Volume 27, Issue 2, Pages 183-196 (July 2007) Downregulation of Wnt Signaling Is a Trigger for Formation of Facultative Heterochromatin and Onset of Cell Senescence in Primary Human Cells  Xiaofen Ye, Brad Zerlanko, Alyssa Kennedy, Gowrishankar Banumathy, Rugang Zhang, Peter D. Adams  Molecular Cell  Volume 27, Issue 2, Pages 183-196 (July 2007) DOI: 10.1016/j.molcel.2007.05.034 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Canonical Wnt Signaling Is Downregulated in Senescent Human Fibroblasts (A) RT-PCR expression analysis of indicated Wnt and GAPDH mRNAs in population doubling (PD) 31 or PD58 WI38 cells, or PD31 cells infected with Ras or control retrovirus. (B) WI38 cells infected with control (young) or Ras (senescent) retrovirus and stained with DAPI to visualize DNA and SAHF and with antibodies to GSK3β. (C) Cell extracts from (B) were immunoprecipitated with indicated antibodies and then western blotted to detect GSK3β. (D) Cell extracts from (B) were immunoprecipitated with indicated antibodies and then incubated with Mg2+ATPγ32P. (E) Immunoprecipitates from (D) were incubated with the substrate phosphopeptide YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE and Mg2+ATPγ32P. Mean of four experiments with standard deviation (SD). (F) Soluble extracts from WI38 cells, PD35 or PD58, were western blotted with antibodies to β-catenin. Molecular Cell 2007 27, 183-196DOI: (10.1016/j.molcel.2007.05.034) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Wnt2 Is Repressed Early during Senescence and Independently of pRB and p53 (A) WI38 cells were passaged from PD33 to PD49 and assayed for the number of cells with HIRA localized to PML bodies by immunofluorescence and Wnt2 and GAPDH mRNAs by RT-PCR. (B) WI38 cells were infected with Ras or control retrovirus and selected in puromycin, total RNA prepared at indicated days after infection, and mRNA abundance determined by RT-PCR. (C) WI38 cells were infected with SV40 large T antigen or control retrovirus and selected and passaged in neomycin, total RNA prepared, and mRNA abundance determined at indicated PD after infection. (D) Western analysis of SV40 large T antigen in cells from (C). (E) Images of cells from (C). Percent of cells with SAHF in each case (scored by epifluorescence of DAPI-stained cells) is indicated. Control cells at PD12 are senescent. SV40 Tag-expressing cells at PD18 are in crisis. Molecular Cell 2007 27, 183-196DOI: (10.1016/j.molcel.2007.05.034) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 GSK3β Kinase Regulates HIRA Localization and Formation of SAHF (A) Primary WI38 cells were treated without or with 50 mM LiCl or 30 μM kenpaullone and scored for HIRA in PML nuclear bodies. Mean of three experiments with SD. (B) Primary WI38 cells were infected with indicated retroviruses and then stained with antibodies to HIRA and with DAPI. (C) WI38 cells were infected with retroviruses encoding GSK3βS9A, Myc-HIRA(520–1017) (HIRA-C) or control. Eight days later, cells were stained with DAPI and antibodies to HIRA. Monoclonal antibody, WC119, used to stain HIRA does not recognize HIRA-C. (D) Quantitation of results from (C). Mean of three independent experiments with SD. Molecular Cell 2007 27, 183-196DOI: (10.1016/j.molcel.2007.05.034) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 HIRA Is a Substrate of GSK3β (A) U2OS cells were transfected with plasmids encoding HA-GSK3β or Myc-HIRA (wild-type, full length), and extracts were prepared and immunoprecipitated and then western blotted with anti-HA or anti-Myc antibodies. (B) Purified recombinant GST or GST fused to residues 421–729 of HIRA (GST-HIRA[421–729]) was incubated with purified GSK3β or buffer. Bound proteins were isolated on glutathione beads and western blotted to detect HIRA and GSK3β. (C) Purified recombinant GST-HIRA(421–729)WT or GST-HIRA(421–729)ΔP was incubated with purified GSK3β and Mg2+ATPγ32P. (D) Purified, recombinant variants of GST-HIRA(421–729) were incubated in the absence or presence of purified GSK3β and Mg2+ATPγ32P. (E) Purified GSK3β was incubated in the absence or presence of a S697 substrate phosphopeptide (RRTLQVSSDPpSMYI) and Mg2+ATPγ32P. (F) WI38 cells were infected with control or HA-HIRA(421–729)WT retrovirus, and extracts were immunoprecipitated with anti-HIRA antibodies and treated with or without λ-phosphatase. (G) WI38 cells were infected with control, HA-HIRA(421–729)WT, or HA-HIRA(421–729)ΔP retrovirus; cells were metabolically labeled with 33P-orthophosphate; and lysates were immunoprecipitated with anti-HA antibodies. (H) WI38 cells were infected with control or indicated HIRA retroviruses and western blotted with a phosphospecific antibody to phosphoS697 of HIRA. (I) WI38 cells were infected with HA-HIRA(421–729) retrovirus in the absence or presence of a GID or control virus. Seven days later, lysates were prepared and western blotted with anti-phosphoS697HIRA. Molecular Cell 2007 27, 183-196DOI: (10.1016/j.molcel.2007.05.034) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Phosphorylation of HIRA Is Required for Its Localization to PML Bodies (A) WI38 cells were infected with HA-HIRA(421–729)WT, HA-HIRA(421–729)S697A, HA-HIRA(421–729)S511A,S515A, or control retroviruses. Eight days later, cells were pre-extracted in buffer containing NP40, fixed, and stained with anti-HA and anti-PML antibodies. (B) Quantitation of (A). Mean of three independent experiments with SD. (C) Cell extracts from (A) were immunoprecipitated and western blotted with anti-HA antibodies and anti-ASF1a antibodies. Molecular Cell 2007 27, 183-196DOI: (10.1016/j.molcel.2007.05.034) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 RNAi-Mediated Knockdown of Wnt2 Triggers Localization of HIRA to PML Bodies and Formation of SAHF (A) Primary WI38 cells were infected with lentiviruses encoding five different shRNAs to Wnt2, or a control lentivirus. The cells were selected in puromycin and 4 days later Wnt2 and GAPDH mRNAs were assayed by RT-PCR. (B) Ten days after infection, the cells from (A) were stained with antibodies to HIRA and with DAPI. Bars are color coded as in (A). Mean of three independent experiments with SD. (C) Images of the cells from (B). (D) Cells from (B) (Control and shWnt2-5) were stained for SA β-gal activity. (E) Cells from (D) were stained with DAPI and antibodies to macroH2A1.2. Molecular Cell 2007 27, 183-196DOI: (10.1016/j.molcel.2007.05.034) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Exogenous Wnt3a Delays Senescence (A) Primary WI38 cells were grown from PD43 to PD63 in control L-medium, Wnt3a-conditioned medium, or standard medium (none) and scored for localization of HIRA to PML nuclear bodies and formation of SAHF. (B) WI38 cells were cultured under standard conditions in Wnt3a-conditioned medium (Wnt3a-CM) or 50 ng/ml recombinant Wnt3a (rWnt3a) and scored for localization of HIRA to PML bodies after 48 hr. Mean of three independent experiments with SD. (C) Primary WI38 cells were infected with a Ras retrovirus, grown in control L-medium or Wnt3a-conditioned medium, and 8 days after infection labeled with 5′-BrdU for 24 hr and assayed for 5′-BrdU-positive cells by FACS. Shown are histograms of 5′-BrdU-positive cells, and the percent of 5′-BrdU-positive cells is indicated. (D) As in (C), but the cells were labeled with 5′-BrdU for 24 or 1 hr (results for the 24 hr pulse are the same results as in [C]). Mock are uninfected cells in standard growth medium. (E) As (C), but the Ras-infected cells were grown in the absence or presence of Wnt3a-conditioned medium (Wnt3a-CM) or 75 ng/ml recombinant Wnt3a (rWnt3a). (F) Human RPE cells were infected with a control or Ras retrovirus and then grown under standard conditions in control L-medium or Wnt3a-conditioned medium for 8 days. (G) As in (F), but the cells were pulse labeled with 5′-BrdU for 1 hr and the percent 5′-BrdU-positive cells determines by FACS. Molecular Cell 2007 27, 183-196DOI: (10.1016/j.molcel.2007.05.034) Copyright © 2007 Elsevier Inc. Terms and Conditions