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A Gain of Function Senescence Bypass Screen Identifies the Homeobox Transcription Factor DLX2 As a Regulator of ATM-P53 Signaling

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A Gain of Function Senescence Bypass Screen Identifies the Homeobox Transcription Factor DLX2 as a Regulator of ATM-P53 Signaling The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Wang, Yifan. 2016. A Gain of Function Senescence Bypass Screen Identifies the Homeobox Transcription Factor DLX2 as a Regulator of ATM-P53 Signaling. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718730 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA ! ! A gain of function senescence bypass screen identifies the Homeobox transcription factor DLX2 as a regulator of ATM-p53 signaling A dissertation presented By Yifan Wang To The Division of Medical Sciences In partial fulfillment of the requirements For the degree of Doctor of Philosophy In the subject of Genetics Harvard University Cambridge, Massachusetts November 2015 © 2015 – Yifan Wang All rights reserved Dissertation advisor: Dr. Stephen Elledge Yifan Wang! ! A gain of function senescence bypass screen identifies the Homeobox transcription factor DLX2 as a regulator of ATM-p53 signaling Abstract Senescence stimuli activate multiple tumor suppressor pathways to initiate cycle arrest and a differentiation program characteristic of senescent cells. We performed a two-stage, gain-of function screen to select for the genes whose enhanced expression can bypass replicative senescence. We uncovered multiple genes known to be involved in p53 and Rb regulation, ATM regulation and two components of the CST complex involved in preventing telomere erosion and additional genes such as REST and FOXO4 that have been implicated in aging. Among the new genes now implicated in senescence we identified DLX2, a Homeobox transcription factor that has been shown to be required for tumor growth, metastasis and associates with poor cancer prognosis. Growth analysis showed that DLX2 expression led to increased cellular replicative lifespan. We found that DLX2 expression inhibited p53 activation, and DLX2 reduced the protein level of upstream activator kinases ATM, DNA-PK. Our data suggest that DLX2 expression reduces the protein components of the TTI1/TTI2/TEL2 complex, a key complex required for the proper folding and stabilization of ATM and DNA-PK and other members of the PIKK family. Over-expression of DLX2 exhibit mutual exclusivity with p53 alteration in cancer patients, suggesting DLX2 may attenuate the p53 pathway during tumor formation. Our functional screen identified novel players that may promote tumorigenesis by regulating the ATM-p53 pathway and senescence. ! ! ! ! ! """! ! Attributions The genome-scale library described in Chapter 2 was designed by Steve Elledge, Mamie Li, and Laura Sack and constructed by Mamie Li and Laura Sack. The customized microarrays used for the primary screen were designed by Michael Schlabach and Qikai Xu. The E6 BJ and E7 BJ fibroblasts used for the screens were generated by Agata Smogorzewska. Qikai Xu and Laura Sack helped with the microarray hybridization of screen samples. The primary screen and subsequent data analysis was performed by Yifan Wang. Chanhee Kang tested the effect of dominant negative p53 on p21. The barcoded lentiviral destination vector collection was designed by Steve Elledge and Yifan Wang. Yifan Wang constructed the vectors, as well as the barcoded gene expression vectors for E6 and E7 sublibrary. The sublibrary screens were performed by Yifan Wang. Qikai Xu performed the ORF-Barcode mapping of the sequencing results. Sublibrary data analysis was performed by Yifan Wang. All other experiments described in chapter 2,3,4 and 5 were designed by Steve Elledge and Yifan Wang, and performed by Yifan Wang. ! ! ! "#! ! Contents Chapter I. Introduction ................................................................................................................ 1 I. An overview ............................................................................................................................. 1 II. Triggers of senescence .......................................................................................................... 2 2-1. Telomere attrition ........................................................................................................... 2 2-2. Oncogene induced senescence (OIS) ............................................................................. 4 2-3. Oxidative stress and cellular senescence ....................................................................... 6 2-4. Metabolic stress ............................................................................................................... 8 2-5. Chromatin perturbation................................................................................................. 9 2-6. Non-telomeric damage .................................................................................................. 10 III. Features and pathways of senescence .............................................................................. 11 3-1. Growth arrest. ............................................................................................................... 11 3-2. Activation of the p53-p21 and the p16-Rb pathway. ................................................. 12 3-3. Senescence Associated Heterochromatin Foci (SAHF) and other epigenetic changes in senescence ......................................................................................................................... 15 3-4. The Senescence Associated Secretory Phenotype (SASP) ......................................... 17 3-5. Senescence Associated-β-Galactosidase (SA-β-GAL) ................................................ 19 3-6. The choice between senescence and apoptosis ............................................................ 19 IV. Physiological and pathological senescence ...................................................................... 21 4-1. Senescence as a barrier to cancer in vivo.................................................................... 21 4-2. Senescence and aging .................................................................................................... 23 4-3. Senescence and wound healing .................................................................................... 26 4-4. Programmed senescence in embryonic development ................................................ 26 4-5. Senescence: antagonistic pleiotropy and multitasking .............................................. 27 V. Conclusion ............................................................................................................................ 28 Chapter II: A gain of function ORFeome screen to identify senescence regulators .............. 30 Abstract ..................................................................................................................................... 30 I. Introduction .......................................................................................................................... 31 II. Screen design and rationale ............................................................................................... 32 III. Results. ............................................................................................................................... 36 3-1. Identification of dominant negative p53 from the screen. ......................................... 36 3-2. Deconvolution of the screen data ................................................................................. 38 Table 2: candidate genes scored from E6 BJ senescence screen .......................................... 42 3-3. The Identification of known p53 and Rb pathway genes .......................................... 44 3-4. Genes scoring in both the E6 and E7 branches. ......................................................... 49 ! ! #! ! Table 3: candidate genes scored from both E6 and E7 screens ............................................... 50 3-5. Telomere maintenance: The CST complex................................................................. 50 3-6. Additional factors linked to senescence or aging identified in both screens. ........... 51 IV. Discussion ........................................................................................................................... 52 V. Material and methods ......................................................................................................... 57 5-1. Cell culture and general procedures. .......................................................................... 57 5-2. Growth curve analysis. ................................................................................................. 58 5-3. ORFeome based senescence bypass primary screen. ................................................. 58 5-4. Primary screen data analysis. ...................................................................................... 59 Chapter III: A barcoded platform for re-screening candidates for senescence bypass ........ 60 Abstract ..................................................................................................................................... 60 I. Introduction .........................................................................................................................
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