Genome-Wide Gain-of-Function Screen Identifies Novel Regulators of Pluripotency

被引:34
作者
Abujarour, Ramzey [1 ]
Efe, Jem [1 ]
Ding, Sheng [1 ]
机构
[1] Scripps Res Inst, Dept Chem, La Jolla, CA 92037 USA
关键词
Embryonic stem cells; Screen; Pluripotency; Mki67ip; Nucleophosmin; EMBRYONIC STEM-CELLS; RNAI SCREEN; MYC TARGET; C-MYC; CANCER; GENES; NUCLEOPHOSMIN; EXPRESSION; NANOG; NETWORK;
D O I
10.1002/stem.472
中图分类号
Q813 [细胞工程];
学科分类号
摘要
Pluripotent stem cells are characterized by the capacity to self-renew and to differentiate into all the cell types of the body. To identify novel regulators of pluripotency, we screened cDNA libraries (>30,000 clones) in P19 embryonal carcinoma cells for factors that modulate the expression of a luciferase reporter driven by the promoter of the pluripotency master regulator Nanog. Ninety confirmed hits activated the reporter and 14 confirmed hits inhibited the reporter by more than two-fold. The identified hits were evaluated by gain-and loss-of-functions approaches. The reporter-activating hits Timp2, Hig2, and Mki67ip promoted embryonic stem (ES) cell self-renewal when episomally overexpressed in ES cells, whereas the reporterinhibiting hits PU.1/Spi1, Prkaca, and Jun induced differentiation of ES cells. Conversely, the knockdown of the activating hits Timp2, Mki67ip, Esrrg, and Dusp7 in ES cells induced differentiation, whereas the knockdown of the reporter-inhibiting hit PU. 1/Spi1 led to inhibition of differentiation. One of the novel hits, the RNA-binding protein Mki67ip was further characterized, and found to be overexpressed in ES cells and in early development and downregulated during differentiation. The knockdown of Mki67ip led to the differentiation of ES cells, decreased growth rate, reduction in pluripotency markers, and induction of lineage-specific markers. In addition, colocalization and coimmunoprecipitation experiments suggest that Mki67ip promotes ES cell self-renewal via a mechanism involving nucleophosmin, a multifunctional nucleolar protein upregulated in stem cells and cancer. STEM CELLS 2010; 28: 1487-1497
引用
收藏
页码:1487 / 1497
页数:11
相关论文
共 33 条
[1]   A bivalent chromatin structure marks key developmental genes in embryonic stem cells [J].
Bernstein, BE ;
Mikkelsen, TS ;
Xie, XH ;
Kamal, M ;
Huebert, DJ ;
Cuff, J ;
Fry, B ;
Meissner, A ;
Wernig, M ;
Plath, K ;
Jaenisch, R ;
Wagschal, A ;
Feil, R ;
Schreiber, SL ;
Lander, ES .
CELL, 2006, 125 (02) :315-326
[2]   N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis [J].
Boon, K ;
Caron, HN ;
van Asperen, R ;
Valentijn, L ;
Hermus, MC ;
van Sluis, P ;
Roobeek, I ;
Weis, I ;
Voûte, PA ;
Schwab, M ;
Versteeg, R .
EMBO JOURNAL, 2001, 20 (06) :1383-1393
[3]   Core transcriptional regulatory circuitry in human embryonic stem cells [J].
Boyer, LA ;
Lee, TI ;
Cole, MF ;
Johnstone, SE ;
Levine, SS ;
Zucker, JR ;
Guenther, MG ;
Kumar, RM ;
Murray, HL ;
Jenner, RG ;
Gifford, DK ;
Melton, DA ;
Jaenisch, R ;
Young, RA .
CELL, 2005, 122 (06) :947-956
[4]   Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells [J].
Chambers, I ;
Colby, D ;
Robertson, M ;
Nichols, J ;
Lee, S ;
Tweedie, S ;
Smith, A .
CELL, 2003, 113 (05) :643-655
[5]   The c-Myc target gene network [J].
Dang, Chi V. ;
O'Donnell, Kathryn A. ;
Zeller, Karen I. ;
Nguyen, Tam ;
Osthus, Rebecca C. ;
Li, Feng .
SEMINARS IN CANCER BIOLOGY, 2006, 16 (04) :253-264
[6]   A Genome-Scale RNAi Screen for Oct4 Modulators Defines a Role of the Paf1 Complex for Embryonic Stem Cell Identity [J].
Ding, Li ;
Paszkowski-Rogacz, Maciej ;
Nitzsche, Anja ;
Slabicki, Mikolaj Michal ;
Heninger, Anne-Kristin ;
de Vries, Ingrid ;
Kittler, Ralf ;
Junqueira, Magno ;
Shevchenko, Andrej ;
Schulz, Herbert ;
Hubner, Norbert ;
Doss, Michael Xavier ;
Sachinidis, Agapios ;
Hescheler, Juergen ;
Iacone, Roberto ;
Anastassiadis, Konstantinos ;
Stewart, A. Francis ;
Pisabarro, M. Teresa ;
Caldarelli, Antonio ;
Poser, Ina ;
Theis, Mirko ;
Buchholz, Frank .
CELL STEM CELL, 2009, 4 (05) :403-415
[7]   Cross-species comparison of human and mouse intestinal polyps reveals conserved mechanisms in adenomatous polyposis coli (APC)-driven tumorigenesis [J].
Gaspar, Claudia ;
Cardoso, Joana ;
Franken, Patrick ;
Molenaar, Lia ;
Morreau, Hans ;
Moeslein, Gabriela ;
Sampson, Julian ;
Boer, Judith M. ;
de Menezes, Renee X. ;
Fodde, Riccardo .
AMERICAN JOURNAL OF PATHOLOGY, 2008, 172 (05) :1363-1380
[8]   MAINTENANCE OF AN EXTRACHROMOSOMAL PLASMID VECTOR IN MOUSE EMBRYONIC STEM-CELLS [J].
GASSMANN, M ;
DONOHO, G ;
BERG, P .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (05) :1292-1296
[9]   Role of nucleophosmin in embryonic development and tumorigenesis [J].
Grisendi, S ;
Bernardi, R ;
Rossi, M ;
Cheng, K ;
Khandker, L ;
Manova, K ;
Pandolfi, PP .
NATURE, 2005, 437 (7055) :147-153
[10]   Nucleophosmin and cancer [J].
Grisendi, Silvia ;
Mecucci, Cristina ;
Falini, Brunangelo ;
Pandolfi, Pier Paolo .
NATURE REVIEWS CANCER, 2006, 6 (07) :493-505