TPR is required for cytoplasmic chromatin fragment formation during senescence

  1. Bethany M Bartlett
  2. Yatendra Kumar
  3. Shelagh Boyle
  4. Tamoghna Chowdhury
  5. Andrea Quintanilla
  6. Charlene Boumendil
  7. Juan Carlos Acosta  Is a corresponding author
  8. Wendy A Bickmore  Is a corresponding author
  1. MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, United Kingdom
  2. Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, United Kingdom
  3. Institute of Biomedicine and Biotechnology of Cantabria (CSIC-Universidad de Cantabria), Spain
  4. Institute of Human Genetics, UMR9002, CNRS – Université de Montpellier, France
7 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
Senescence-specific accessible chromatin sites dependent on TPR are near senescence-associated secretory phenotype (SASP) genes and are enriched in binding sites for SASP-related transcription factors.

(A) Model of the nuclear pore showing the location of TPR in the nuclear basket and heterochromatin exclusion at the pore. (B) Schematic of experimental protocol for senescence induction in IMR90 …

Figure 1—figure supplement 1
TPR-dependent senescence-specific accessible chromatin peaks are enriched in H3K27ac and associated with genes relevant to inflammation.

Related to Figure 1. (A) Volcano plot of differential accessibility analysis of day 8 (d8) ATAC-seq peaks in RAS siCTRL vs STOP siCTRL. The horizontal dashed line indicates an adjusted p-value (FDR) …

Figure 2 with 1 supplement
Prolonged loss of TPR during senescence blocks NF-κB activation.

(A) TPR and NF-κB immunostaining in control (STOP) and oncogene-induced senescence (OIS) (RAS) cells after 4-hydroxytamoxifen (4-OHT) and siRNA (control and TPR) treatment for 8 days. Scale bar: 10 …

Figure 2—source data 1

Quantification of NF-κB nucleocytoplasmic ratios and statistical analysis for data in Figure 2B and F, and for biological replicates in Figure 2—figure supplement 1A and D.

Median NF-κB nucleocytoplasmic ratios (n/c) and number of cells analysed for day 8 (d8) STOP or RAS cells subject to knockdown with control (CTRL) or TPR siRNAs, and for experiments where these cells were treated with conditioned media (CM) from either STOP or RAS cells. Kruskal-Wallis testing was used to determine statistical significance for each replicate (p-value in parentheses) followed by Dunn’s post hoc testing. p-values after Benjamini and Hochberg correction.

https://cdn.elifesciences.org/articles/101702/elife-101702-fig2-data1-v3.docx
Figure 2—source data 2

Uncropped and labelled gels for Figure 2.

https://cdn.elifesciences.org/articles/101702/elife-101702-fig2-data2-v3.pdf
Figure 2—source data 3

Raw unedited gels for Figure 2.

https://cdn.elifesciences.org/articles/101702/elife-101702-fig2-data3-v3.zip
Figure 2—figure supplement 1
TPR depletion blocks NF-κB activation during senescence.

Related to Figure 2. (A) Quantification of NF-κB nucleocytoplasmic ratios by immunofluorescence in STOP and RAS cells after 4-hydroxytamoxifen (4-OHT) and siRNA treatment for 8 days. Kruskal-Wallis …

Figure 3 with 2 supplements
Decreased NF-κB activation upon TPR knockdown precedes the senescence-associated secretory phenotype (SASP).

(A) Schematic showing positive feedback loop in SASP signalling. Secreted IL-1α and IL-1β bind IL-1R1 at the cell membrane, leading to increased NF-κB activation and increased IL-1α and IL-1β …

Figure 3—source data 1

Quantification of NF-κB nucleocytoplasmic ratios, nuclear intensity, and statistical analysis for data in Figure 3C and D and for biological replicates in Figure 3—figure supplement 1A and B.

Median NF-κB nucleocytoplasmic ratios (n/c) and number of cells analysed for day 3 (d3) and d5 STOP or RAS cells subject to knockdown with control (CTRL) or TPR siRNAs. Kruskal-Wallis testing was used to determine statistical significance (p-value in parentheses) followed by Dunn’s post hoc testing. p-Values after Benjamini and Hochberg correction. NA: Kruskal-Wallis test showed no significant differences so it is not appropriate to carry out pairwise testing.

https://cdn.elifesciences.org/articles/101702/elife-101702-fig3-data1-v3.docx
Figure 3—source data 2

Uncropped and labelled gels for Figure 3.

https://cdn.elifesciences.org/articles/101702/elife-101702-fig3-data2-v3.zip
Figure 3—source data 3

Raw unedited gels for Figure 3.

https://cdn.elifesciences.org/articles/101702/elife-101702-fig3-data3-v3.zip
Figure 3—figure supplement 1
Decreased NF-κB activation upon TPR knockdown at days 3 and 5.

Related to Figure 3. (A and B) Quantification of (A) nucleocytoplasmic ratios of NF-κB or (B) nuclear NF-κB intensity from a biological replicate of the experiment shown in Figure 3B–D. (n) …

Figure 3—figure supplement 2
TPR knockdown does not affect chromatin accessibility at day 3 (d3).

(A) Heatmap showing ATAC-seq signal in control (STOP) and oncogene-induced senescence (OIS) (RAS) cells 3 days after 4-hydroxytamoxifen (4-OHT) treatment and transfected with either CTRL or TPR …

Figure 4 with 1 supplement
Decreased STING expression and TBK1 activation upon TPR knockdown during early stages of oncogene-induced senescence (OIS).

(A) Volcano plot of differential expression analysis of RNA isolated from RAS cells at day 3 (d3) of OIS and treated with siTPR vs siCTRL. Genes showing a significant change in expression in RAS, …

Figure 4—source data 1

Statistical analysis for STING1 qPCR data in Figure 4B and for cGAMP ELISA data in Figure 4D.

One-way ANOVA was used to determine statistical significance followed by Šídák’s multiple comparisons test.

https://cdn.elifesciences.org/articles/101702/elife-101702-fig4-data1-v3.docx
Figure 4—source data 2

Uncropped and labelled gels for Figure 4.

https://cdn.elifesciences.org/articles/101702/elife-101702-fig4-data2-v3.zip
Figure 4—figure supplement 1
Decreased abundance of mRNAs for intronless genes and for STING1 in RAS cells upon TPR knockdown at day 3 (d3).

Related to Figure 4. (A) Volcano plots of differential expression analysis of d3 STOP (top) and RAS (bottom) cells treated with TPR vs CTRL siRNAs with intronless genes coloured pink. Horizontal …

TPR and HMGA1 are required for the induction of cytoplasmic chromatin fragments (CCFs) during the early phase of oncogene-induced senescence (OIS).

(A) Mean percentage of cells containing CCFs in STOP and RAS cells at day 3 (d3) or d5 of OIS and treated with either control (siCTRL) or TPR siRNAs. Data points are for three biological replicates. …

Figure 5—source data 1

Statistical analysis for cytoplasmic chromatin fragments (CCF) and senescence-associated heterochromatic foci (SAHF) data in Figure 5A, G, and H.

Data were fitted to a generalised linear model before carrying out pairwise comparisons between samples. 500 cells were assessed per sample for each replicate of each experiment.

https://cdn.elifesciences.org/articles/101702/elife-101702-fig5-data1-v3.docx
Author response image 1
Author response image 2

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens)IMR90 STOP cellsAcosta et al., 2013Generated in the J-C Acosta lab
Cell line (Homo sapiens)IMR90 RAS cellsAcosta et al., 2013Generated in the J-C Acosta lab
Antibodyanti-β-actin−HRP (mouse monoclonal)Sigma-AldrichA3854WB (1:80000)
Antibodyanti-GAPDH (mouse monoclonal)Abcamab125247, RRID:AB 11129118WB (1:5000)
Antibodyanti-phospho-Histone H2AX
(Ser139) (mouse monoclonal)
Merck05–636IF (1:1000)
Antibodyanti-H3K27me2/me3 (mouse monoclonal)Active Motif#39536 RRID:AB_2793247IF (1:1000)
Antibodyanti-H3K9me3 (rabbit polyclonal)Abcamab8898 RRID:AB_306848IF (1:2000)
Antibodyanti-IKKα (rabbit polyclonal)Cell Signaling Technology#2682 RRID:AB_331626WB (1:1000)
Antibodyanti phospho-IKKα/β
(Ser176/180) (rabbit monoclonal)
Cell Signaling Technology#2697 RRID:AB_2079382WB (1:1000)
Antibodyanti-NF-κB p65 (mouse monoclonal)Santa Cruzsc-8008 RRID:AB_628017WB (1:1000), IF (1:100)
Antibodyanti-NF-κB p65 (rabbit
recombinant monoclonal)
Cell Signaling Technology#8242 RRID:AB_10859369IF (1:500)
Antibodyanti-phospho- NF-κB p65
(Ser536) (rabbit recombinant
monoclonal)
Cell Signaling Technology#3033 RRID:AB_331284WB (1:500)
Antibodyanti-POM121 (rabbit polyclonal)GenetexGTX102128 RRID:AB_10732546IF (1:500)
Antibodyanti-STING (rabbit monoclonal)Cell Signaling Technology#13647 RRID:AB_2732796WB (1:2000)
Antibodyanti-phosphoTBK1 (Ser172) (rabbit monoclonal)Cell Signaling Technology#5483 RRID:AB_10693472WB (1:1000)
Antibodyanti-TPR (rabbit polyclonal)Abcamab84516IF (1:500)
Antibodyanti-vinculin (rabbit polyclonal)Abcamab91459 RRID:AB_2050446WB (1:5000)
Antibodyanti-rabbit IgG (H+L) secondary,
Alexa Fluor 488 (goat polyclonal)
InvitrogenA11034IF (1:1000)
Antibodyanti-mouse IgG (H+L) secondary,
Alexa Fluor 568 (donkey polyclonal)
InvitrogenA10037IF (1:1000)
Antibodyanti-rabbit IgG, HRP-linked (goat polyclonal)Cell Signaling Technology#7074 RRID:AB_2099233WB (1:2000)
Antibodyanti-mouse IgG, HRP-linked (horse polyclonal)Cell Signaling Technology#7076 RRID:AB_330924WB (1:2000)
Sequence-based reagentsiCTRLDharmaconD-001810-10-59ON-TARGETplus siRNA pool
Sequence-based reagentsiTPRDharmaconL-010548–00ON-TARGETplus siRNA pool
Sequence-based reagentsiHMGA1DharmaconL-004597–00ON-TARGETplus siRNA pool
Sequence-based reagentSTING1_FwdDou et al., 2017RT-qPCR primerATATCTGCGGCTGATCCTGC
Sequence-based reagentSTING1_RevDou et al., 2017RT-qPCR primerTTGTAAGTTCGAATCCGGGC
Sequence-based reagentGAPDH_FwdDou et al., 2017RT-qPCR primerCAGCCTCAAGATCATCAGCA
Sequence-based reagentGAPDH_RevDou et al., 2017RT-qPCR primerTGTGGTCATGAGTCCTTCCA
Commercial assay or kitPierce BCA protein
analysis kit
Thermo Fisher23225Methods: Immunoblotting
Commercial assay or kitSuperSignal West Femto
maximum sensitivity
substrate kit
Thermo Fisher10095983Methods: Immunoblotting
Commercial assay or kit2’3’-cGAMP ELISA kitCayman Chemical501700Methods: 2’3’-cGAMP ELISA
Commercial assay or kitRNeasy mini kitQiagen74104Methods: RT-qPCR and RNA
seq­­ library preparation
Commercial assay or kitNEBNext Ultra II
Directional RNA library
prep kit
New England BiolabsE7760Methods: RNA seq­­
library preparation
Commercial assay or kitNEBNext Poly(A) mRNA
Magnetic Isolation Module
New England BiolabsE7490Methods: RNA seq­­
library preparation
Chemical compound, drug4-hydroxytamoxifenSigmaH7904
OtherH3K27ac ChIP-seqParry et al., 2018NCBI GEO: GSE103590See Figure 1—figure supplement 1
OtherATAC-seqThis paperNCBI GEO: GSE264390See Methods
OtherRNA-seqThis paperNCBI GEO: GSE264387See Methods
Software, algorithmCellProfilerStirling et al., 2021RRID:SCR_007358
Software, algorithmMicromanagerhttps://micromanager.orgVersion 1.4
Software, algorithmFastQCRRID:SCR_014583
Software, algorithmcutadaptMartin, 2011RRID:SCR_011841
Software, algorithmbowtie2Langmead and Salzberg, 2012RRID:SCR_016368
Software, algorithmMACS2Zhang et al., 2008;

https://pypi.org/project/MACS2/
Software, algorithmHOMERHeinz et al., 2010RRID:SCR_010881
Software, algorithmedgeRRobinson et al., 2010RRID:SCR_012802
Software, algorithmlimmaRitchie et al., 2015RRID:SCR_010943
Software, algorithmdeepToolsRamírez et al., 2016RRID:SCR_016366
Software, algorithmGREATMcLean et al., 2010RRID:SCR_005807
Software, algorithmHISAT2Kim et al., 2019RRID:SCR_015530
Software, algorithmGATKVan der Auwera
and O’Connor, 2020
RRID:SCR_015530
Software, algorithmsubreadLiao et al., 2014RRID:SCR_009803
Software, algorithmDeSeq2Love et al., 2014RRID:SCR_015687
Software, algorithmclusterProfilerWu et al., 2021RRID:SCR_016884
Software, algorithmggplot2Wickham, 2016RRID:SCR_014601
Software, algorithmTEtranscriptsJin et al., 2015RRID:SCR_023208

Additional files

Supplementary file 1

Supplementary file 1a, b, and c are tables summarising data in the manuscript.

(a) Table summarising day 8 ATAC-seq changes in peak accessibility. Number of ATAC-peaks with significant changes generated from comparisons between samples using the limma package with an adjusted p-value cut-off of 0.05. Peaks significantly upregulated in RAS siCTRL compared to STOP siCTRL (SEN+) were further divided into TPR-dependent (SEN+TPR+) and TPR-independent (SEN+TPR-) as shown. (b) Table indicating the proximity of senescence-associated secretory phenotype (SASP) gene promoters to TPR-dependent, senescence-dependent ATAC-seq peaks. Distance (in bp) between TPR+SEN+ ATAC-seq peaks from the transcription start site (TSS) of genes involved in positive regulation of the inflammatory response, cytokine activity, and cytokine receptors. (c) Table summarising day 3 ATAC-seq changes in peak accessibility. Number of peaks with significant changes generated from comparisons between samples using the limma package with an adjusted p-value cut-off of 0.05.

https://cdn.elifesciences.org/articles/101702/elife-101702-supp1-v3.docx
MDAR checklist
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