Figure 3. 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) % Quantitation of A–F from three replicate experiments. * indicates p<0.05 relative to Control or p53 shRNAs. (H) Cell proliferation assayed by crystal violet incorporation (OD units) in HFFs treated as in A–F. * indicates p<0.001 relative to Ctl or p53 shRNAs. (I–L) SA-βgal assays of HFFs stably transduced with control or p16 (Haga et al., 2007) shRNAs subsequently transduced with CAPERα or TBX3 shRNAs. (M) % Quantitation of I-L from three replicate experiments. * indicates p<0.05 relative to Ctl shRNA. (N) Cell proliferation assayed by crystal violet incorporation (OD units) in HFFs treated as in I–L. * indicates p<0.01 relative to Ctl shRNA. (O) ChIP-PCR with antibodies listed at top on three regions upstream of the CDKN2A-p16 transcriptional start site (TSS); position relative to (TSS) is indicated in parentheses at left of panels. PCR of input material used for the ChIP is shown under ‘Input’. The shRNA transduced is listed above each lane (HFF Tx). TBX3 knockdown decreases binding of TBX3 (lanes 8) and CAPERα (lanes 11) to all three regions. CAPERα knockdown has minimal effect on TBX3 binding (lanes 9). Knockdown of either TBX3 or CAPERα decreases the repressive chromatin mark H3K9me3 (lanes14, 15) and increases the activating chromatin mark H3K4me3 (lanes 17, 18). TBX3, CAPERα = human; Tbx3, Caperα = mouse.

DOI: http://dx.doi.org/10.7554/eLife.02805.009

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 been previously employed by numerous investigators (Masutomi et al., 2003; Boehm et al., 2005; Haga et al., 2007; Hong et al., 2009; Elzi et al., 2012). TBX3, CAPERα = human; Tbx3, Caperα = mouse.

DOI: http://dx.doi.org/10.7554/eLife.02805.010

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 genome browser. Chromatin states in various cell types based are noted by colored bars below. Of these 7 regions, 3 were bound by both TBX3 and CAPERα: regions 3, 4 and 5 (data are presented in Figure 3O). TBX3, CAPERα = human; Tbx3, Caperα = mouse.

DOI: http://dx.doi.org/10.7554/eLife.02805.011

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 below each panel and correspond to regions 3, 4 and 5 in Figure 3—figure supplement 2. TBX3, CAPERα = human; Tbx3, Caperα = mouse.

DOI: http://dx.doi.org/10.7554/eLife.02805.012

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) ChIP/PCR of HFF chromatin showing lack of TBX3 and CAPERα binding to known regulatory elements (Baksh et al., 2002; Wang et al., 2005; Louie et al., 2010) of: (B) CDKN1A-p21 (location relative to transcription start site is noted in parentheses at the bottom of the panels (C) CDK4 (D) CDK2 (E) CDKN1B. TBX3, CAPERα = human; Tbx3, Caperα = mouse.

DOI: http://dx.doi.org/10.7554/eLife.02805.013