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Loss of Tumor Suppressor STAG2 Promotes Telomere Recombination and Extends the Replicative Lifespan of Normal Human Cells

Daniloski, Zharko; Smith, Susan
Sister chromatids are held together by cohesin, a tripartite ring with a peripheral SA1/2 subunit, where SA1 is required for telomere cohesion and SA2 for centromere cohesion. The STAG2 gene encoding SA2 is often inactivated in human cancer, but not in in a manner associated with aneuploidy. Thus, how these tumors maintain chromosomal cohesion and how STAG2 loss contributes to tumorigenesis remain open questions. Here we show that, despite a loss in centromere cohesion, sister chromatids in STAG2 mutant tumor cells maintain cohesion in mitosis at chromosome arms and telomeres. Telomere maintenance in STAG2 mutant tumor cells occurred by either telomere recombination or telomerase activation mechanisms. Notably, these cells were refractory to telomerase inhibitors, indicating recombination can provide an alternative means of telomere maintenance. STAG2 silencing in normal human cells that lack telomerase led to increased recombination at telomeres, delayed telomere shortening, and postponed senescence onset. Insofar as telomere shortening and replicative senescence prevent genomic instability and cancer by limiting the number of cell divisions, our findings suggest that extending the lifespan of normal human cells due to inactivation of STAG2 could promote tumorigenesis by extending the period during which tumor-driving mutations occur. Cancer Res; 77(20); 5530-42. (c)2017 AACR.
PMCID:5645240
PMID: 28819029
ISSN: 1538-7445
CID: 2742662

Cell cycle-regulated ubiquitination of tankyrase 1 by RNF8 and ABRO1/BRCC36 controls the timing of sister telomere resolution

Tripathi, Ekta; Smith, Susan
Timely resolution of sister chromatid cohesion in G2/M is essential for genome integrity. Resolution at telomeres requires the poly(ADP-ribose) polymerase tankyrase 1, but the mechanism that times its action is unknown. Here, we show that tankyrase 1 activity at telomeres is controlled by a ubiquitination/deubiquitination cycle depending on opposing ubiquitin ligase and deubiquitinase activities. In late S/G2 phase, the DNA damage-responsive E3 ligase RNF8 conjugates K63-linked ubiquitin chains to tankyrase 1, while in G1 phase such ubiquitin chains are removed by BRISC, an ABRO1/BRCC36-containing deubiquitinase complex. We show that K63-linked ubiquitin chains accumulate on tankyrase 1 in late S/G2 to promote its stabilization, association with telomeres, and resolution of cohesion. Timing of this posttranslational modification coincides with the ATM-mediated DNA damage response that occurs on functional telomeres following replication in G2. Removal of ubiquitin chains is controlled by ABRO1/BRCC36 and occurs as cells exit mitosis and enter G1, ensuring that telomere cohesion is not resolved prematurely in S phase. Our studies suggest that a cell cycle-regulated posttranslational mechanism couples resolution of telomere cohesion with completion of telomere replication to ensure genome integrity.
PMCID:5694945
PMID: 27993934
ISSN: 1460-2075
CID: 2369282

Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing

Lin, Jiangguo; Countryman, Preston; Chen, Haijiang; Pan, Hai; Fan, Yanlin; Jiang, Yunyun; Kaur, Parminder; Miao, Wang; Gurgel, Gisele; You, Changjiang; Piehler, Jacob; Kad, Neil M; Riehn, Robert; Opresko, Patricia L; Smith, Susan; Tao, Yizhi Jane; Wang, Hong
Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA-DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA-DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding.
PMCID:5291270
PMID: 27298259
ISSN: 1362-4962
CID: 2145082

The PARsylation activity of tankyrase in adipose tissue modulates systemic glucose metabolism in mice

Zhong, Linlin; Ding, Yun; Bandyopadhyay, Gautam; Waaler, Jo; Borgeson, Emma; Smith, Susan; Zhang, Mingchen; Phillips, Susan A; Mahooti, Sepi; Mahata, Sushil K; Shao, Jianhua; Krauss, Stefan; Chi, Nai-Wen
AIMS/HYPOTHESIS: Tankyrase (TNKS) is a ubiquitously expressed molecular scaffold that is implicated in diverse processes. The catalytic activity of TNKS modifies substrate proteins through poly-ADP-ribosylation (PARsylation) and is responsive to cellular energetic state. Global deficiency of the TNKS protein in mice accelerates glucose utilisation and raises plasma adiponectin levels. The aim of this study was to investigate whether the PARsylation activity of TNKS in adipocytes plays a role in systemic glucose homeostasis. METHODS: To inhibit TNKS-mediated PARsylation, we fed mice with a diet containing the TNKS-specific inhibitor G007-LK. To genetically inactivate TNKS catalysis in adipocytes while preserving its function as a molecular scaffold, we used an adipocyte-selective Cre transgene to delete TNKS exons that encoded the catalytic domain at the C-terminus. Tissue-specific insulin sensitivity in mice was investigated using hyperinsulinaemic-euglycaemic clamps. To model adipose-liver crosstalk ex vivo, we applied adipocyte-conditioned media to hepatocytes and assessed the effect on gluconeogenesis. RESULTS: The TNKS inhibitor G007-LK improved glucose tolerance and insulin sensitivity and promptly increased plasma adiponectin levels. In female mice, but not in male mice, adipocyte-selective genetic inactivation of TNKS catalysis improved hepatic insulin sensitivity and post-transcriptionally increased plasma adiponectin levels. Both pharmacological and genetic TNKS inhibition in female mouse-derived adipocytes induced a change in secreted factors to decrease gluconeogenesis in primary hepatocytes. CONCLUSIONS/INTERPRETATION: Systemic glucose homeostasis is regulated by the PARsylation activity of TNKS in adipocytes. This regulation is mediated in part by adipocyte-secreted factors that modulate hepatic glucose production. Pharmacological TNKS inhibition could potentially be used to improve glucose tolerance.
PMID: 26631215
ISSN: 1432-0428
CID: 1863522

Loss of ATRX Suppresses Resolution of Telomere Cohesion to Control Recombination in ALT Cancer Cells

Ramamoorthy, Mahesh; Smith, Susan
The chromatin-remodeler ATRX is frequently lost in cancer cells that use ALT (alternative lengthening of telomeres) for telomere maintenance, but its function in telomere recombination is unknown. Here we show that loss of ATRX suppresses the timely resolution of sister telomere cohesion that normally occurs prior to mitosis. In the absence of ATRX, the histone variant macroH2A1.1 binds to the poly(ADP-ribose) polymerase tankyrase 1, preventing it from localizing to telomeres and resolving cohesion. The resulting persistent telomere cohesion promotes recombination between sister telomeres, while it suppresses inappropriate recombination between non-sisters. Forced resolution of sister telomere cohesion induces excessive recombination between non-homologs, genomic instability, and impaired cell growth, indicating the ATRX-macroH2A1.1-tankyrase axis as a potential therapeutic target in ALT tumors.
PMCID:4573400
PMID: 26373281
ISSN: 1878-3686
CID: 1778202

Persistent telomere cohesion triggers a prolonged anaphase

Kim, Mi Kyung; Smith, Susan
Telomeres use distinct mechanisms (not used by arms or centromeres) to mediate cohesion between sister chromatids. However, the motivation for a specialized mechanism at telomeres is not well understood. Here we show, using fluorescence in situ hybridization and live-cell imaging, that persistent sister chromatid cohesion at telomeres triggers a prolonged anaphase in normal human cells and cancer cells. Excess cohesion at telomeres can be induced by inhibition of tankyrase 1, a poly(ADP-ribose) polymerase that is required for resolution of telomere cohesion, or by overexpression of proteins required to establish telomere cohesion, the shelterin subunit TIN2 and the cohesin subunit SA1. Regardless of the method of induction, excess cohesion at telomeres in mitosis prevents a robust and efficient anaphase. SA1- or TIN2-induced excess cohesion and anaphase delay can be rescued by overexpression of tankyrase 1. Moreover, we show that primary fibroblasts, which accumulate excess telomere cohesion at mitosis naturally during replicative aging, undergo a similar delay in anaphase progression that can also be rescued by overexpression of tankyrase 1. Our study demonstrates that there are opposing forces that regulate telomere cohesion. The observation that cells respond to unresolved telomere cohesion by delaying (but not completely disrupting) anaphase progression suggests a mechanism for tolerating excess cohesion and maintaining telomere integrity. This attempt to deal with telomere damage may be ultimately futile for aging fibroblasts but useful for cancer cells.
PMCID:3873891
PMID: 24173716
ISSN: 1059-1524
CID: 781822

SA1 binds directly to DNA through its unique AT-hook to promote sister chromatid cohesion at telomeres

Bisht, Kamlesh K; Daniloski, Zharko; Smith, Susan
Sister chromatid cohesion relies on cohesin, a complex comprising a tri-partite ring and a peripheral subunit Scc3, which is found as two related isoforms SA1 and SA2 in vertebrates. There is a division of labor between the vertebrate cohesin complexes; SA1-cohesin is required at telomeres and SA2-cohesin at centromeres. Depletion of SA1 has dramatic consequences for telomere function and genome integrity, but the mechanism by which SA1-cohesin mediates cohesion at telomeres is not well understood. Here we dissect the individual contribution of SA1 and the ring subunits to telomere cohesion and show that telomeres rely heavily on SA1 and to a lesser extent on the ring for cohesion. Using chromatin immunoprecipitation we show that SA1 is highly enriched at telomeres, is decreased at mitosis when cohesion is resolved, and is increased when cohesion persists. Overexpression of SA1 alone was sufficient to induce cohesion at telomeres, independent of the cohesin ring and dependent on its unique (not found in SA2) N-terminal domain, which we show binds to telomeric DNA through an AT-hook motif. We suggest that a specialized cohesion mechanism may be required to accommodate the high level of DNA replication-associated repair at telomeres.
PMCID:3730250
PMID: 23729739
ISSN: 0021-9533
CID: 484052

GDP-Mannose-4,6-Dehydratase Is a Cytosolic Partner of Tankyrase 1 That Inhibits Its Poly(ADP-Ribose) Polymerase Activity

Bisht, Kamlesh K; Dudognon, Charles; Chang, William G; Sokol, Ethan S; Ramirez, Alejandro; Smith, Susan
Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) that participates in a broad range of cellular activities due to interaction with multiple binding partners. Tankyrase 1 recognizes a linear six-amino-acid degenerate motif and, hence, has hundreds of potential target proteins. Binding of partner proteins to tankyrase 1 usually results in their poly(ADP-ribosyl)ation (PARsylation) and can lead to ubiquitylation and proteasomal degradation. However, it is not known how tankyrase 1 PARP activity is regulated. Here we identify GDP-mannose 4,6-dehydratase (GMD) as a binding partner of tankyrase 1. GMD is a cytosolic protein required for the first step of fucose synthesis. We show that GMD is complexed to tankyrase 1 in the cytosol throughout interphase, but its association with tankyrase 1 is reduced upon entry into mitosis, when tankyrase 1 binds to its other partners TRF1 (at telomeres) and NuMA (at spindle poles). In contrast to other binding partners, GMD is not PARsylated by tankyrase 1. Indeed, we show that GMD inhibits tankyrase 1 PARP activity in vitro, dependent on the GMD tankyrase 1 binding motif. In vivo, depletion of GMD led to degradation of tankyrase 1, dependent on the catalytic PARP activity of tankyrase 1. We speculate that association of tankyrase 1 with GMD in the cytosol sequesters tankyrase 1 in an inactive stable form that can be tapped by other target proteins as needed.
PMCID:3434517
PMID: 22645305
ISSN: 0270-7306
CID: 173024

Tankyrase 1 regulates centrosome function by controlling CPAP stability

Kim, Mi Kyung; Dudognon, Charles; Smith, Susan
CPAP-a gene mutated in primary microcephaly-is required for procentriole formation. Here we show that CPAP degradation and function is controlled by the poly(ADP-ribose) polymerase tankyrase 1. CPAP is PARsylated by tankyrase 1 in vitro and in vivo. Overexpression of tankyrase 1 leads to CPAP proteasomal degradation, preventing centriole duplication, whereas depletion of tankyrase 1 stabilizes CPAP in G1, generating elongated procentrioles and multipolarity. Tankyrase 1 localizes to centrosomes exclusively in G1, coinciding with CPAP degradation. Hence, tankyrase 1-mediated PARsylation regulates CPAP levels during the cell cycle to limit centriole elongation and ensure normal centrosome function.
PMCID:3410387
PMID: 22699936
ISSN: 1469-221x
CID: 174386

mRNA Decay Factor AUF1 Maintains Normal Aging, Telomere Maintenance, and Suppression of Senescence by Activation of Telomerase Transcription

Pont, Adam R; Sadri, Navid; Hsiao, Susan J; Smith, Susan; Schneider, Robert J
Inflammation is associated with DNA damage, cellular senescence, and aging. Cessation of the inflammatory cytokine response is mediated in part through cytokine mRNA degradation facilitated by RNA-binding proteins, including AUF1. We report a major function of AUF1-it activates telomerase expression, suppresses cellular senescence, and maintains normal aging. AUF1-deficient mice undergo striking telomere erosion, markedly increased DNA damage responses at telomere ends, pronounced cellular senescence, and rapid premature aging that increases with successive generations, which can be rescued in AUF1 knockout mice and their cultured cells by resupplying AUF1 expression. AUF1 binds and strongly activates the transcription promoter for telomerase catalytic subunit Tert. In addition to directing inflammatory cytokine mRNA decay, AUF1 destabilizes cell-cycle checkpoint mRNAs, preventing cellular senescence. Thus, a single gene, AUF1, links maintenance of telomere length and normal aging to attenuation of inflammatory cytokine expression and inhibition of cellular senescence.
PMCID:3966316
PMID: 22633954
ISSN: 1097-2765
CID: 173022