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NuRD–ZNF827 recruitment to telomeres creates a molecular scaffold for homologous recombination

Nature Structural & Molecular Biology volume 21, pages 760–770 (2014)Cite this article

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Abstract

Alternative lengthening of telomeres (ALT) is a homologous recombination (HR)-dependent mechanism for de novo synthesis of telomeric DNA in mammalian cells. Nuclear receptors are bound to the telomeres of cells that use ALT. Here we demonstrate that nuclear receptors recruit ZNF827, a zinc-finger protein of unknown function, which recruits the nucleosome remodeling and histone deacetylation (NuRD) complex via binding to an N-terminal RRK motif within ZNF827. This results in decreased shelterin binding, hypoacetylation of telomeric chromatin, enhanced telomere-telomere interactions and recruitment of HR proteins, and it is critically important for cell viability and proliferation. We propose that NuRD–ZNF827 recruitment to human telomeres causes remodeling of telomeric chromatin and creates an environment that promotes telomere-telomere recombination and integrates and controls multiple mechanistic elements of ALT activity.

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Figure 1: Nuclear-receptor recruitment of NuRD to ALT telomeres is cell cycle regulated.
Figure 2: The N-terminal RRK motif of ZNF827 is required for nuclear receptor–dependent NuRD recruitment to ALT telomeres.
Figure 3: NuRD–ZNF827 deacetylates histones and prevents shelterin binding at ALT telomeres.
Figure 4: NuRD–ZNF827 recruitment to telomeres promotes ALT activity.
Figure 5: ALT-specific telomere-telomere interactions are caused by NuRD–ZNF827.
Figure 6: ZNF827 knockdown increases the telomeric DDR and causes senescence and apoptosis in ALT cells.
Figure 7: NuRD–ZNF827 recruitment to telomeres drives HR and protects against the DDR in ALT cells.

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Acknowledgements

This work was supported by an Australian Postgraduate Award (to D.C.), a Cancer Institute of New South Wales (NSW) Research Scholar Award (to D.C.), National Health and Medical Research Council of Australia project grant 1009231 (to R.R.R. and H.A.P.), Cancer Council NSW project grant 1069550 (to H.A.P.), Cancer Council NSW program grant PG11-08 (to R.R.R.) and a Cancer Institute NSW Fellowship (to H.A.P.).

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Authors and Affiliations

  1. Telomere Length Regulation Group, Children's Medical Research Institute, Westmead, New South Wales, Australia

    Dimitri Conomos & Hilda A Pickett

  2. Sydney Medical School, University of Sydney, New South Wales, Australia

    Dimitri Conomos, Roger R Reddel & Hilda A Pickett

  3. Cancer Research Unit, Children's Medical Research Institute, Westmead, New South Wales, Australia

    Roger R Reddel

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  1. Dimitri Conomos
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Contributions

All authors conceived and designed the project. Experiments were conducted by D.C. and H.A.P. All authors analyzed data and authored the manuscript. We thank J. Mackay (University of Sydney) for advice and helpful discussion.

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Correspondence to Roger R Reddel or Hilda A Pickett.

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Integrated supplementary information

Supplementary Figure 1 Cell cycle regulation of NuRD recruitment by nuclear receptors.

(a) Western blot analysis of panel of mortal, telomerase-positive and ALT cell lines showing global expression levels of nuclear receptors and NuRD components.

(b) Telomere-ChIP against nuclear receptors in synchronized WI38-VA13/2RA cells. Quantitation of the percentage of telomeric DNA pulled down is presented in the accompanying graph (mean ± range; n = 2 biological replicates).

(c) Western blot analysis of synchronized WI38-VA13/2RA cells showing global expression levels of nuclear receptors and NuRD components.

(d) Telomere-ChIP against nuclear receptors in HT1080 hTR and HT1080 hTR TCA cells. Quantitation is presented in the accompanying graph (mean ± range; n = 2 biological replicates; *P<0.05 by two tailed t test).

(e) Western blot analysis of HT1080 hTR and HT1080 hTR TCA cells showing global expression levels of nuclear receptors as well as NuRD components.

Supplementary Figure 2 ZNF827 binding to telomeres is cell cycle regulated and ALT specific.

(a) Telomere-ChIP against ZNF827 in the HT1080 hTR and HT1080 hTR TCA cell lines. Quantitation of the percentage of telomeric DNA pulled down is presented in the accompanying graph (mean ± range; n = 2 biological replicates; *P<0.05 by two tailed t test).

(b) Reverse Co-IP of NuRD and shelterin components from WI38-VA13/2RA cells overexpressing ZNF827 followed by western blot analysis to detect ZNF827.

(c) Indirect immunofluorescence against the NuRD component RBAP46 (red) and Myc-tagged ZNF827 (purple) coupled with telomere-FISH (green) and DAPI (blue) in WI38-VA13/2RA nuclei 72 h following knockdown of both TR4 and COUP-TF2. Bar represents 10 μm.

(d) Real-time RT-PCR showing ZNF827 transcript levels in WI38-VA13/2RA cells 72 h post-transfection with siRNA against different nuclear receptors (mean ± 1 s.d.; n = 3 technical replicates; **P<0.005 by two tailed t test).

(e) Real-time RT-PCR showing ZNF827 transcript levels in WI38-VA13/2RA cells 48 h after overexpression of different nuclear receptors (mean ± 1 s.d.; n = 3 technical replicates; **P<0.005 by two tailed t test).

(f) Real-time RT-PCR showing ZNF827 transcript levels in a panel of mortal, telomerase-positive and ALT cell lines (mean ± 1 s.d.; n = 3 technical replicates).

(g) Telomere-ChIP against ZNF827 in synchronized WI38-VA13/2RA cells. Quantitation is presented in the accompanying graph (mean ± range; n = 2 biological replicates).

(h) Real-time RT-PCR showing ZNF827 transcript levels in synchronized WI38-VA13/2RA cells (mean ± 1 s.d.; n = 3 technical replicates).

(i) Real-time RT-PCR showing ZNF827 transcript levels in HT1080 hTR and HT1080 hTR TCA cells (mean ± 1 s.d.; n = 3 technical replicates; ***P<0.0001 by two tailed t test).

Supplementary Figure 3 NuRD–ZNF827 remodels ALT telomeric chromatin.

(a) Telomere-ChIP against histones and histone marks in WI38-VA13/2RA cells 72 h after simultaneous siRNA-mediated knockdown of TR4 and COUP-TF2. Quantitation of the percentage of telomeric DNA pulled down normalized to the appropriate histone (either H3 or H4) is presented in the two accompanying graphs (mean ± range; n = 2 biological replicates; *P<0.05 by two tailed t test).

(b) Telomere-ChIP against histones and histone marks in a panel of mortal, telomerase-positive and ALT cell lines. Quantitation normalized to the appropriate histone is presented in the two accompanying graphs (mean ± range; n = 2 biological replicates).

Supplementary Figure 4 ALT phenotypic characteristics are affected by ZNF827 expression.

(a) Real-time RT-PCR confirming knockdown of ZNF827 transcript levels 72 h post-transfection with siRNA in WI38-VA13/2RA and IIICF/c cells (mean ± 1 s.d.; n = 3 technical replicates; **P<0.005 and ***P<0.0001 by two tailed t test).

(b) Western blot analysis of WI38-VA13/2RA and IIICF/c cells 48 h after overexpression of ZNF827.

(c) Western blot analysis of HT1080 hTR cells 48 h after overexpression of ZNF827.

(d) CO-FISH of metaphases from HT1080 hTR cells 48 h post-ZNF827 overexpression. Leading (red; Texas Red-conjugated (TTAGGG)3) and lagging (green; Alexa Fluor 488-conjugated (CCCTAA)3) telomere-FISH coupled with DAPI (blue) counterstaining. Bar represents 10 μm.

(e) Quantitation of the percentage of chromosome ends with two colors on only one sister chromatid observed 48 h after ZNF827 overexpression in WI38-VA13/2RA cells via CO-FISH (mean ± 1 s.d.; n = 3 biological replicates, quantifying >2,000 chromosome ends per replicate; *P<0.05 by two tailed t test).

(f) Quantitation of the percentage of fragile telomeres observed 48 h after ZNF827 overexpression in WI38-VA13/2RA cells via CO-FISH (mean ± 1 s.d.; n = 3 biological replicates, quantifying >2,000 chromosome ends per replicate; *P<0.05 by two tailed t test).

(g) Quantitation of the percentage of chromosome ends associating with ECTR DNA observed 48 h after ZNF827 overexpression in WI38-VA13/2RA cells via CO-FISH (mean ± 1 s.d.; n = 3 biological replicates, quantifying >2,000 chromosome ends per replicate; *P<0.05 by two tailed t test).

Supplementary Figure 5 ALT-specific telomere bridges caused by the NuRD–ZNF827 complex lead to prolonged prometaphase.

(a) Quantitation of the percentage of (pro)metaphases with telomere bridges in the HT1080 hTR and HT1080 hTR TCA cell lines (mean ± 1 s.d.; n = 3 biological replicates, quantifying ~50 (pro)metaphases per replicate; ***P<0.0001 by two tailed t test).

(b) Quantitation of the percentage of WI38-VA13/2RA cells with telomere bridges 48 h after ZNF827 overexpression and 24 h after treatment with various HDAC inhibitors (mean ± 1 s.d.; n = 3 biological replicates, quantifying ~50 cells per replicate; *P<0.05 by two tailed t test).

(c) Telomere-ChIP against NuRD components in WI38-VA13/2RA cells 24 h after TSA treatment. Quantitation of the percentage of telomeric DNA pulled down is presented in the accompanying graph (mean ± range; n = 2 biological replicates; *P<0.05 by two tailed t test).

(d) Quantitation of the percentage of WI38-VA13/2RA cells with telomere bridges with or without the use of standard cytogenetic methods (mean ± 1 s.d.; n = 3 biological replicates, quantifying ~50 cells per replicate; ***P<0.0001 by two tailed t test).

(e) Quantitation of the percentage of cells with telomere bridges 48 h after ZNF827 overexpression in synchronized WI38-VA13/2RA cells (mean ± 1 s.d.; n = 3 biological replicates, quantifying ~50 cells per replicate; *P<0.05 and **P<0.005 by two tailed t test).

(f) Western blot analysis of WI38-VA13/2RA cells following siRNA-mediated knockdown (72 h) and overexpression (48 h) of ZNF827 showing the global expression of cell cycle markers.

(g) Quantitation of the percentage of HT1080 hTR, WI38-VA13/2RA and IIICF/c cells in each stage of the cell cycle following siRNA-mediated knockdown (72 h) and overexpression (48 h) of ZNF827 (mean ± 1 s.d.; n = 3 biological replicates).

Supplementary Figure 6 Validation of ZNF827 knockdown in cell-line panel.

Real-time RT-PCR confirming the knockdown of ZNF827 transcript levels 72 h post-transfection with siRNA in the entire panel of cell lines shown in Figure 6a (mean ± 1 s.d.; n = 3 technical replicates; *P<0.05 by two tailed t test).

Supplementary Figure 7 Original western blots for main figures.

(a) Complete western blots of nuclear receptors and NuRD components in WI38-VA13/2RA cells after nuclear receptor knockdown shown in Figure 1d. The molecular weight standards for all blots are shown on the left.

(b) Complete western blot of HT1080 hTR and WI38-VA13/2RA cells after ZNF827 overexpression shown in Figure 2b. The molecular weight standards for all blots are shown on the left.

(c) Complete western blots of NuRD and shelterin components for Co-IP of ZNF827 from WI38-VA13/2RA cells overexpressing ZNF827 shown in Figure 2g. The molecular weight standards for all blots are shown on the left.

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Supplementary Table 1

List of primary antibodies used in this study (XLSX 15 kb)

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Conomos, D., Reddel, R. & Pickett, H. NuRD–ZNF827 recruitment to telomeres creates a molecular scaffold for homologous recombination. Nat Struct Mol Biol 21, 760–770 (2014). https://doi.org/10.1038/nsmb.2877

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