5 μM) for 12 h, were analyzed with comet assay. Similar results were observed in two biological replicates. d DSB-inducing agents increase α-satellite transcription. RNAs from HeLa cells treated with DMSO, Phleo (80 μg/ml, 24 h), MMC (indicated, 12 h), or Cispl (indicated, 12 h) were analyzed with real-time PCR. Results for other primers are recorded in Figs. S6d-f. Biological replates (n = 5 for MMC and n = 3 for others). e Etoposide increases α-satellite transcription in a time-dependent manner. RNAs from HeLa cells treated with DMSO or etoposide (2.5 μM) as indicated were analyzed with real-time PCR. Results for other primers are recorded in Fig. S6g. n = 3 biological replicates. f CRISPR-Cas9 generates DSBs specifically on the centromere. Sat-gRNA plasmids were transfected into HeLa cells for 24 h and immunostaining was performed. ~16% of cells had centromeric γH2AX foci. g DSBs generated by CRISPR-Cas9 increase α-satellite transcription. RNAs in (e) were subjected to real-time PCR analysis. n = 4 biological replicates. h ATM inhibition decrease etoposide- induced γH2AX levels. HeLa cells were treated with DMSO, etoposide (30 μM), etoposide plus KU-55933 (10 μM), or etoposide plus caffeine (2 μM) for 12 h and then stained with the indicated antibodies. Similar results were observed in two biological repeats. Quantification was based on one single experiment. Each dot represents one cell. DMSO n = 32, Etop n = 18, Etop+Ku n = 21, Etop+Caff n = 27. i ATM inhibition does not affect etoposide-induced α-satellite transcription. RNAs in (h) were analyzed with real-time PCR. n = 3 biological replicates. All data here are presented as mean values +/− SEM expect for (h, +/− SD). Two-sided Student’s T-test (a, b, d, e, g) and ANOVA followed by pairwise comparisons using Tukey’s test for (h, i). ns, not significant (P > 0.1). Numeric values for P < 0.1 are shown. Source data are provided as Source Data file.
Fig. 4
Fig. 4. DSB-induced α-satellite transcription depends on TopI and induced RNAs are predominantly derived from α-satellite high-order-repeats (HORs) and spread across the nucleus.
a TopI inhibition dramatically decreases total α-SatRNA levels in etoposide-treated HeLa cells. RNAs from HeLa cells treated with DMSO, CPT (2.5 μM), Etoposide (Etop, 30 μM), or CPT plus Etoposide for 12 h were subjected to real-time PCR analysis. n = 3 biological replicates. b TopI inhibition dramatically reduces total α-SatRNA levels in etoposide-treated RPE-1 cells. RNAs from RPE-1 cells treated with DMSO, CPT (2.5 μM), Etoposide (30 μM), or Etoposide plus CPT (E + C) for 12 h were subjected to real-time PCR analysis. n = 3 biological replicates. c TopI inhibition abates the transcriptional activity in both unperturbed and etoposide-treated HeLa cells. HeLa cells were treated with DMSO, CPT (2.5 μM), Etoposide (Etop, 30 μM), or Etoposide plus CPT for 12 h and EU was added 1 h before harvest. EU-RNAs were purified for real-time PCR analysis. n = 3 biological replicates. d IAA-induced TopI-mAID degradation decreases etoposide-induced α-SatRNA levels. HeLa cells of TopI-mAID were treated with IAA (1 μM) and etoposide. RNAs were extracted for real-time PCR analysis. n = 4 biological replicates. e TopI is not required for the transcription of ribosomal and telomeric repeats in etoposide-treated cells. RNAs from HeLa cells treated with DMSO, Etoposide (Etop, 30 μM), or Etoposide plus CPT (2.5 μM) for 12 h, were subjected to real-time PCR analysis. n = 3 biological replicates. f RNA-seq analyses of α-satRNAs. RNAs from RPE-1 cells treated with DMSO, CPT (2.5 μM), Etoposide (Etop, 30 μM), or CPT plus etoposide for 12 h, were subjected to RNA-Seq analysis. g TopI inhibition globally decreases the levels of α-satellite high-order repeat (HOR) RNAs. Average fold change for HOR transcripts upon chemical treatment in (f) is shown. n = 2 biological replicates. h, i Fluorescence in situ hybridization analysis of α-satRNAs. HeLa cells were treated with DMSO, CPT (2.5 μM), Etoposide (Etop, 30 μM), or etoposide plus CPT for 12 h and then subjected to RNA-FISH analysis using probe-1 (h) and probe-2 (i). Arrows in (i) indicate the inside or outside localization of t α-satRNA foci. n = 3 biological replicates. All Data here are presented as mean values with +/− SEM. Two-sided Student’s T-test for (a–d, h, i) and ANOVA followed by pairwise comparisons using Tukey’s test for (e). ns, not significant (P > 0.1). Numeric values for P < 0.1 are shown. Source data are provided as Source Data file.
Fig. 5
Fig. 5. TopI-dependent satellite transcription is conserved across eukaryotes.
a TopI inhibition decreases satellite transcription in mouse cells. RNAs from Mouse 3T3 cells treated with CPT (2.5 μM) were analyzed with real-time PCR. n = 4 biological replicates. b TopI knockdown decreases satellite transcription in mouse cells. RNAs from mouse 3T3 cells transfected with mTopI siRNA (#1 and #2) were analyzed with real-time PCR. n = 3 biological replicates. c TopI is required for DSB-induced satellite transcription in mouse cells. RNAs from mouse 3T3 cells treated with DMSO, CPT (2.5 μM), Etoposide (Etop, 30 μM), or Etoposide plus CPT (E+C) for 12 h, were analyzed with real-time PCR. n = 3 biological replicates. d TopI inhibition decreases satellite transcription in Drosophila cells. RNAs from Drosophila S2 cells treated with DMSO or CPT (10 μM) for 6 or 12 h were analyzed with real-time. n = 4 biological replicates. e TopI is required for DSB-induced satellite transcription in Drosophila cells. RNAs extracted from Drosophila S2 cells treated with DMSO, CPT (10 μM), Etoposide (Etop, 30 μM), triptolide (Trip, 4 μM) or Etoposide plus CPT (E+C) for 12 h. n = 3 biological replicates. f TopI promotes satellite transcription in Drosophila larvae tissues. RNAs from Drosophila larvae wing imaginal discs with mock or siTopI (#3 and #5) treatment were analyzed with real-time PCR. n = 3 biological replicates. g, h Satellite transcription is gradually elevated in a TopI-dependent manner in Drosophila tumor tissues. RNAs from control, notch-driven (g) or lethal (s) giant larvae (lgl) (h) Drosophila solid-tumor tissues with mock or siTopI treatment were analyzed with real-time PCR. n = 3 technical replicates. All Data here are presented as mean values +/− SEM. Two-sided Student’s T-test for (a, b, d, f, g, h) and ANOVA followed by pairwise comparisons using Tukey’s test for (c, e). ns, not significant (P > 0.1). Numeric values for P < 0.1 are shown. Source data are provided as Source Data file.
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