Coordinated control of senescence by lncRNA and a novel T-box3 co-repressor complex
- Weis Center for Research, Geisinger Clinic, United States
- University of Utah, United States
- Huntsman Cancer Institute, University of Utah, United States
- Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, United States
Figures
Figure 1 with 1 supplement
CAPERα and TBX3 directly interact via the TBX3 repressor domain.
(A) Representative spectrum for CAPERα identified in anti-TBX3 co-IP of HEK293 cell lysates. Mass spec analysis identified six specific CAPERα peptides, providing 8.5% sequence coverage of the …
Figure 1—figure supplement 1
Missense mutation of the C-terminus of Tbx3 disrupts interaction with CAPERα.
(A) Anti-Tbx3 IB of exon 7 missense (ex7) and wt proteins expressed in HEK293 cells also transfected with anti-TBX3 shRNA. The overexpressed proteins are produced (red arrowhead). (B) anti-CAPERα …
Figure 2 with 3 supplements
Knockdown of endogenous CAPERα and TBX3 in primary human fibroblasts and mouse embryos induces premature senescence and disrupts expression of cell cycle and senescence regulators.
(A–C) Representative bright field images of senescence associated β-galactosidase (SA-βG) assays of HFFs transduced with control, TBX3 shRNA A or CAPERα shRNA A. Only occasional cells in the control …
Figure 2—figure supplement 1
Effective knockdown of endogenous CAPERα in primary human foreskin fibroblasts using viral shRNA transduction.
(A) RT-PCR analysis of CAPERα and HPRT transcript levels in HFFs transduced with two different retroviruses producing anti-CAPERα shRNAs (CAP sh A and B) and control shRNA virus (Ctl sh). Red …
Figure 2—figure supplement 2
Effective knockdown of endogenous TBX3 in primary human foreskin fibroblasts using viral shRNA transduction.
(A) RT-PCR analysis of TBX3 and HPRT transcript levels in primary human foreskin fibroblasts (HFFs) transduced with control (Ctl sh) or TBX3 (TBX3 shA) shRNA retrovirus. (B) RT-PCR analysis of TBX3 …
Figure 2—figure supplement 3
Tbx3 null murine embryonic fibroblasts (MEFS) have altered lamin β1 localization, nuclear disruption and mislocalized Caperα.
. (A–B′) Representative WT and Tbx3 null MEFs cells stained for laminβ1 at passage (P) 4 (A and B) and P1 (A′ and B′); note nuclear distortion and rupture in senescing Tbx3 null MEFs as early as P1. …
Figure 3 with 4 supplements
RB and p16 mediate senescence after CAPERα/TBX3 loss of function and CAPERα/TBX3 regulates chromatin structure of CDKN2A-p16.
(A–F) SA-βgal assays of HFFs stably transduced with control (Ctl) or p53 (Masutomi et al., 2003) or RB (Boehm et al., 2005) shRNAs subsequently transduced with CAPERα or TBX3 shRNAs. (G) % …
Figure 3—figure supplement 1
Effective knockdown of p53, RB and p16 in HFFs.
(A–C) RT-PCR analysis of p53 (A), RB (B) and p16 (C) transcript levels relative to HPRT after shRNA-mediated KD in HFFs. The shRNAs employed for these knockdowns were obtained from Addgene and have …
Figure 3—figure supplement 2
UCSC Genome Browser view of the CDKN2A locus and 5′ regions screened for binding by CAPERα and TBX3.
Seven regions tested upstream of CDKN2A-p16 promoter by ChIP with anti-TBX3 and anti-CAPERα antibodies. Amplicons are numbered black boxes 1–7 ‘Your Seq’ at top superimposed on window from UCSC …
Figure 3—figure supplement 3
CDKN2a-p16 H3K27 trimethylation markedly decreases in HFFS after knockdown of CAPERα or TBX3 consistent with activation of CDKN2a-p16 expression.
ChIP-PCR of CDKN2A-p16 regulatory elements with anti-H3K27me3 in control, TBX3 or CAPERα shRNA-transduced HFFs. Locations of amplicons relative to transcription start site are noted in parentheses …
Figure 3—figure supplement 4
Testing CAPERα and TBX3 binding to p14, p21, CDK2, CDK4, and CDKN1B regulatory elements.
(A) ChIP-PCR of CDKN2A-p14 promoter with antibodies listed at top in control (C) and TBX3 siRNA (C′) transduced HFFs. Red arrowhead indicates loss of CAPERα binding after TBX3 knockdown. (B–E) …
Figure 4 with 1 supplement
CAPERα/TBX3 directly represses expression of the long noncoding RNA UCA1.
(A–C) Gel showing RT-PCR analysis of TBX3, CAPERα, and HPRT expression in control, TBX3 and CAPERα siRNA-transfected HEK293 cells. The siRNAs effectively decreased transcript levels of their …
Figure 4—figure supplement 1
Validation of differential display findings.
(A) Additional representative differential display gels with transcripts unchanged or independently affected by knockdown of CAPERα or TBX3 in HEK293 cells. (B) RT-PCR validating differential …
Figure 5 with 2 supplements
UCA1 expression is sufficient to induce senescence and required for normal execution of oncogene-induced senescence.
(A) UCA1 and HPRT transcripts assessed by RT-PCR in control and UCA1-overexpressing HFFs. (B and C) Representative bright field images of SA-βgal assay of cultured HFFs transfected with control and U…
Figure 5—figure supplement 1
Western blots showing changes in protein levels in response to UCA1 overexpression in HFFs.
pcDNA3.1 are control transfected cells and UCA1 were transfected with UCA1 expression plasmid in pcDNA3.1 (as in Figure 5A). Note increased p16 and p21 levels and hypophosphorylation of RB. TBX3, …
Figure 5—figure supplement 2
ChIP-PCR assay for H3K9 acetylation of known regulatory elements of prosenescence and cell cycle genes whose expression is dyregulated after UCA1 overexpression.
Input, rabbit IgG negative control ChIP, and H3K9acetylation ChIP in control “C” or UCA1 “U” transfected HFFs for gene regulatory regions as labeled at bottom (primer sequences listed in ChIP …
Figure 6 with 6 supplements
UCA1 stabilizes CDKN2A-p16 mRNA levels during senescence by sequestering hnRNP A1.
(A) Graphs of transcript levels assayed by RT-qPCR in HFFs transfected with control (blue) or UCA1 (red) expression plasmids and treated with Actinomycin (D). Y axis shows % mRNA level relative to …
Figure 6—figure supplement 1
Graphs showing best fit lines, their equations, and R values used to calculate estimated mRNA half-life values shown in Figure 6A.
TBX3, CAPERα = human; Tbx3, Caperα = mouse.
Figure 6—figure supplement 2
Northern blot assay of p16INK mRNA levels in the absence and presence of UCA1.
(A) Top panel shows Northern blot of HFF cells transfected with control plasmid pcDNA3.1 and treated with Actinomycin D for the times (hr) indicated at top. (A′) The ethidum bromide stained gel …
Figure 6—figure supplement 3
Graphs showing best fit lines, their equations and R values used to calculate estimated half-life values after hnRNP A1 siRNA knockdown shown in Figure 6B.
(A) Western blot assaying hnRNP A1 protein levels in HFFs after transfection of control or anti-hnRNP A1 siRNA. (B) Graphs of best fit lines, equations and R values for half-lives shown in Figure 6B.…
Figure 6—figure supplement 4
RNA Immunoprecipitation analysis of hnRNP A1 interactions with Myc and p14ARF mRNAs.
RIP-PCR of MYC and CDKN2A-p14 mRNAs shows they are bound by hnRNP A1 but these interactions are unaffected by OIS/RAS, UCA1 overexpression, or knockdown of TBX3 or CAPERα. TBX3, CAPERα = human; …
Figure 6—figure supplement 5
RIP-PCR of HFF lysates using antibodies listed at top.
Only hnRNP A1 (A) and hnRNP D (B) bind UCA1 lncRNA, while TUG1 and H19 lncRNAs are bound by other hnRNPs. TBX3, CAPERα = human; Tbx3, Caperα = mouse.
Figure 6—figure supplement 6
RIP-PCR indicates that RB, p21, and CDK6 mRNAs do not interact with hnRNP A1 in PS or RAS HFFs.
TBX3, CAPERα = human; Tbx3, Caperα = mouse.
Figure 7 with 4 supplements
Disruption of the CAPERα/TBX3 repressor by OIS activates CDKN2A-p16 and UCA1 to trigger a senescence transcriptional response.
(A) ChIP-PCR of regions upstream of the CDKN2A-p16 transcriptional start site (position relative to TSS in parentheses) in PS and RAS HFFs; the −-3706–3308 amplicon is a negative control. OIS …
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Figure 7—source data 1
Differentially expressed genes after knockdown of CAPERα in HFFs detected by RNA-Seq.
- https://doi.org/10.7554/eLife.02805.027
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Figure 7—source data 2
Differentially expressed genes after knockdown of TBX3 in HFFs detected by RNA-Seq.
- https://doi.org/10.7554/eLife.02805.028
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Figure 7—source data 3
Determining the statistical significance of shared differentially expressed genes using the hypergeometric test, as implemented in the R statistical language (phyper).
- https://doi.org/10.7554/eLife.02805.029
Figure 7—figure supplement 1
Repression of CDKN2A-p16 and DUSP4 by CAPERα /TBX3 correlates with chromatin architecture and is relieved during oncogene induced senescence.
(A) ChIP-PCR to assess chromatin marks on CDKN2A-p16 regulatory elements in PS and RAS HFFs; antibodies are listed at top. (B) ChIP-PCR of DUSP4 promoter in PS and RAS HFFs; antibodies are listed at …
Figure 7—figure supplement 2
CAPERα relocalization due to oncogene-induced senescence is independent of PML bodies.
Immunocytochemical assay for endogenous CAPERα (green), PML (red), and DNA (DAPI, blue) in PS and RAS HFFs. TBX3, CAPERα = human; Tbx3, Caperα = mouse.
Figure 7—figure supplement 3
Validation of RNA-Seq identified expression changes induced by CAPERα and TBX3 KD.
qPCR validation of a subset of transcripts with altered expression detected by genome wide RNA-Seq on cDNA prepared from CAPERα (red) and TBX3 (blue) KD, and RAS HFFs (green). Downregulated …
Figure 7—figure supplement 4
IL6 and HDAC9 are direct targets of CAPERα/TBX3.
ChIP-PCR with antibodies listed at top showing CAPERα/TBX3 directly binds IL6 (and HDAC9) control elements. Effects of TBX3 or CAPER KD on chromatin marks are shown compared with control KD. …
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