Premature Senescence and Increased TGFβ Signaling in the Absence of Tgif1

Transforming growth factor β (TGFβ) signaling regulates cell cycle progression in several cell types, primarily by inducing a G1 cell cycle arrest. Tgif1 is a transcriptional corepressor that limits TGFβ responsive gene expression. Here we demonstrate that primary mouse embryo fibroblasts (MEFs) lacking Tgif1 proliferate slowly, accumulate increased levels of DNA damage, and senesce prematurely. We also provide evidence that the effects of loss of Tgif1 on proliferation and senescence are not limited to primary cells. The increased DNA damage in Tgif1 null MEFs can be partially reversed by culturing cells at physiological oxygen levels, and growth in normoxic conditions also partially rescues the proliferation defect, suggesting that in the absence of Tgif1 primary MEFs are less able to cope with elevated levels of oxidative stress. Additionally, we show that Tgif1 null MEFs are more sensitive to TGFβ-mediated growth inhibition, and that treatment with a TGFβ receptor kinase inhibitor increases proliferation of Tgif1 null MEFs. Conversely, persistent treatment of wild type cells with low levels of TGFβ slows proliferation and induces senescence, suggesting that TGFβ signaling also contributes to cellular senescence. We suggest that in the absence of Tgif1, a persistent increase in TGFβ responsive transcription and a reduced ability to deal with hyperoxic stress result in premature senescence in primary MEFs

Overlap of TGFβ-mediated transcriptional changes with those in <i>Tgif1</i> null MEFs.

<p>A) Data from the comparison of <i>Tgif1</i> null to wild type P3 MEFs was compared to that from MEFs treated with TGFβ for 10 hours (from GSE15871). Total numbers of probe-sets with significant changes, and the overlaps are shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035460#pone-0035460-g005" target="_blank">Figure 5</a>. Chi squared analysis was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035460#pone-0035460-g005" target="_blank">Figure 5</a> and is shown above. B) Data from the comparison of <i>Tgif1</i> null to wild type P3 MEFs was compared to MEFs treated with TNFα for 4 hours (from GSE3700). Data was analyzed and is presented as in A. C) Twelve genes represented in the overlap shown in panel B were analyzed by qRT-PCR. Six genes each were selected from those that went up in both and those that went down in both. Expression was analyzed in triplicate cultures of passage 3 <i>Tgif1</i> null MEFs treated with a TGFβ receptor kinase inhibitor (1 µM SB-431542), or left untreated. Data is shown as mean (+ s.d.) with the value in the untreated cells set to 1 in each case. * p<0.05, ** p<0.01, *** p<0.001, as determined by the Student's T test. Shown below is the fold change (on a linear scale) in the comparison of the <i>Tgif1</i> null to wild type P3 array data for each gene.</p

Definition of pRB- and p53-Dependent and -Independent Steps in HIRA/ASF1a-Mediated Formation of Senescence-Associated Heterochromatin Foci▿ †

Cellular senescence is an irreversible proliferation arrest triggered by short chromosome telomeres, activated oncogenes, and cell stress and mediated by the pRB and p53 tumor suppressor pathways. One of the earliest steps in the senescence program is translocation of a histone chaperone, HIRA, into promyelocytic leukemia (PML) nuclear bodies. This relocalization precedes other markers of senescence, including the appearance of specialized domains of facultative heterochromatin called senescence-associated heterochromatin foci (SAHF) and cell cycle exit. SAHF represses expression of proliferation-promoting genes, thereby driving exit from the cell cycle. HIRA bound to another histone chaperone, ASF1a, drives formation of SAHF. Here, we show that HIRA's translocation to PML bodies occurs in response to all senescence triggers tested. Dominant negative HIRA mutants that block HIRA's localization to PML bodies prevent formation of SAHF, as does a PML-RARα fusion protein which disrupts PML bodies, directly supporting the idea that localization of HIRA to PML bodies is required for formation of SAHF. Significantly, translocation of HIRA to PML bodies occurs in the absence of functional pRB and p53 tumor suppressor pathways. However, our evidence indicates that downstream of HIRA's localization to PML bodies, the HIRA/ASF1a pathway cooperates with pRB and p53 to make SAHF, with the HIRA/ASF1a and pRB pathways acting in parallel. We present evidence that convergence of the HIRA/ASF1a and pRB pathways occurs through a DNAJ-domain protein, DNAJA2

Increased DNA damage in MEFs lacking Tgif1.

<p>Passage 2 wild type and <i>Tgif1</i> null cells were analyzed by comet assay, under denaturing conditions. The percentage of total DNA in the tail was quantified for at least 50 cells per condition. A) The percentage of DNA in the tail is plotted for each of two independent batches of wild type and <i>Tgif1</i> null MEFs. Data is plotted as median, 25^th and 75^th percentiles (box) and 5^th and 95^th percentiles (whiskers). p-values determined by the Student's T test are shown. B) The data shown in A, binned into 5% blocks, are plotted to show the distribution. C) Cells were treated with 100 µM H₂O₂ for 20 minutes and analyzed by comet assay at time-points thereafter over a 160 minute time-course. Data are presented as in A, with p-values for comparisons between wild type and Tgif1 null shown where significant. D) representative images of mitotic cells with DNA bridges are shown for <i>Tgif1</i> null cells. Cells analyzed for α-tubulin (green), pHH3 (red), and Hoechst (blue) are shown, together with a merged image of all three colors. Images were captured at 40×. Scale bar = 25 µM. E) Wild type and <i>Tgif1</i> null MEFs were grown on a 3T3 protocol in a standard incubator (5% CO₂ in air [20% O₂]), or in a chamber with 5% CO₂ and 3% O₂. Growth is plotted as relative cumulative cell number, with the starting 300,000 cells at P2 set equal to 1.</p

TGFβ induces growth inhibition and senescence.

<p>A) The TGFβ data-set from GSE15871 was compared to probe-set changes between P3 and P5 wild type MEFs. Data was analyzed and is presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035460#pone-0035460-g005" target="_blank">Figures 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035460#pone-0035460-g007" target="_blank">7</a>. B) The culture conditions over passages 2 to 5 are shown schematically: Arrows below indicate times at which TGFβ (1 pM or 3 pM) was added for the 3T3 assay. The time of addition of TGFβ and EdU is shown above, and the time at which the SAβG assay was performed is shown below. Relative cell proliferation was measured at passage 3 and 5 in cells grown under standard conditions, followed by a single 24 hour treatment with TGFβ at the indicated concentration. The percentage of EdU positive cells is shown. C) Cell proliferation was determined in a 3T3 assay and is shown as cumulative increase in cell number. Cells were cultured under standard conditions or with the addition of 1 or 3 pM TGFβ at the times indicated in panel B. D) The percentage (average + s.d. of triplicate cultures) of wild type cells with positive SAβG staining is shown at passage 5, after continued treatment with 1 pM or 3 pM TGFβ, or under standard conditions. Significant differences between control and plus TGFβ are shown: * p<0.05, ** p<0.01, as determined by the Student's T test. E) A tentative model describing the involvement of Tgif1 and TGFβ in the pathways leading to cellular senescence.</p

<i>Tgif1</i> null MEFs have increased DNA damage foci.

<p>A) Representative images of passage 5 cells stained with an antibody against γH2AX (green) and Hoechst (blue) are shown. Images were captured at 20×. Scale bar = 50 µM. B) The distribution of the number of γH2AX damage foci per nucleus is shown for passage 4 and 5 wild type and <i>Tgif1</i> null cells. Significance values were determined by Chi squared test, comparing the distribution in <i>Tgif1</i> null to that expected based on the wild type. C) The distributions of the number of γH2AX damage foci per nucleus in passage 4 cells were compared to those from cells that had been grown in 3% oxygen from passage 2 to 4. Data is presented and analyzed as in B. Note that the P4 MEFs analyzed for damage foci in 20% oxygen in panels B and C are the same. p-values for the comparisons of wild type to <i>Tgif1</i> null and 3% to 20% oxygen are shown below.</p