Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb 1;69(3):371-384.e6.
doi: 10.1016/j.molcel.2018.01.012.

SLFN11 Blocks Stressed Replication Forks Independently of ATR

Junko Murai  1 , Sai-Wen Tang  2 , Elisabetta Leo  2 , Simone A Baechler  2 , Christophe E Redon  2 , Hongliang Zhang  2 , Muthana Al Abo  2 , Vinodh N Rajapakse  2 , Eijiro Nakamura  3 , Lisa M Miller Jenkins  4 , Mirit I Aladjem  2 , Yves Pommier  5
Affiliations

SLFN11 Blocks Stressed Replication Forks Independently of ATR

Junko Murai et al. Mol Cell. .

Abstract

SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.

Keywords: ATAC-seq; ATR; CHK1; PARP inhibitors; SLFN11; camptothecin; cell cycle checkpoints; hydroxyurea; prexasertib (LY2606368); replication origin.

PubMed Disclaimer

Conflict of interest statement

DECLARATION OF INTEREST

The authors declare no competing interests.

Figures

Figure 1
Figure 1. SLFN11 requires its ATPase activity to block replication independently of ATR and does not affect γ H2AX and ATR activation
(A–F) Functional analyses of SLFN11 in CCRF-CEM parental and SLFN11-del cells. (A) Representative flow cytometry cell cycle data in response to CPT (100 nM), ATRi (AZD6738 or VE-821; 1 μM) and roscovitine (20 μM). The percentage of highly replicating cells is annotated (red dashed boxes and numbers; average of 3 independent experiments). PI: propidium iodide. (B) γH2AX measured by immunofluorescence 4 hours after drug treatment [100 nM CPT, 1 μM ATRi (VE-821)]. Error bars represent mean ± standard error of the mean (SEM, n = 113–238). ***p ≤ 0.0001 (two-tailed unpaired t test). (C) Western blot for phospho-S345 CHK1 and total CHK1. VE: VE-821, AZD: AZD6738. (D) Viability curves for CPT alone (circles) and with ATRi (triangles) (mean ± SD, n = 3). Data are representative of three independent experiments. (E) Representative flow cytometry cell cycle data after 24 hour CPT (100 nM) treatments. (F) Representative Western blot for CHK1 activation and degradation, and cyclin A levels using whole cell extracts. (G–H) SLFN11 expression in the indicated K562 cell lines measured by Western blotting (G) and immunofluorescence without pre-extraction (H). (I) Drug resistance of the ATPase-defective SLFN11 cells (mean ± SD, n = 3). Data are representative of two independent experiments. (J) Representative cell cycle analyses after 4 hours treatments [250 nM CPT, 1 μM ATRi (VE-821)] shown as (A). (See also Figure S1)
Figure 2
Figure 2. SLFN11 is recruited to chromatin and the nuclear periphery via RPA1, where it blocks replication in response to replication stress
(A) Western blots of chromatin bound fraction (CB) and whole cell extract (WCE) in CCRF-CEM parental cells [100 nM CPT, 0.5 mM HU]. (B–C) Immunofluorescence analyses in CCRF-CEM SLFN11-del and parental cells treated as indicated [100 nM CPT, 1 μM ATRi (AZD6738)] for 4 h. (B left) Drug treatment and replication labelling scheme and representative confocal microscopy images; DNA replication foci (EdU, purple), chromatin bound SLFN11 (green) and DAPI (blue). (B right) Correlations between EdU and SLFN11 signals in individual cells for the indicated treatments. Thresholds for EdU (10) and SLFN11 (5) were set from distributions in the control experiment. The percentage of cells in each segment is annotated as bold red numbers (n = 108–110). Results are representative of two independent experiments. (C) Representative images of chromatin-bound SLFN11 (green), phospho-RPA2 (S4/S8) (red) and DAPI (blue) (left), and tracing of the distribution of signals along the white dashed arrow shown in the merged panel (right). (D) Representative confocal microscopy images for chromatin bound SLFN11 (green), phospho-RPA2 (S4/S8) (red) and DAPI (blue) in DU145 parental cells. Representative tracings are shown at right. Cells were transfected with control siRNA (siControl) or siRNA for RPA1 or CtIP (siRPA1; siCtIP) for 48 hours before 4 hour CPT treatments (100 nM). (E) Efficiency of the siRNAs determined by Western blot with whole cell extract. (F) Percentage of cells having SLFN11 signals exclusively at the nuclear periphery after CPT treatment in siControl and siCtIP treated cells (mean ± SD; n = 4; 60–117 cells with SLFN11 signal at nuclear periphery were examined in each sample). ***p ≤ 0.0001 (two-tailed unpaired t test). (See also Figure S2)
Figure 3
Figure 3. SLFN11 localizes to replication foci and binds MCM3 and DHX9 in response to CPT and the ATPase domain of SLFN11 is required to block replication but not for chromatin recruitment
(A) SLFN11 binds chromatin independently of its ATPase domain. Western blots of chromatin bound fraction (CB) and whole cell extract (WCE) (CPT 250 nM). Histone H3 and GAPDH were used as control. (B–C) Immunofluorescence of DNA replication foci (EdU) (purple) and chromatin-bound SLFN11 (green). Treatments were for 4 hours [250 nM CPT and 1 μM ATR inhibitor (ATRi) AZD6738]. EdU was added 30 min before cell collection. (B) Representative confocal microscopy images. (C) Percentage of EdU-positive cells in the SLFN11-positive cells (mean ± SD, n = 3; >30 SLFN11-positive cells were examined in each sample). (D) Binding of SLFN11 to MCM3 and DHX9. Immunoprecipitation with anti-SLFN11 antibody (E-4) was coupled with mass spectrometry (IP-MS) using nuclear fractions of DU145 parental cells (left). Confirmation of the IP-MS results using antibodies against SLFN11 (E-4), DHX9 and MCM3 (right). Ctrl: control, PMS: peptide spectrum match. Whole data are listed in Table S1 (control) and S2 (CPT-treated). (See also Figure S3, Table S1 and S2)
Figure 4
Figure 4. SLFN11 binding to chromatin does not interfere with CDC45 and PCNA loading but blocks RPA extension and replication in response to CPT
(A, C and E) Representative confocal microscopy images for replication foci (EdU), CDC45 and SLFN11 (A), PCNA and EdU (C), and RPA2 and EdU (E)]. CCRF-CEM SLFN11-del and parental cells were treated for 4 hours [100 nM CPT, 1 μM ATR inhibitor (ATRi) (AZD6738) and 20 μM roscovitine (Ros)]. (B, D and F) Quantification of A, C, and D, respectively, and correlations between EdU and the indicated chromatin-bound proteins. Data are shown as Figure 2B. n = 104–113 (B), n = 150–158 (D), n = 101–102 (F). Data are representative of two independent experiments. (See also Figures S4 and S5)
Figure 5
Figure 5. SLFN11 does not interfere with the initiation of DNA replication but opens chromatin in the vicinity of the initiation sites in response to CPT
(A) Sequencing tracks of nascent strand DNA-seq for the TOP1 and GAPDH loci in CCRF-CEM SLFN11-del (blue) and parental cells (red). Drug treatments were for 4 hours [100 nM CPT, 2 μM ATRi (VE-821)]. Arrows indicate center of peaks. gDNA: genomic DNA. (B) Bar graphs summarizing the number of high intensity replication initiation peaks determined by NS-seq. Calculations used MACS2 broad peak analysis and peaks with signal value >50 are plotted. (C) Representative sequencing tracks of ATAC-seq for the TOP1 and GAPDH loci in CCRF-CEM SLFN11-del (blue) and parental cells (red). Cells were treated with 100 nM CPT. Each height is adjusted by total read number after trimming in each sample (Table S3). Arrows indicate center of peaks. (D) Bar graphs representing signal values for all peaks of each ATAC-seq data calculated using MACS2 broad peak analysis (mean ± SEM). The number of peak in each condition is shown in parentheses. ***p ≤ 0.0001 (two-tailed unpaired t test). (E) Enrichment of the active histone mark H3K9Ac at TOP1 and GAPDH loci is similar in CCRF-CEM SLFN11-del (blue) and parental (red) cells with or without CPT treatment (100 nM, 4h) analyzed by chromatin immunoprecipitation (ChIP) assay. Values (bound/input, mean ± SD, n = 3–4) represent fold enrichments normalized to background level for anti-H3K9Ac antibody (arrow in each panel). (F) Co-localization between NS-seq peaks as regions of interest (ROI) and ATAC-seq peak regions as features in CCRF-CEM SLFN11-del (left) and parental cells (right) using the Coloweb co-localization analysis tool. Peak data were obtained using the SICER algorithm. Windows were centered on peaks from NS-seq (ROI). The percentage of ROI collocated with the feature are shown above in each panel. A ROI is counted as co-located if it has at least one feature within the window size (20 kb). (G) Sequencing tracks of ATAC-seq for representative initiation loci (TOP1, HBB, JUNB and CTCF) in K562 cells. The indicated cells were treated as indicated (CPT 1 μM) for 4 hours. Each height is adjusted by total read number after trimming of each sample (Table S3). (See also Figures S6)
Figure 6
Figure 6. SLFN11 binds stressed replication forks in response to CHK1 inhibition and blocks replication and RPA extension
(A–F) Immunofluorescence analysis for DNA replication (EdU) (purple), chromatin-bound SLFN11 (green), CDC45 (red) and DAPI (blue). CCRF-CEM parental and SLFN11-del cells were treated for 4 hours with CHK1 inhibitor (CHK1i, LY2606368, 10 nM]. EdU was added 30 min before cell collection. (A) Representative confocal microscopy images. (B–D) Distribution of mean signal intensity of individual nuclei for SLFN11 (B), CDC45 (C), and EdU (D). Mean ± SEM. (E) Correlation between CDC45 and SLFN11 signals (R: Pearson correlation coefficient; red line: linear regression curve). (F) Relationship between EdU and SLFN11 signals [n = 109–114 (B–F)]. Data are representative of two independent experiments. ***p ≤ 0.0001, **p ≤ 0.001 (two-tailed unpaired t test). (G–I) Immunofluorescence analyses for chromatin bound RPA2 (green), CDC45 (red) and DAPI (blue). CCRF-CEM parental and SLFN11-del cells were treated as in panels (A–F). (G) Representative confocal microscopy images. (H) Distribution of mean signal intensity of individual nuclei for RPA2 (mean ± SEM; n = 116–202). (I) Co-localization of CDC45 with RPA2. Calculations were derived from 5–7 images (130 μm × 130 μm) containing ~50 cells/image (mean ± SD). ***p ≤ 0.0001 (two-tailed unpaired t test). (J) Western blot analysis with the indicated antibodies for input and captured proteins isolated by iPOND. The indicated cells were treated with CHK1i (100 nM) for 2 hours, and pulse-labeled with EdU for 10 min just before collecting cells (scheme on the left). (See also Figures S7)
Figure 7
Figure 7. Molecular model of SLFN11-induced replication fork block in response to replication stress
(A) Replication without replicative stress. (B) Replication stress induced by CPT in SLFN11-positive cells. (C) Unscheduled origin firing induced by CPT+ATR inhibitor or by CHK1 inhibitor in SLFN11-positive (top) and SLFN11-negative cells (bottom) (see Discussion for details).
See this image and copyright information in PMC

Comment in

References

    1. Aladjem MI, Redon CE. Order from clutter: selective interactions at mammalian replication origins. Nat Rev Genet 2016 - PMC - PubMed
    1. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, Wilson CJ, Lehar J, Kryukov GV, Sonkin D, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012;483:603–607. - PMC - PubMed
    1. Branzei D, Foiani M. Regulation of DNA repair throughout the cell cycle. Nature reviews Molecular cell biology. 2008;9:297–308. - PubMed
    1. Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods. 2013;10:1213–1218. - PMC - PubMed
    1. Feijoo C, Hall-Jackson C, Wu R, Jenkins D, Leitch J, Gilbert DM, Smythe C. Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing. J Cell Biol. 2001;154:913–923. - PMC - PubMed

Publication types