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SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations

Nature Structural & Molecular Biology volume 27, pages 438–449 (2020)Cite this article

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A Publisher Correction to this article was published on 14 May 2020

An Author Correction to this article was published on 14 May 2020

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Abstract

The SLX4 tumor suppressor is a scaffold that plays a pivotal role in several aspects of genome protection, including homologous recombination, interstrand DNA crosslink repair and the maintenance of common fragile sites and telomeres. Here, we unravel an unexpected direct interaction between SLX4 and the DNA helicase RTEL1, which, until now, were viewed as having independent and antagonistic functions. We identify cancer and Hoyeraal–Hreidarsson syndrome-associated mutations in SLX4 and RTEL1, respectively, that abolish SLX4–RTEL1 complex formation. We show that both proteins are recruited to nascent DNA, tightly co-localize with active RNA pol II, and that SLX4, in complex with RTEL1, promotes FANCD2/RNA pol II co-localization. Importantly, disrupting the SLX4–RTEL1 interaction leads to DNA replication defects in unstressed cells, which are rescued by inhibiting transcription. Our data demonstrate that SLX4 and RTEL1 interact to prevent replication–transcription conflicts and provide evidence that this is independent of the nuclease scaffold function of SLX4.

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Fig. 1: SLX4 interacts with RTEL1.
Fig. 2: Mapping the interaction domains of SLX4 and RTEL1.
Fig. 3: SLX4 promotes replication fork progression during S phase through interaction with RTEL1.
Fig. 4: SLX4 promotes replication fork progression independently of its associated SSEs.
Fig. 5: SLX4 and RTEL1 interaction promotes FANCD2/RNA polymerase II co-localization and prevents conflicts between replication and transcription.
Fig. 6: Transcription inhibition rescues replication defects caused by loss of SLX4–RTEL1 complex formation.

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Data availability

The data supporting the findings of this study are available with the online version of the article. The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium via the PRIDE partner repository with dataset identifiers PXD012426 and PXD012425. NGS data (PERMED 1069 = isolate P1, PERMED 1115 = isolate P2) have been deposited in the Sequence Read Archive under BioProject PRJNA611917 and can be retrieved at https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA611917.

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Acknowledgements

A.T. and P.H.L.G. express their gratitude to M. Boiero Sanders and C. Machu for their help in the analysis of microscopy data. We thank M. Modesti for supplying the GFP nanobody and all members of the 3R community of CRCM for helpful discussions. We also thank S. Granjeaud for helpful discussions on statistical analyses.

Work in the laboratory of P.H.L.G. was funded by the Institut National du Cancer (INCa-PLBio2016-159), Siric-Cancéropôle PACA (AAP Projets émergents 2015). A.T. was supported by INCa-PLBio2016-159 and Z.H. was supported by INCa-PLBio2016-159 and Fondation ARC. Work in the laboratory of D.B. was funded by SIRIC (INCa-DGOS-Inserm 6038), Label Ligue (EL2016 DB), Ruban Rose and Fondation Groupe EDF. The laboratory of P.K. was supported by INCa (INCa-PLBio2016-159) and INCa-DGOS-Inserm 12551, and E.D. was supported by INCa (PLBio2016-144). V.N.’s research is supported by ERC starting grant agreement no. 638898. Work in the laboratory of R.G. was funded by FRISBI (ANR-10-INSB-05-01) and ANR CHIPSET (ANR-15-CE11-0008-01). RTEL1-related work in the laboratories of A.L.-V., P.R. and I.C. was partially supported by a joint grant from the Agence Nationale pour la Recherche (ANR-14-CE10-0006-01). The IBiSA Marseille Proteomic platform is funded by Institut Paoli-Calmettes, National Institute of Cancer and Aix-Marseille University. J.P.B. is a scholar of Institut Universitaire de France.

We wish to thank Y. Liu and I. Hickson for sharing unpublished results.

Author information

Author notes

  1. A. Takedachi

    Present address: Department of Chemistry, Faculty of Science, Fukuoka University, Fukuoka, Japan

  2. Z. Hasanova

    Present address: Institute of Molecular Genetics, Prague, Czech Republic

  3. These authors contributed equally: A. Takedachi, E. Despras.

Authors and Affiliations

  1. Centre de Recherche en Cancérologie de Marseille, CRCM, Inserm, CNRS, Aix-Marseille Université, Institut Paoli-Calmettes, Marseille, France

    A. Takedachi, S. Scaglione, J. H. Guervilly, M. Blin, S. Audebert, L. Camoin, Z. Hasanova, A. Guille, D. Churikov, M. Chaffanet, J. P. Borg, F. Bertucci, D. Birnbaum & P. H. L. Gaillard

  2. Inovarion, Paris, France

    A. Takedachi

  3. CNRS UMR9019, Université Paris-Saclay, Equipe labellisée Ligue contre le Cancer, Gustave Roussy, Villejuif, France

    E. Despras & P. L. Kannouche

  4. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette cedex, France

    R. Guérois

  5. Institut Curie, PSL Research University, CNRS, UMR3244, Paris, France

    M. Schertzer & A. Londoño-Vallejo

  6. Sorbonne Universités, UPMC Univ. Paris 06, CNRS, UMR3244, Paris, France

    M. Schertzer & A. Londoño-Vallejo

  7. Sorbonne Université, Muséum National d’Histoire Naturelle, UMR CNRS 7590, IRD, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, IMPMC, Paris, France

    I. Callebaut

  8. CNRS UMR9019, Université Paris-Saclay, Gustave Roussy, Villejuif, France

    V. Naim

  9. INSERM UMR 1163, Laboratory of Genome Dynamics in the Immune System, Equipe Labellisée La Ligue contre le Cancer, Paris Descartes-Sorbonne Paris Cité University, Imagine Institute, Paris, France

    P. Revy

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  1. A. Takedachi
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Contributions

A.T. performed experiments for proteomic analyses, biochemical analyses of the SLX4-RTEL1 interaction, colony survival assays and the analysis of FANCD2 foci formation. A.T. generated all cell lines producing YFP- or Flag-HA-tagged recombinant proteins used in the current study. E.D. performed all DNA fiber analyses and PLA experiments. S.S. generated plasmids used in Y2H and designed and performed all Y2H experiments. R.G. performed all structural analyses with the help of I.C. and helped in the design of in vitro biochemical studies. J.H.G. worked on the detection of the endogenous SLX4–RTEL1 complex, generated reagents and helped with data analysis. M.B. performed the pulldowns and characterization of Flag-HA-SLX4–SMX. S.A. and L.C. carried out all proteomic analyses, with insight and expertise from J.P.B. A.G., M.C., F.B. and D.B. ran the NGS of biological samples and bioinformatic analyses of the SLX4 patient-derived mutations. P.R. generated the fibroblast cell line from the RTEL1-deficient patient P7 and helped with the design and data interpretation of experiments with HHS-associated RTEL1 mutations. Z.H., D.C. and V.N. helped in the design of cellular studies and data analysis. M.S. and A.L.-V. generated the RTEL1 antibody and contributed to the design of experiments and data interpretation. P.H.L.G. produced recombinant proteins and performed in vitro binding assays and wrote the manuscript. S.S. and R.G. made equal contributions to the study. The manuscript was reviewed by all authors. A.T., E.D., P.L.K. and P.H.L.G. conceived and planned the study.

Corresponding author

Correspondence to P. H. L. Gaillard.

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The authors declare no competing interests.

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Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 RTEL1 is a binding partner of SLX4.

(a) Mass spectrometry analysis of proteins that co-purify with YFP-SLX4. The silver stained SDS PAGE gel provides a qualitative assessment of the various samples that were analysed by tandem mass spectrometry (See Supplementary Note for details on Mass spectrometry and data analysis methods). A summary table of all known SLX4 interactors that were identified in the YFP-SLX4 pull down is shown in Extended Data Fig. 2. The mass spectrometry data are provided in Supplementary Table 1. (b) Co-immunofluorescence of YFP-SLX4 and endogenous RTEL1. (c) Immunoblots and ponceau stained proteins of the cell lysates used for the immunoprecipitates shown in Fig. 1b. SLX4 runs just above the 250 kDa mark while RTEL1 runs between the 130 and 250 kDa mark. (d) Top: Schematic representation of the YFP-tagged SLX4 fragments used for mapping the RTEL1-binding domain in SLX4. Bottom: Co-immunoprecipitation of endogenous RTEL1 with YFP-SLX4 fragments. The fragments in red are those that define the minimal RTEL1 binding domain. (e) Comparative mass spectrometry analysis of proteins that co-purify with a YFP-SLX4577-795 fragment, either wild type or containing the D614G or L618P cancer associated mutations, overproduced in HeLa Flp-In TRex cells. The upper panel is a silver stained SDS PAGE gel providing a qualitative assessment of the various samples that were analysed by tandem mass spectrometry. Below are immunoblots with antibodies against SLX4 and RTEL1. (See Supplementary Note for details on Mass spectrometry and data analysis methods). The list of proteins that were identified in all three runs of the wild-type YFP-SLX4577-795 sample but in none of the runs of the D614G and L618P mutated samples or the HeLa “Fit0” negative control is shown in Extended Data Fig. 3 and the mass spectrometry data full report in Supplementary Table 2. Source data including uncropped images of the immunoblots in panels c–e are available with the paper online.

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Extended Data Fig. 2 List of known SLX4 interactors identified in YFP-SLX4 pull down.

List of all known SLX4 interactors (light green) and RTEL1 (dark green) that were identified in the YFP-SLX4 pull down shown in Extended Data Fig. 1a. The table shows a spectral counting based on the number of peptide-to-spectrum matching (PSM) events. (see Supplementary Note for Mass spectrometry and data analysis methods and Supplementary Table 1 for the full data report).

Extended Data Fig. 3 List of SLX4 partners impacted by the D614G and L618P SLX4 mutations.

List of all proteins identified in all three runs of the wild-type YFP-SLX4577-795 sample but in none of the runs of the D614G and L618P mutated samples or the HeLa “Fit0” (HeLa Flp-In T-REX cells with no SLX4 cDNA integrated at the FRT site) negative control (see Supplementary Table 2 for the mass spectrometry data full report). The table shows a spectral counting based on the number of peptide-to-spectrum matching (PSM) events. (see Supplementary Note for Mass spectrometry and data analysis methods and Supplementary Table 1 for the data full report).

Extended Data Fig. 4 SLX4 binds HD1 of RTEL1.

(a) Multiple sequence alignment of RTEL1 homologs focused on the region 888-1156 of human RTEL1. Top two sequences report the secondary structures (H for helix) and disorder status (D) predicted by PSIPRED and SPOTD algorithms, respectively (See Methods). Blue boxes indicate the delimitation of the canonical harmonin/PAH domains HD1 and HD2 and the red box spots out the extension required for interaction with SLX4. For species having diverged before the emergence of bony fishes, the second harmonin domain is not present. NCBI RefSeq identifiers are given within brackets. (b) E. coli produced 6His-tagged HD1a (RTEL1885-975), HD1b (RTEL1885-990) and HD2a (RTEL11046-1142) fragments were used in a Ni + +-pull down in vitro assay to monitor their interaction with a GST-tagged SLX4577-1042 (Helix+BTB) fragment. The first and last lanes represent the inputs of the Ni + +-pull down assays. B: Ni + +-beads, Ft: Flow through. The pelleted beads were resuspended in a volume of Laemmli buffer equivalent to the initial volume of the binding assay. Identical volumes of the GST-tagged SLX4577-1042 (Helix+BTB) fragment (diluted to the final concentration used in the binding assay), the B and the Ft samples were loaded on the gel. (c) Schematic representation of the RTEL1 fragment (Top) used in Y2H to assess direct binding to the SLX4577-1042 fragment. K897E: Hoyeraal-Hreidarsson syndrome (HHS) associated mutation. Bottom panel shows Y2H to assess direct binding between the RTEL1 fragments and SLX4577-1042 (Helix+BTB) fragment. (d) Schematic representation of the YFP-tagged RTEL1 fragments (Top) used in the YFP-pull down to assess binding to endogenous SLX4 (Bottom). All indicated RTEL1 point mutations are from Hoyeraal-Hreidarsson syndrome (HHS) patients31. Uncropped images of the immunoblots in panels b,d and Y2H in c are available as Source data.

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Extended Data Fig. 5 Interaction between SLX4 and RTEL1 is required for proper replication fork progression but not for ICL repair.

(a) Colony survival assay with mock-depleted (siLUC) and SLX4-depleted (siUTR) HeLa “Fit0” and HeLa Flp-In T-REX cells expressing WT or mutated Flag-HA-SLX4 as indicated treated with MMC for 24 hrs. Values represent the means and SEM from three independent experiments. The Immunoblots were carried out with antibodies against SLX4 and XPF. n/a: lanes that are not relevant to the colony survival assay. A portion of the corresponding Ponceau stained membrane is shown under the immunoblots. SLX4 runs just above the 250 kDa mark while XPF runs slightly above the 100 kDa mark. (b) Analysis of replication fork dynamics in HeLa cells depleted for SLX4 or RTEL1, as described in Fig. 3a. NT: non-targeting control siRNA. Data are shown in boxplots (median, first and third quartile) with 5th-95th percentile whiskers (+: mean, n: number of unbroken signals analysed). Statistical significance was assessed with the Mann-Whitney test (ns: not significant, ***: p < 0.001, ****: p < 0.0001). The immunoblots were performed with antibodies against SLX4, RTEL1 and ß-actin used as internal loading control. The arrow indicates the SLX4 band. (c) as in b in U2OS “Fit0” cells depleted for SLX4 or RTEL1. LUC: control siRNA. (d) Control immunoblots and the corresponding Ponceau stained membrane for Fig. 3b showing the relative levels of endogenous SLX4 (lane 1 before depletion; lanes 2 to 8 after depletion) and recombinant WT or mutated SLX4 proteins expressed in cells depleted for endogenous SLX4 (lanes 3 to 8). SLX4 runs just above the 250 kDa mark while XPF runs slightly above the 100 kDa mark. n/a: lanes that are not relevant to the data shown in Fig. 3b. (e) as in b in U2OS “Fit0” and U2OS Flp-In T-REX cells stably expressing DOX-inducible WT or mutated Flag-HA-SLX4 as indicated. siSLX4UTR was used to deplete endogenous SLX4. Uncropped images of the immunoblots in panels a-d and data for graphs in panels a,b,c,e are available as Source data.

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Extended Data Fig. 6 SLX4-RTEL1 complex formation is dispensable for their recruitment on nascent DNA.

(a) PLA between SLX4 (HA) and RTEL1 was performed in HeLa Flp-In T-REX cells expressing Flag-HA-SLX4 before HA counterstaining (in green and red, respectively). PLA spots per HA-positive cells are plotted (red bars: median with interquartile range). Parental HeLa “Fit0” cells were used as a negative control and PLA spots were counted in random nuclei for this condition (grey distribution with orange bars). Kruskal-Wallis test (n > 55, ****: p < 0.0001). Representative single cells with different HA contents are shown (scale bar: 10 μm). (b) Nascent DNA strands were pulse-labelled with 5-ethynyl-2-deoxyuridine (EdU). Biotin was conjugated to EdU by click chemistry after cell fixation. In situ proximity ligation assay (PLA) was performed between endogenous RTEL1 and EdU, using an anti-biotin antibody, before EdU counterstaining (in green and red, respectively). Reactions omitting one of the primary antibodies (Ab) were used as negative controls. The number of PLA spots per EdU-positive cells is plotted, except in the RTEL1 Ab only negative control in which PLA spots were counted in random nuclei (red or orange bars: median with interquartile range, n > 79). Statistical significance was tested with the Kruskal-Wallis test (****: p < 0.0001). Representative nuclei are shown (scale bar: 10 μm). (c) As in b in SV40-immortalised patient fibroblasts expressing WT or R957W RTEL1. The immunoblot was performed with antibodies against RTEL1 and GAPDH used as internal loading control. Uncropped images of the immunoblots in panel c and data for graphs in panels a-c are available as Source data.

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Extended Data Fig. 7 SLX4-RTEL1 interaction is needed for tight colocalization between FANCD2 and RNA Pol II.

(a) Number of SLX4 foci detected by anti-HA immunofluorescence in U2OS Flp-In T-REX cells producing the indicated Flag-HA-SLX4 proteins. (b) Representative images of the immunofluorescence data quantified in Fig. 5a and Extended Data Fig. 7a. (c) Representative fields for the PLA FANCD2/RNA pol II pS2 shown in Fig. 5c. Scale bar: 10 μm. (d) PLA between SLX4 (HA) and RNA pol II pS2 was performed in U2OS Flp-In T-REX cells expressing Flag-HA-SLX4 before HA counterstaining. Single-cell HA intensity (n > 152, left panel) and PLA spots per HA-positive cells (right panel) are plotted (n > 109, red bars: median with interquartile range). Parental U2OS “Fit0” cells were used as a negative control and PLA spots were counted in random nuclei for this condition (grey distribution with orange bars). Kruskal-Wallis test (ns: not significant, ****: p < 0.0001). (e) PLA between endogenous RTEL1 and RNA pol II pS2 was performed in SV40-immortalised patient fibroblasts expressing WT or R957W RTEL1. Reactions omitting one of the primary antibodies (Ab) were used as negative controls. Kruskal-Wallis test (ns: not significant, ****: p < 0.0001). Data for graphs in panels a,d,e are available as Source data.

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Extended Data Fig. 8 Transcription is toxic to replication in absence of SLX4-RTEL1 complex formation.

(a) Supporting data for the DNA fiber assay shown in Fig. 6a. (b) HeLa “Fit0” cells were depleted for SLX4 or RTEL1. 1 µM triptolide was added to the culture medium for 3 h before and during the IdU and CldU pulses to inhibit transcription initiation. Replication fork dynamics was analysed as in Fig. 3a. Mann-Whitney test, ns: not significant, **: p < 0.01, ****: p < 0.0001). Uncropped images of the immunoblots and data for graphs in panels a and b are available as Source data.

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Supplementary information

Supplementary Information

Supplementary Note

Reporting Summary

Supplementary Table 1

Full data report from the mass spectrometry analysis of Extended Data Fig. 1a.

Supplementary Table 2

Full data report from the mass spectrometry analysis of Extended Data Fig. 1e.

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Takedachi, A., Despras, E., Scaglione, S. et al. SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations. Nat Struct Mol Biol 27, 438–449 (2020). https://doi.org/10.1038/s41594-020-0419-3

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