High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells
Identifieur interne : 000433 ( Pmc/Corpus ); précédent : 000432; suivant : 000434High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells
Auteurs : Yo Saito ; Tohru Fujiwara ; Keiichi Ohashi ; Yoko Okitsu ; Noriko Fukuhara ; Yasushi Onishi ; Kenichi Ishizawa ; Hideo HarigaeSource :
- PLoS ONE [ 1932-6203 ] ; 2015.
Abstract
Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA) library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with
Url:
DOI: 10.1371/journal.pone.0137079
PubMed: 26325290
PubMed Central: 4556642
Links to Exploration step
PMC:4556642Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells</title><author><name sortKey="Saito, Yo" sort="Saito, Yo" uniqKey="Saito Y" first="Yo" last="Saito">Yo Saito</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Fujiwara, Tohru" sort="Fujiwara, Tohru" uniqKey="Fujiwara T" first="Tohru" last="Fujiwara">Tohru Fujiwara</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff002"><addr-line>Molecular Hematology/Oncology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Ohashi, Keiichi" sort="Ohashi, Keiichi" uniqKey="Ohashi K" first="Keiichi" last="Ohashi">Keiichi Ohashi</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Okitsu, Yoko" sort="Okitsu, Yoko" uniqKey="Okitsu Y" first="Yoko" last="Okitsu">Yoko Okitsu</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Fukuhara, Noriko" sort="Fukuhara, Noriko" uniqKey="Fukuhara N" first="Noriko" last="Fukuhara">Noriko Fukuhara</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Onishi, Yasushi" sort="Onishi, Yasushi" uniqKey="Onishi Y" first="Yasushi" last="Onishi">Yasushi Onishi</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Ishizawa, Kenichi" sort="Ishizawa, Kenichi" uniqKey="Ishizawa K" first="Kenichi" last="Ishizawa">Kenichi Ishizawa</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff003"><addr-line>Department of Hematology and Cell Therapy, Yamagata University Faculty of Medicine, Yamagata, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Harigae, Hideo" sort="Harigae, Hideo" uniqKey="Harigae H" first="Hideo" last="Harigae">Hideo Harigae</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff002"><addr-line>Molecular Hematology/Oncology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26325290</idno><idno type="pmc">4556642</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556642</idno><idno type="RBID">PMC:4556642</idno><idno type="doi">10.1371/journal.pone.0137079</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000433</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000433</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells</title><author><name sortKey="Saito, Yo" sort="Saito, Yo" uniqKey="Saito Y" first="Yo" last="Saito">Yo Saito</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Fujiwara, Tohru" sort="Fujiwara, Tohru" uniqKey="Fujiwara T" first="Tohru" last="Fujiwara">Tohru Fujiwara</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff002"><addr-line>Molecular Hematology/Oncology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Ohashi, Keiichi" sort="Ohashi, Keiichi" uniqKey="Ohashi K" first="Keiichi" last="Ohashi">Keiichi Ohashi</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Okitsu, Yoko" sort="Okitsu, Yoko" uniqKey="Okitsu Y" first="Yoko" last="Okitsu">Yoko Okitsu</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Fukuhara, Noriko" sort="Fukuhara, Noriko" uniqKey="Fukuhara N" first="Noriko" last="Fukuhara">Noriko Fukuhara</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Onishi, Yasushi" sort="Onishi, Yasushi" uniqKey="Onishi Y" first="Yasushi" last="Onishi">Yasushi Onishi</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Ishizawa, Kenichi" sort="Ishizawa, Kenichi" uniqKey="Ishizawa K" first="Kenichi" last="Ishizawa">Kenichi Ishizawa</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff003"><addr-line>Department of Hematology and Cell Therapy, Yamagata University Faculty of Medicine, Yamagata, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Harigae, Hideo" sort="Harigae, Hideo" uniqKey="Harigae H" first="Hideo" last="Harigae">Hideo Harigae</name><affiliation><nlm:aff id="aff001"><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff002"><addr-line>Molecular Hematology/Oncology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA) library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with <italic>GATA-2</italic> intronic enhancer (+9.9 kb/1S); therefore, we established a cell line capable of stably expressing +9.9 kb/1S-Luciferase. Subsequently, we screened 995 transcription factor genes and revealed that <italic>CITED2</italic> acts as a GATA-2 activator in human hematopoietic cells. These results provide novel insights into and further identify the regulatory mechanism of GATA-2.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Orkin, Sh" uniqKey="Orkin S">SH Orkin</name></author><author><name sortKey="Zon, Li" uniqKey="Zon L">LI Zon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Evans, T" uniqKey="Evans T">T Evans</name></author><author><name sortKey="Reitman, M" uniqKey="Reitman M">M Reitman</name></author><author><name sortKey="Felsenfeld, G" uniqKey="Felsenfeld G">G Felsenfeld</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yamamoto, M" uniqKey="Yamamoto M">M Yamamoto</name></author><author><name sortKey="Ko, Lj" uniqKey="Ko L">LJ Ko</name></author><author><name sortKey="Leonard, Mw" uniqKey="Leonard M">MW Leonard</name></author><author><name sortKey="Beug, H" uniqKey="Beug H">H Beug</name></author><author><name sortKey="Orkin, Sh" uniqKey="Orkin S">SH Orkin</name></author><author><name sortKey="Engel, Jd" uniqKey="Engel J">JD Engel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Harigae, H" uniqKey="Harigae H">H Harigae</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bresnick, Eh" uniqKey="Bresnick E">EH Bresnick</name></author><author><name sortKey="Lee, Hy" uniqKey="Lee H">HY Lee</name></author><author><name sortKey="Fujiwara, T" uniqKey="Fujiwara T">T Fujiwara</name></author><author><name sortKey="Johnson, Kd" uniqKey="Johnson K">KD Johnson</name></author><author><name sortKey="Keles, S" uniqKey="Keles S">S Keles</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Martin, Di" uniqKey="Martin D">DI Martin</name></author><author><name sortKey="Zon, Li" uniqKey="Zon L">LI Zon</name></author><author><name sortKey="Mutter, G" uniqKey="Mutter G">G Mutter</name></author><author><name sortKey="Orkin, Sh" uniqKey="Orkin S">SH Orkin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lim, Kc" uniqKey="Lim K">KC Lim</name></author><author><name sortKey="Lakshmanan, G" uniqKey="Lakshmanan G">G Lakshmanan</name></author><author><name sortKey="Crawford, Se" uniqKey="Crawford S">SE Crawford</name></author><author><name sortKey="Gu, Y" uniqKey="Gu Y">Y Gu</name></author><author><name sortKey="Grosveld, F" uniqKey="Grosveld F">F Grosveld</name></author><author><name sortKey="Engel, Jd" uniqKey="Engel J">JD Engel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tsai, Fy" uniqKey="Tsai F">FY Tsai</name></author><author><name sortKey="Keller, G" uniqKey="Keller G">G Keller</name></author><author><name sortKey="Kuo, Fc" uniqKey="Kuo F">FC Kuo</name></author><author><name sortKey="Weiss, M" uniqKey="Weiss M">M Weiss</name></author><author><name sortKey="Chen, J" uniqKey="Chen J">J Chen</name></author><author><name sortKey="Rosenblatt, M" uniqKey="Rosenblatt M">M Rosenblatt</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tsai, Fy" uniqKey="Tsai F">FY Tsai</name></author><author><name sortKey="Orkin, Sh" uniqKey="Orkin S">SH Orkin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ezoe, S" uniqKey="Ezoe S">S Ezoe</name></author><author><name sortKey="Matsumura, I" uniqKey="Matsumura I">I Matsumura</name></author><author><name sortKey="Nakata, S" uniqKey="Nakata S">S Nakata</name></author><author><name sortKey="Gale, K" uniqKey="Gale K">K Gale</name></author><author><name sortKey="Ishihara, K" uniqKey="Ishihara K">K Ishihara</name></author><author><name sortKey="Minegishi, N" uniqKey="Minegishi N">N Minegishi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rodrigues, Np" uniqKey="Rodrigues N">NP Rodrigues</name></author><author><name sortKey="Janzen, V" uniqKey="Janzen V">V Janzen</name></author><author><name sortKey="Forkert, R" uniqKey="Forkert R">R Forkert</name></author><author><name sortKey="Dombkowski, Dm" uniqKey="Dombkowski D">DM Dombkowski</name></author><author><name sortKey="Boyd, As" uniqKey="Boyd A">AS Boyd</name></author><author><name sortKey="Orkin, Sh" uniqKey="Orkin S">SH Orkin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Minegishi, N" uniqKey="Minegishi N">N Minegishi</name></author><author><name sortKey="Ohta, J" uniqKey="Ohta J">J Ohta</name></author><author><name sortKey="Suwabe, N" uniqKey="Suwabe N">N Suwabe</name></author><author><name sortKey="Nakauchi, H" uniqKey="Nakauchi H">H Nakauchi</name></author><author><name sortKey="Ishihara, H" uniqKey="Ishihara H">H Ishihara</name></author><author><name sortKey="Hayashi, N" uniqKey="Hayashi N">N Hayashi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pan, X" uniqKey="Pan X">X Pan</name></author><author><name sortKey="Minegishi, N" uniqKey="Minegishi N">N Minegishi</name></author><author><name sortKey="Harigae, H" uniqKey="Harigae H">H Harigae</name></author><author><name sortKey="Yamagiwa, H" uniqKey="Yamagiwa H">H Yamagiwa</name></author><author><name sortKey="Minegishi, M" uniqKey="Minegishi M">M Minegishi</name></author><author><name sortKey="Akine, Y" uniqKey="Akine Y">Y Akine</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Moriguchi, T" uniqKey="Moriguchi T">T Moriguchi</name></author><author><name sortKey="Yamamoto, M" uniqKey="Yamamoto M">M Yamamoto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sanalkumar, R" uniqKey="Sanalkumar R">R Sanalkumar</name></author><author><name sortKey="Johnson, Kd" uniqKey="Johnson K">KD Johnson</name></author><author><name sortKey="Gao, X" uniqKey="Gao X">X Gao</name></author><author><name sortKey="Boyer, Me" uniqKey="Boyer M">ME Boyer</name></author><author><name sortKey="Chang, Yi" uniqKey="Chang Y">YI Chang</name></author><author><name sortKey="Hewitt, Kj" uniqKey="Hewitt K">KJ Hewitt</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Snow, Jw" uniqKey="Snow J">JW Snow</name></author><author><name sortKey="Trowbridge, Jj" uniqKey="Trowbridge J">JJ Trowbridge</name></author><author><name sortKey="Johnson, Kd" uniqKey="Johnson K">KD Johnson</name></author><author><name sortKey="Fujiwara, T" uniqKey="Fujiwara T">T Fujiwara</name></author><author><name sortKey="Emambokus, Ne" uniqKey="Emambokus N">NE Emambokus</name></author><author><name sortKey="Grass, Ja" uniqKey="Grass J">JA Grass</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Snow, Jw" uniqKey="Snow J">JW Snow</name></author><author><name sortKey="Trowbridge, Jj" uniqKey="Trowbridge J">JJ Trowbridge</name></author><author><name sortKey="Fujiwara, T" uniqKey="Fujiwara T">T Fujiwara</name></author><author><name sortKey="Emambokus, Ne" uniqKey="Emambokus N">NE Emambokus</name></author><author><name sortKey="Grass, Ja" uniqKey="Grass J">JA Grass</name></author><author><name sortKey="Orkin, Sh" uniqKey="Orkin S">SH Orkin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Johnson, Kd" uniqKey="Johnson K">KD Johnson</name></author><author><name sortKey="Hsu, Ap" uniqKey="Hsu A">AP Hsu</name></author><author><name sortKey="Ryu, Mj" uniqKey="Ryu M">MJ Ryu</name></author><author><name sortKey="Wang, J" uniqKey="Wang J">J Wang</name></author><author><name sortKey="Gao, X" uniqKey="Gao X">X Gao</name></author><author><name sortKey="Boyer, Me" uniqKey="Boyer M">ME Boyer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dickinson, Re" uniqKey="Dickinson R">RE Dickinson</name></author><author><name sortKey="Griffin, H" uniqKey="Griffin H">H Griffin</name></author><author><name sortKey="Bigley, V" uniqKey="Bigley V">V Bigley</name></author><author><name sortKey="Reynard, Ln" uniqKey="Reynard L">LN Reynard</name></author><author><name sortKey="Hussain, R" uniqKey="Hussain R">R Hussain</name></author><author><name sortKey="Haniffa, M" uniqKey="Haniffa M">M Haniffa</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hsu, Ap" uniqKey="Hsu A">AP Hsu</name></author><author><name sortKey="Sampaio, Ep" uniqKey="Sampaio E">EP Sampaio</name></author><author><name sortKey="Khan, J" uniqKey="Khan J">J Khan</name></author><author><name sortKey="Calvo, Kr" uniqKey="Calvo K">KR Calvo</name></author><author><name sortKey="Lemieux, Je" uniqKey="Lemieux J">JE Lemieux</name></author><author><name sortKey="Patel, Sy" uniqKey="Patel S">SY Patel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ostergaard, P" uniqKey="Ostergaard P">P Ostergaard</name></author><author><name sortKey="Simpson, Ma" uniqKey="Simpson M">MA Simpson</name></author><author><name sortKey="Connell, Fc" uniqKey="Connell F">FC Connell</name></author><author><name sortKey="Steward, Cg" uniqKey="Steward C">CG Steward</name></author><author><name sortKey="Brice, G" uniqKey="Brice G">G Brice</name></author><author><name sortKey="Woollard, Wj" uniqKey="Woollard W">WJ Woollard</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Guiu, J" uniqKey="Guiu J">J Guiu</name></author><author><name sortKey="Shimizu, R" uniqKey="Shimizu R">R Shimizu</name></author><author><name sortKey="D Altri, T" uniqKey="D Altri T">T D'Altri</name></author><author><name sortKey="Fraser, St" uniqKey="Fraser S">ST Fraser</name></author><author><name sortKey="Hatakeyama, J" uniqKey="Hatakeyama J">J Hatakeyama</name></author><author><name sortKey="Bresnick, Eh" uniqKey="Bresnick E">EH Bresnick</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Trompouki, E" uniqKey="Trompouki E">E Trompouki</name></author><author><name sortKey="Bowman, Tv" uniqKey="Bowman T">TV Bowman</name></author><author><name sortKey="Lawton, Ln" uniqKey="Lawton L">LN Lawton</name></author><author><name sortKey="Fan, Zp" uniqKey="Fan Z">ZP Fan</name></author><author><name sortKey="Wu, Dc" uniqKey="Wu D">DC Wu</name></author><author><name sortKey="Dibiase, A" uniqKey="Dibiase A">A DiBiase</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fujimaki, S" uniqKey="Fujimaki S">S Fujimaki</name></author><author><name sortKey="Harigae, H" uniqKey="Harigae H">H Harigae</name></author><author><name sortKey="Sugawara, T" uniqKey="Sugawara T">T Sugawara</name></author><author><name sortKey="Takasawa, N" uniqKey="Takasawa N">N Takasawa</name></author><author><name sortKey="Sasaki, T" uniqKey="Sasaki T">T Sasaki</name></author><author><name sortKey="Kaku, M" uniqKey="Kaku M">M Kaku</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zeng, W" uniqKey="Zeng W">W Zeng</name></author><author><name sortKey="Chen, G" uniqKey="Chen G">G Chen</name></author><author><name sortKey="Kajigaya, S" uniqKey="Kajigaya S">S Kajigaya</name></author><author><name sortKey="Nunez, O" uniqKey="Nunez O">O Nunez</name></author><author><name sortKey="Charrow, A" uniqKey="Charrow A">A Charrow</name></author><author><name sortKey="Billings, Em" uniqKey="Billings E">EM Billings</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kumar, Ms" uniqKey="Kumar M">MS Kumar</name></author><author><name sortKey="Hancock, Dc" uniqKey="Hancock D">DC Hancock</name></author><author><name sortKey="Molina Arcas, M" uniqKey="Molina Arcas M">M Molina-Arcas</name></author><author><name sortKey="Steckel, M" uniqKey="Steckel M">M Steckel</name></author><author><name sortKey="East, P" uniqKey="East P">P East</name></author><author><name sortKey="Diefenbacher, M" uniqKey="Diefenbacher M">M Diefenbacher</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Endo, K" uniqKey="Endo K">K Endo</name></author><author><name sortKey="Harigae, H" uniqKey="Harigae H">H Harigae</name></author><author><name sortKey="Nagai, T" uniqKey="Nagai T">T Nagai</name></author><author><name sortKey="Fujie, H" uniqKey="Fujie H">H Fujie</name></author><author><name sortKey="Meguro, K" uniqKey="Meguro K">K Meguro</name></author><author><name sortKey="Watanabe, N" uniqKey="Watanabe N">N Watanabe</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dorfman, Dm" uniqKey="Dorfman D">DM Dorfman</name></author><author><name sortKey="Wilson, Db" uniqKey="Wilson D">DB Wilson</name></author><author><name sortKey="Bruns, Ga" uniqKey="Bruns G">GA Bruns</name></author><author><name sortKey="Orkin, Sh" uniqKey="Orkin S">SH Orkin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Koeffler, Hp" uniqKey="Koeffler H">HP Koeffler</name></author><author><name sortKey="Billing, R" uniqKey="Billing R">R Billing</name></author><author><name sortKey="Lusis, Aj" uniqKey="Lusis A">AJ Lusis</name></author><author><name sortKey="Sparkes, R" uniqKey="Sparkes R">R Sparkes</name></author><author><name sortKey="Golde, Dw" uniqKey="Golde D">DW Golde</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fujiwara, T" uniqKey="Fujiwara T">T Fujiwara</name></author><author><name sortKey="Fukuhara, N" uniqKey="Fukuhara N">N Fukuhara</name></author><author><name sortKey="Funayama, R" uniqKey="Funayama R">R Funayama</name></author><author><name sortKey="Nariai, N" uniqKey="Nariai N">N Nariai</name></author><author><name sortKey="Kamata, M" uniqKey="Kamata M">M Kamata</name></author><author><name sortKey="Nagashima, T" uniqKey="Nagashima T">T Nagashima</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fujiwara, T" uniqKey="Fujiwara T">T Fujiwara</name></author><author><name sortKey="Yokoyama, H" uniqKey="Yokoyama H">H Yokoyama</name></author><author><name sortKey="Okitsu, Y" uniqKey="Okitsu Y">Y Okitsu</name></author><author><name sortKey="Kamata, M" uniqKey="Kamata M">M Kamata</name></author><author><name sortKey="Fukuhara, N" uniqKey="Fukuhara N">N Fukuhara</name></author><author><name sortKey="Onishi, Y" uniqKey="Onishi Y">Y Onishi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fujiwara, T" uniqKey="Fujiwara T">T Fujiwara</name></author><author><name sortKey="O Geen, H" uniqKey="O Geen H">H O'Geen</name></author><author><name sortKey="Keles, S" uniqKey="Keles S">S Keles</name></author><author><name sortKey="Blahnik, K" uniqKey="Blahnik K">K Blahnik</name></author><author><name sortKey="Linnemann, Ak" uniqKey="Linnemann A">AK Linnemann</name></author><author><name sortKey="Kang, Ya" uniqKey="Kang Y">YA Kang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cortes Lavaud, X" uniqKey="Cortes Lavaud X">X Cortes-Lavaud</name></author><author><name sortKey="Maicas, M" uniqKey="Maicas M">M Maicas</name></author><author><name sortKey="Vazquez, I" uniqKey="Vazquez I">I Vazquez</name></author><author><name sortKey="Vicente, C" uniqKey="Vicente C">C Vicente</name></author><author><name sortKey="Urquiza, L" uniqKey="Urquiza L">L Urquiza</name></author><author><name sortKey="Garcia Sanchez, Ma" uniqKey="Garcia Sanchez M">MA Garcia-Sanchez</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wozniak, Rj" uniqKey="Wozniak R">RJ Wozniak</name></author><author><name sortKey="Boyer, Me" uniqKey="Boyer M">ME Boyer</name></author><author><name sortKey="Grass, Ja" uniqKey="Grass J">JA Grass</name></author><author><name sortKey="Lee, Y" uniqKey="Lee Y">Y Lee</name></author><author><name sortKey="Bresnick, Eh" uniqKey="Bresnick E">EH Bresnick</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Khandekar, M" uniqKey="Khandekar M">M Khandekar</name></author><author><name sortKey="Brandt, W" uniqKey="Brandt W">W Brandt</name></author><author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y Zhou</name></author><author><name sortKey="Dagenais, S" uniqKey="Dagenais S">S Dagenais</name></author><author><name sortKey="Glover, Tw" uniqKey="Glover T">TW Glover</name></author><author><name sortKey="Suzuki, N" uniqKey="Suzuki N">N Suzuki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kranc, Kr" uniqKey="Kranc K">KR Kranc</name></author><author><name sortKey="Schepers, H" uniqKey="Schepers H">H Schepers</name></author><author><name sortKey="Rodrigues, Np" uniqKey="Rodrigues N">NP Rodrigues</name></author><author><name sortKey="Bamforth, S" uniqKey="Bamforth S">S Bamforth</name></author><author><name sortKey="Villadsen, E" uniqKey="Villadsen E">E Villadsen</name></author><author><name sortKey="Ferry, H" uniqKey="Ferry H">H Ferry</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, Y" uniqKey="Chen Y">Y Chen</name></author><author><name sortKey="Haviernik, P" uniqKey="Haviernik P">P Haviernik</name></author><author><name sortKey="Bunting, Kd" uniqKey="Bunting K">KD Bunting</name></author><author><name sortKey="Yang, Yc" uniqKey="Yang Y">YC Yang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kranc, Kr" uniqKey="Kranc K">KR Kranc</name></author><author><name sortKey="Bamforth, Sd" uniqKey="Bamforth S">SD Bamforth</name></author><author><name sortKey="Braganca, J" uniqKey="Braganca J">J Bragança</name></author><author><name sortKey="Norbury, C" uniqKey="Norbury C">C Norbury</name></author><author><name sortKey="Van Lohuizen, M" uniqKey="Van Lohuizen M">M van Lohuizen</name></author><author><name sortKey="Bhattacharya, S" uniqKey="Bhattacharya S">S Bhattacharya</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ding, X" uniqKey="Ding X">X Ding</name></author><author><name sortKey="Lin, Q" uniqKey="Lin Q">Q Lin</name></author><author><name sortKey="Ensenat Waser, R" uniqKey="Ensenat Waser R">R Ensenat-Waser</name></author><author><name sortKey="Rose John, S" uniqKey="Rose John S">S Rose-John</name></author><author><name sortKey="Zenke, M" uniqKey="Zenke M">M Zenke</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Korthuis, Pm" uniqKey="Korthuis P">PM Korthuis</name></author><author><name sortKey="Berger, G" uniqKey="Berger G">G Berger</name></author><author><name sortKey="Bakker, B" uniqKey="Bakker B">B Bakker</name></author><author><name sortKey="Rozenveld Geugien, M" uniqKey="Rozenveld Geugien M">M Rozenveld-Geugien</name></author><author><name sortKey="Jaques, J" uniqKey="Jaques J">J Jaques</name></author><author><name sortKey="De Haan, G" uniqKey="De Haan G">G de Haan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kamata, M" uniqKey="Kamata M">M Kamata</name></author><author><name sortKey="Okitsu, Y" uniqKey="Okitsu Y">Y Okitsu</name></author><author><name sortKey="Fujiwara, T" uniqKey="Fujiwara T">T Fujiwara</name></author><author><name sortKey="Kanehira, M" uniqKey="Kanehira M">M Kanehira</name></author><author><name sortKey="Nakajima, S" uniqKey="Nakajima S">S Nakajima</name></author><author><name sortKey="Takahashi, T" uniqKey="Takahashi T">T Takahashi</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, CA USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26325290</article-id><article-id pub-id-type="pmc">4556642</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0137079</article-id><article-id pub-id-type="publisher-id">PONE-D-15-19817</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells</article-title><alt-title alt-title-type="running-head">siRNA Screening to Identify Upstream Gene of GATA-2</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Saito</surname><given-names>Yo</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Fujiwara</surname><given-names>Tohru</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref><xref ref-type="aff" rid="aff002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Ohashi</surname><given-names>Keiichi</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Okitsu</surname><given-names>Yoko</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Fukuhara</surname><given-names>Noriko</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Onishi</surname><given-names>Yasushi</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Ishizawa</surname><given-names>Kenichi</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref><xref ref-type="aff" rid="aff003"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Harigae</surname><given-names>Hideo</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref><xref ref-type="aff" rid="aff002"><sup>2</sup></xref><xref rid="cor001" ref-type="corresp">*</xref></contrib></contrib-group><aff id="aff001"><label>1</label><addr-line>Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></aff><aff id="aff002"><label>2</label><addr-line>Molecular Hematology/Oncology, Tohoku University Graduate School of Medicine, Sendai, Japan</addr-line></aff><aff id="aff003"><label>3</label><addr-line>Department of Hematology and Cell Therapy, Yamagata University Faculty of Medicine, Yamagata, Japan</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Akagi</surname><given-names>Tadayuki</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>Kanazawa University, JAPAN</addr-line></aff><author-notes><fn fn-type="conflict" id="coi001"><p><bold>Competing Interests: </bold>This study was supported by KANAE Foundation for the Promotion of Medical Science (TF) (<ext-link ext-link-type="uri" xlink:href="http://www.kanae-zaidan.com">http://www.kanae-zaidan.com</ext-link>) which receives financial support from Sanofi KK, and by a research grant from Chugai Pharmaceutical Co., Ltd. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.</p></fn><fn fn-type="con" id="contrib001"><p>Conceived and designed the experiments: TF HH. Performed the experiments: YS TF KO. Analyzed the data: YS TF. Contributed reagents/materials/analysis tools: TF NF Y. Okitsu Y. Onishi KI HH. Wrote the paper: TF HH.</p></fn><corresp id="cor001">* E-mail: <email>harigae@med.tohoku.ac.jp</email></corresp></author-notes><pub-date pub-type="epub"><day>1</day><month>9</month><year>2015</year></pub-date><pub-date pub-type="collection"><year>2015</year></pub-date><volume>10</volume><issue>9</issue><elocation-id>e0137079</elocation-id><history><date date-type="received"><day>27</day><month>5</month><year>2015</year></date><date date-type="accepted"><day>12</day><month>8</month><year>2015</year></date></history><permissions><copyright-year>2015</copyright-year><copyright-holder>Saito et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited</license-p></license></permissions><self-uri content-type="pdf" xlink:type="simple" xlink:href="pone.0137079.pdf"></self-uri><abstract><p>Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA) library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with <italic>GATA-2</italic> intronic enhancer (+9.9 kb/1S); therefore, we established a cell line capable of stably expressing +9.9 kb/1S-Luciferase. Subsequently, we screened 995 transcription factor genes and revealed that <italic>CITED2</italic> acts as a GATA-2 activator in human hematopoietic cells. These results provide novel insights into and further identify the regulatory mechanism of GATA-2.</p></abstract><funding-group><funding-statement>This study was supported by KANAE Foundation for the Promotion of Medical Science (TF) (<ext-link ext-link-type="uri" xlink:href="http://www.kanae-zaidan.com">http://www.kanae-zaidan.com</ext-link>) which receives financial support from Sanofi KK, and by a research grant from Chugai Pharmaceutical Co., Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</funding-statement></funding-group><counts><fig-count count="6"></fig-count><table-count count="1"></table-count><page-count count="15"></page-count></counts><custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name><meta-value>All relevant data are within the paper and its Supporting Information files.</meta-value></custom-meta></custom-meta-group></article-meta><notes><title>Data Availability</title><p>All relevant data are within the paper and its Supporting Information files.</p></notes></front><body><sec sec-type="intro" id="sec001"><title>Introduction</title><p>Hematopoiesis is a complex process controlled by numerous transcription factors that regulate and coordinate the expression of lineage-specific genes [<xref rid="pone.0137079.ref001" ref-type="bibr">1</xref>]. Previous baseline studies have suggested that the GATA family of transcription factors, which act in developmental regulation, is directly involved in hematopoiesis [<xref rid="pone.0137079.ref002" ref-type="bibr">2</xref>–<xref rid="pone.0137079.ref005" ref-type="bibr">5</xref>]. GATA-1, GATA-2, and GATA-3 are known as the hematopoietic GATA factors, given their important roles in this process [<xref rid="pone.0137079.ref001" ref-type="bibr">1</xref>, <xref rid="pone.0137079.ref004" ref-type="bibr">4</xref>–<xref rid="pone.0137079.ref007" ref-type="bibr">7</xref>]. Among them, GATA-2 is required for the maintenance and expansion of hematopoietic stem cells (HSCs) and/or multipotent progenitors during early hematopoiesis [<xref rid="pone.0137079.ref005" ref-type="bibr">5</xref>, <xref rid="pone.0137079.ref008" ref-type="bibr">8</xref>–<xref rid="pone.0137079.ref011" ref-type="bibr">11</xref>].</p><p>To date, the mechanisms underlying GATA-2 transcription have been extensively analyzed. Two first exons/promoters of the gene, named 1S and 1G, have been identified in both mice and humans [<xref rid="pone.0137079.ref012" ref-type="bibr">12</xref>, <xref rid="pone.0137079.ref013" ref-type="bibr">13</xref>]. Transcripts involving the 1G promoter are commonly found in tissues expressing GATA-2, whereas 1S transcripts are believed to play an important role in hematopoietic cells [<xref rid="pone.0137079.ref012" ref-type="bibr">12</xref>, <xref rid="pone.0137079.ref013" ref-type="bibr">13</xref>]. During erythroid differentiation, GATA-2 levels decline concomitantly with an increase in GATA-1 levels [<xref rid="pone.0137079.ref005" ref-type="bibr">5</xref>]. GATA-1 represses <italic>Gata2</italic> transcription by displacing GATA-2 from the sites at −77, −3.9, −2.8, −1.8, and +9.5 kilobase (kb) relative to the 1S promoter, which are known as GATA switch sites [<xref rid="pone.0137079.ref005" ref-type="bibr">5</xref>, <xref rid="pone.0137079.ref014" ref-type="bibr">14</xref>]. However, despite the compelling evidence supporting the locations and characteristics of these GATA switch sites, targeted individual deletions of the −1.8, −2.8, and −3.9 kb sites lead to minor increases in <italic>Gata2</italic> expression in murine hematopoietic precursors [<xref rid="pone.0137079.ref015" ref-type="bibr">15</xref>–<xref rid="pone.0137079.ref017" ref-type="bibr">17</xref>]. On the other hand, deletion of the +9.5 site leads to delayed embryonic lethality compared with global <italic>Gata2</italic> knockout [<xref rid="pone.0137079.ref018" ref-type="bibr">18</xref>]. Noticeably, in humans, the heterozygous mutation of the intronic enhancer at +9.9 kb, which corresponds to +9.5 kb in mice, has been found in patients with GATA-2 deficiency (MonoMAC syndrome) [<xref rid="pone.0137079.ref018" ref-type="bibr">18</xref>], which is characterized by a predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) [<xref rid="pone.0137079.ref019" ref-type="bibr">19</xref>–<xref rid="pone.0137079.ref021" ref-type="bibr">21</xref>]. Thus, the 1S promoter and +9.5 kb enhancer regions could be considered as important regulatory regions for <italic>GATA-2</italic> expression.</p><p>Several transcription factors involved in various signaling pathways, such as the Wnt and Notch pathways, are known to participate in GATA-2 regulation [<xref rid="pone.0137079.ref022" ref-type="bibr">22</xref>, <xref rid="pone.0137079.ref023" ref-type="bibr">23</xref>]; however, relatively less is known about how these transcriptional molecular mechanism associate with GATA-2 expression. Given the pathophysiological links between GATA-2 and aplastic anemia, MonoMAC syndrome, and lung cancer [<xref rid="pone.0137079.ref019" ref-type="bibr">19</xref>–<xref rid="pone.0137079.ref021" ref-type="bibr">21</xref>, <xref rid="pone.0137079.ref024" ref-type="bibr">24</xref>–<xref rid="pone.0137079.ref026" ref-type="bibr">26</xref>], it is extremely important to clarify and comprehensively understand the details regarding the mechanisms behind the upstream transcription of <italic>GATA-2</italic>. With this in mind, we conducted a high-throughput screening analysis based on a small interfering RNA (siRNA) library to provide a novel insights into the factors involved in the regulation of GATA-2 expression.</p></sec><sec sec-type="materials|methods" id="sec002"><title>Materials and Methods</title><sec id="sec003"><title>Cell culture</title><p>The human erythroleukemia cell lines YN-1 [<xref rid="pone.0137079.ref027" ref-type="bibr">27</xref>] and K562 [<xref rid="pone.0137079.ref028" ref-type="bibr">28</xref>], human myeloid leukemia cell line KG1a [<xref rid="pone.0137079.ref029" ref-type="bibr">29</xref>], human T cell leukemia cell line Jurkat, human monocytoid cell line U937, and the human pre-B cell leukemia cell line NALM6 were cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Miami, FL, USA) and 1% penicillin/streptomycin (Sigma-Aldrich). Stable YN-1 cells expressing pGL4.20 (<italic>GATA-2</italic> +9.9/1S; described below) were cultured in RPMI-1640 containing 10% FBS, 1% penicillin/streptomycin, and 1 μg/ml puromycin (Sigma-Aldrich). K562 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA); other cell lines (YN-1, KG1a, Jurkat, U937, and NALM6) were obtained from the Cell Resource Center for Biomedical Research at Tohoku University (<ext-link ext-link-type="uri" xlink:href="http://www2.idac.tohoku.ac.jp/dep/ccr/">www2.idac.tohoku.ac.jp/dep/ccr/</ext-link>/).</p></sec><sec id="sec004"><title>Plasmids</title><p><italic>GATA-2</italic> sequences were cloned from bacterial artificial chromosome DNA (RP11-475N22: Empire Genomics, Buffalo, NY, USA). Primers linked to restriction enzyme sites were used to amplify the <italic>GATA-2</italic> genomic region to be included in the plasmids (primer sequences are available upon request). DNA sequence analysis was used to confirm the integrity of the cloned sequences. The luciferase reporter vectors, pGL3 (Luc) and pGL4.20 (Luc2/puro) and renilla vectors pRL and pGL4.74 were purchased from Promega (Madison, WI, USA). All restriction enzymes described below were purchased from Toyobo (Osaka, Japan).</p><p>The 1S-Luc construct was created by amplifying a 500-base pair (bp) sequence of the <italic>GATA-2</italic> 1S promoter region using the appropriate primer pair (<xref rid="pone.0137079.s003" ref-type="supplementary-material">S1 Table</xref>). This sequenced region was subsequently digested with MluI and BglII, and the digested products were purified and ligated into the pGL3 basic vector, which had been previously digested and purified using the same pair of restriction enzymes. The (−1)Luc and (−1.8)Luc plasmids were created using the NheI and BglII restriction enzymes by the same protocol used for 1SLuc. (−3.4)Luc, (−4.6)Luc, and (−4.6)Luc2 and using GeneArt SeamLess cloning (Invitrogen, Carlsbad, CA, USA). The <italic>GATA-2</italic> +9.9 kb site was inserted upstream of the 1SLuc, (−3.4)Luc, or (−4.6)Luc sequence. GATA and/or E-box mutated constructs were generated using a QuickChange™ Site-Directed Mutagenesis Kit (Agilent technologies, Santa Clara, CA, USA).</p><p>For GATA-2 overexpression, <italic>GATA-2</italic> mRNA was cloned into the pBABE-puro vector (Addgene Plasmid 1764; Addgene Cambridge, MA, USA).</p></sec><sec id="sec005"><title>Promoter assay</title><p>To evaluate the <italic>GATA-2</italic> transcriptional activity, aliquots of YN-1 cells were transfected with 1 μg of <italic>GATA-2</italic> promoter construct and 100 ng of the pRL or the pGL4.74 [<italic>hRluc</italic>/TK] vector (Promega) via FuGene HD (Promega). The cells were harvested 24 h after plasmid transfection, and firefly and <italic>Renilla</italic> luciferase activity levels in the cell extracts were determined using a Dual-Luciferase Reporter Assay System (Promega) or a ONE-Glo Luciferase Assay System, with GloMax 20/20 Luminometer (Promega).</p></sec><sec id="sec006"><title>Retroviral gene transfer</title><p>Retroviral GATA-2 expression was conducted as described previously [<xref rid="pone.0137079.ref030" ref-type="bibr">30</xref>].</p></sec><sec id="sec007"><title>siRNA screening</title><p>For siRNA screening, we targeted 995 genes encoding transcription factors from the siPerfect transcription factor library (RNAi, Tokyo, Japan). The library was arranged in 13 96-well plates (Thermo Scientific, Yokohama, Japan), with each well containing 5 pmol of a pool of 21-mer siRNA duplexes. Lamin A siRNA and nontarget siRNA were used as the positive and negative controls, respectively. Next, 1 × 10<sup>4</sup> stable YN-1 cells expressing pGL4.20 (<italic>GATA-2</italic> +9.9/1S) in 100 μl of RPMI containing puromycin were transferred to each of the 96 wells in each plate (Berthold, Land Baden-Württemberg, Germany). We added the transfection reagent GenomONE-Si (Ishihara Sangyo, Osaka, Japan) to the siRNA library plates according to the manufacturer’s protocol. Subsequently, we added 0.9 μl of siRNA transfection mixture per well (final concentration in wells, 5nM). siRNA transfection was conducted twice (at 0 and 24 h). At 48 h, we assessed the luciferase activity of each well using a ONE-Glo Luciferase Assay System (Promega) and Berthold Centro LB960 luminometer (Berthold). For the luciferase activity assay, we added 100 μl of ONE-Glo Reagent to each well on the plates and evaluated firefly luciferase activity with a luminometer after incubating the plates for 3 min at room temperature.</p></sec><sec id="sec008"><title>siRNA-mediated knockdown</title><p>For siRNA-mediated knockdown, 1 × 10<sup>6</sup> YN-1 cells with stable expression of pGL4.20 (<italic>GATA-2</italic> +9.9/1S) were transfected with an Amaxa Nucleofector (Nucleofector solution V, Nucleofector program T-016; Lonza Group, Basel, Switzerland). The antisense siRNA sequences specific for human Brahma related gene 1 (<italic>BRG1</italic>) were: GCACACCGCUGCAGAACAA, CCAAGCCGGUCGUGAGUGA, GCGACUCACUGACGGAGAA, and GACCAGCACUCCCAAGGUU. For human CBP/p300-Interacting Transactivator with Glu/Asp-Rich C-Terminal Domain, 2 (<italic>CITED2</italic>) knockdown, we used the sequences GUUCUUAUGUCCUUGGUGA, CAACCAGUAUUUCAACCAU, GAAAUGGGUUUGGACCGCA, and CUGCAGGCCACCAGAUGAA (Dharmacon, Lafayette, CO, USA). The siGENOME non-targeting siRNA pool #1 (Thermo Scientific Dharmacon) was used as a negative control. siRNA sequences specific for <italic>GATA-2</italic> knockdown and the corresponding negative control were as described previously [<xref rid="pone.0137079.ref031" ref-type="bibr">31</xref>].</p></sec><sec id="sec009"><title>CRISPR/Cas9-based genome editing</title><p>To knockout endogenous CITED2 in YN-1 cells, the GeneArt CRISPR Nuclease Vector with CD4 enrichment kit (GE Life Sciences/Dharmacon) was used. The target sequence for nuclease recruitment was CATATGATGGCCATGAACCACGG (+25 to +47 from the translation initiation site, according to NCBI accession number NM_001168389). The plasmid was transfected using an Amaxa Nucleofector (Nucleofector solution V, Nucleofector program T-016; Lonza Group), and CD4-positive cells were subsequently cloned.</p></sec><sec id="sec010"><title>Real-time quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR)</title><p>Real-time quantitative RT-PCR was conducted using the SYBR Green master mix (Qiagen, Venlo, The Netherlands) according to a previously described protocol [<xref rid="pone.0137079.ref032" ref-type="bibr">32</xref>]. All primer sequences are listed in <xref rid="pone.0137079.s003" ref-type="supplementary-material">S1 Table</xref>.</p></sec><sec id="sec011"><title>Western blotting analysis</title><p>Whole-cell lysates were prepared by boiling 1 × 10<sup>7</sup> cells/ml in sodium dodecyl sulfate (SDS) buffer [<xref rid="pone.0137079.ref032" ref-type="bibr">32</xref>]. Samples (5 μl) were resolved using SDS-polyacrylamide gel electrophoresis and analyzed with specific antibodies.</p></sec><sec id="sec012"><title>Antibodies</title><p>Antibodies specific for GATA-2 (H-116, sc-9008) and CITED2 (JA22, sc-21795) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A specific alpha-tubulin antibody was purchased from Calbiochem (Darmstadt, Germany). CITED2-specific antibody (EPR3416, ab108345) and rabbit control IgG were purchased from abcam (Cambridge, MA, USA).</p></sec><sec id="sec013"><title>Statistics</title><p>Statistical significance was assessed with a two-sided Student’s <italic>t</italic> test.</p></sec></sec><sec id="sec014"><title>Results and Discussion</title><sec id="sec015"><title><italic>GATA-2</italic> 1S transcript is highly expressed in the YN-1 cell line</title><p>To conduct high-throughput siRNA screening, the selection of a hematopoietic cell line that expresses high levels of the target gene, in this case <italic>GATA-2</italic>, is desirable. In this study, we selected six hematopoietic cell lines, YN-1, K562, KG1a, Jurkat, U937, and NALM6, as candidate lines. Real-time quantitative RT-PCR and western blot analyses revealed that GATA-2 was abundantly expressed on both the gene and protein levels in YN-1, K562, and KG1a cell lines, but was almost undetectable in Jurkat, U937, and Nalm6 cells (<xref rid="pone.0137079.g001" ref-type="fig">Fig 1A and 1B</xref>). The validity of the GATA-2 antibody was confirmed by both GATA-2 overexpression and knockdown in K562 cells (<xref rid="pone.0137079.s001" ref-type="supplementary-material">S1 Fig</xref>). As previously described, GATA-2 transcription involves two different exons, a distal (1S) and a proximal (1G) promoter; the former is considered to be particularly important in hematopoietic cells [<xref rid="pone.0137079.ref005" ref-type="bibr">5</xref>, <xref rid="pone.0137079.ref012" ref-type="bibr">12</xref>, <xref rid="pone.0137079.ref013" ref-type="bibr">13</xref>], although it remained unknown regarding the contribution of 1S and 1G transcripts to the absolute level of GATA-2 expression in hematopoietic cells. Therefore, we conducted a quantitative RT-PCR analysis of each transcript. The <italic>GATA-2</italic> 1G transcript was expressed in YN-1, K562, and KG1a cells (<xref rid="pone.0137079.g001" ref-type="fig">Fig 1C</xref>); noticeably, GATA-2 1S was the most abundantly expressed in YN-1 cells (<xref rid="pone.0137079.g001" ref-type="fig">Fig 1C</xref>). KG1a cells were derived from a human patient who presented with acute myelogenous leukemia characterized by an undifferentiated blast cell phenotype [<xref rid="pone.0137079.ref029" ref-type="bibr">29</xref>]. Therefore, the cells might have retained the characteristics of HSCs and thereby expressed GATA-2. On the other hand, the K562 and YN-1 lines were derived from a patient with chronic myelogenous leukemia [<xref rid="pone.0137079.ref027" ref-type="bibr">27</xref>, <xref rid="pone.0137079.ref028" ref-type="bibr">28</xref>]. Although GATA-2 expression has already been confirmed in K562 cells [<xref rid="pone.0137079.ref028" ref-type="bibr">28</xref>, <xref rid="pone.0137079.ref032" ref-type="bibr">32</xref>], the relative expression levels of 1S and 1G transcript had not yet been characterized. In addition, GATA-2 expression had not been evaluated in YN-1 cells, and the cause of the abundant expression of <italic>GATA-2</italic> 1S in this cell line remained unknown. In the current study, we selected the YN-1 cell line for siRNA screening.</p><fig id="pone.0137079.g001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0137079.g001</object-id><label>Fig 1</label><caption><title><italic>GATA-2</italic> 1S expression in YN-1 cells.</title><p>Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting were performed to measure GATA-2 expression in six hematopoietic cell lines. (A) Quantitative RT-PCR for <italic>GATA-2</italic> mRNA (mean ± standard error [SE], n = 3). GAPDH mRNA was used as a control. The <italic>GATA-2</italic> mRNA expression level in YN-1 cells was set to 1. (B) Anti-GATA-2 western blotting analysis of whole-cell extracts from six cell lines. Alpha-tubulin was used as a loading control. The asterisk demotes cross-reactive band. (C) Quantitative RT-PCR for <italic>GATA-2</italic> 1S or 1G mRNA (mean ± SE, n = 3). <italic>GAPDH</italic> mRNA was used as a control. The <italic>GATA-2</italic><italic>1S</italic> and <italic>1G</italic> mRNA expression levels in YN-1 cells were set to 1.</p></caption><graphic xlink:href="pone.0137079.g001"></graphic></fig></sec><sec id="sec016"><title><italic>GATA-2</italic> 1S promoter fused to +9.9 kb enhancer induced high promoter activity</title><p>We conducted a transient luciferase promoter analysis to determine the optimal configuration for siRNA screening. Based on the importance of 1S promoter for GATA-2 expression in hematopoietic cells [<xref rid="pone.0137079.ref012" ref-type="bibr">12</xref>, <xref rid="pone.0137079.ref013" ref-type="bibr">13</xref>], we selected 1S promoter as a potential promising tool to identify novel upstream factor of GATA-2 in hematopoietic cells. As shown in <xref rid="pone.0137079.g002" ref-type="fig">Fig 2A</xref>, the 1S promoter exhibited a modest promoter activity, and the addition of a +9.9 kb enhancer to this promoter (+9.9/1S) resulted in a higher luciferase activity. On the other hand, addition of -4.6 to -0.5 kb fragment to the 1S promoter led to lower promoter activity compared with that of the 1S promoter alone (<xref rid="pone.0137079.g002" ref-type="fig">Fig 2A</xref>). The reason for lower promoter activity following addition of −4.6 to −1 kb sequence could be partially explained by the repressive effect of CEBPA (CCAAT/enhancer binding protein, alpha) through its binding site at −1.2 and −2.4 kb sites (<xref rid="pone.0137079.g002" ref-type="fig">Fig 2A</xref>) [<xref rid="pone.0137079.ref033" ref-type="bibr">33</xref>]. Low promoter activity based on a sequence alteration at −1 kb, which does not include CEBPA binding sites (<xref rid="pone.0137079.g002" ref-type="fig">Fig 2A</xref>), might imply the presence of other unrecognized repressive elements at this position. Regarding the functional validation of the sequence +9.9/1S, we introduced a mutation within the GATA-binding element and/or E-box motifs and demonstrated that the GATA-binding domain plays an important role in luciferase activity induced by the +9.9/1S construct (<xref rid="pone.0137079.g002" ref-type="fig">Fig 2B</xref>). Wozniak et al. [<xref rid="pone.0137079.ref034" ref-type="bibr">34</xref>] analyzed the murine +9.5/1S construct in detail; these authors demonstrated that the +9.5/1S construct showed a high luciferase activity, and the disruption of the GATA binding element within +9.5 kb eliminated its enhancer activity. Therefore, we selected the +9.9 kb/1S-luciferase construct for the screening.</p><fig id="pone.0137079.g002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0137079.g002</object-id><label>Fig 2</label><caption><title><italic>GATA-2</italic> 1S promoter activity is associated with the +9.9 kb enhancer.</title><p>(A) The enhancer activities of the constructs, which included <italic>GATA-2</italic> upstream or intron regions with various combinations of <italic>GATA</italic> binding sites, CCAAT/enhancer binding protein, alpha (<italic>CEBPA</italic>) binding sites, and an E-box. YN-1 cells were transiently cotransfected with Renilla (pRL) and luciferase vectors (Luc). The luciferase vector was either empty or contained the <italic>GATA-2 1S</italic> promoter cloned upstream of luciferase (1SLuc), with or without the upstream 1S promoter sequence and/or +9.9 kb site. Luciferase activity was measured 24 h after the first transfection. Results are presented as ratios of luciferase activities (means of Firefly/Renilla ± standard error, n = 3). (B) Impact of the GATA-binding site and/or E-box site mutation on the +9.9/1S promoter activity. We generated wild-type, E-box only, GATA-binding site only and double-mutation constructs fused to a luciferase reporter gene and performed a transient transfection assay in YN-1 cells (mean ± standard deviation, n = 3). * p<0.05.</p></caption><graphic xlink:href="pone.0137079.g002"></graphic></fig></sec><sec id="sec017"><title>Establishment of YN-1 cells with stable luciferase activity under the control of +9.9 kb/1S</title><p>A luciferase plasmid was introduced into the YN-1 cells, which were subsequently selected using puromycin to ensure stable expression. We used an empty luciferase vector (Luc2) and a vector containing a +9.9/1S or −4.6 kb sequence. Similar to the results of the transient luciferase assay (<xref rid="pone.0137079.g002" ref-type="fig">Fig 2</xref>), the luciferase activity was highest in the YN-1 cells expressing the +9.9 kb/1S construct (<xref rid="pone.0137079.g003" ref-type="fig">Fig 3A</xref>). Next, to test whether an already known GATA-2 regulator might affect the luciferase activity, we knocked down <italic>BRG1</italic> [<xref rid="pone.0137079.ref015" ref-type="bibr">15</xref>]. Previous reports have shown that <italic>Brg1</italic> knockdown in G1E cells repressed the expression of GATA-2, through its effect on the +9.5 enhancer [<xref rid="pone.0137079.ref015" ref-type="bibr">15</xref>]. As shown in <xref rid="pone.0137079.g003" ref-type="fig">Fig 3B–3E</xref>, knockdown of <italic>BRG1</italic> resulted in a significant downregulation of <italic>GATA-2</italic> expression and luciferase activity.</p><fig id="pone.0137079.g003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0137079.g003</object-id><label>Fig 3</label><caption><title>Establishment of YN-1 cells showing stable luciferase activity.</title><p>(A) YN-1 cells were transfected with pGL4.20 (GATA-2 +9.9/1S or -4.6) and selected using puromycin. (B) Small-interfering-RNA (siRNA)-mediated knockdown of Brahma related gene 1 (<italic>BRG1</italic>) was conducted in YN-1 cells stably expressing luciferase activity under the control of +9.9/1S. Transfection was conducted twice via electroporation, at 24-h intervals. The cells were harvested for assays 48 h after the first transfection, and quantitative reverse transcriptase-polymerase chain reaction for Brahma related gene 1 (BRG1) was conducted (mean ± standard error [SE], n = 3, p < 0.001). (C-E) <italic>BRG1</italic> knockdown resulted in the significant downregulation of both <italic>GATA-2</italic> expression (mean ± SE, n = 3, p < 0.001) (C), <italic>GATA-2</italic> 1S transcript (mean ± SE, n = 3, p < 0.001) (D), and luciferase activity (mean ± SE, n = 3, p = 0.0044) (E). * p<0.05.</p></caption><graphic xlink:href="pone.0137079.g003"></graphic></fig></sec><sec id="sec018"><title>High-throughput siRNA screening identified <italic>GATA-2</italic> transcription regulators</title><p>To discover novel regulators of GATA-2 expression, we conducted siRNA-based high-throughput screening. As shown in <xref rid="pone.0137079.g004" ref-type="fig">Fig 4A</xref>, YN-1 cells expressing +9.9 kb/1S-Luc2 were transfected twice with siRNA at 24-h intervals, and luciferase activity was assessed after 48 h. We used an siRNA library that contained a total 995 genes involved in transcriptional regulation. To test whether our transfection protocol could efficiently knockdown the target genes, siRNA directed against Lamin A was included as a positive control, and knockdown of the encoding gene was confirmed via quantitative RT-PCR (<xref rid="pone.0137079.g004" ref-type="fig">Fig 4B</xref>).</p><fig id="pone.0137079.g004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0137079.g004</object-id><label>Fig 4</label><caption><title>High-throughput small interfering RNA (siRNA) screening to identify regulators of <italic>GATA-2</italic> transcription.</title><p>(A) Schematic representation of the high-throughput siRNA screening. YN-1 cells with stable +9.9/1S-<italic>Luc2</italic> expression were selected using puromycin. Subsequently, the cells were transfected twice at 24-h intervals. Luciferase activity was assessed 48 h after the first transfection. We used an siPerfect siRNA library (RNAi) that targeted 995 transcription factor genes. (B) We used <italic>Lamin A</italic> siRNA to validate the efficiency of the transfection protocol (mean ± standard error, n = 3, p < 0.001). * p<0.05.</p></caption><graphic xlink:href="pone.0137079.g004"></graphic></fig><p>Following the screening analysis, the top 10% of the genes that significantly (p < 0.05) repressed luciferase activity after their knockdown were selected from each plate (n = 83: <xref rid="pone.0137079.t001" ref-type="table">Table 1</xref>, <xref rid="pone.0137079.s004" ref-type="supplementary-material">S2 Table</xref>), which included potential GATA-2 regulators of GATA-1 and GFI1B. On the other hand, however, the analysis did not identify known GATA-2 regulators, such as TAL1 [<xref rid="pone.0137079.ref032" ref-type="bibr">32</xref>, <xref rid="pone.0137079.ref035" ref-type="bibr">35</xref>], presumably due to the limited screening sensitivity. Subsequently, we narrowed down the genes, based on their potential importance in hematopoiesis. In the present study, we focused on the gene CITED2 (Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2).</p><table-wrap id="pone.0137079.t001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0137079.t001</object-id><label>Table 1</label><caption><title>Candidates of GATA-2 activators.</title></caption><alternatives><graphic id="pone.0137079.t001g" xlink:href="pone.0137079.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col></colgroup><thead><tr><th align="left" rowspan="1" colspan="1">Plate No.</th><th align="left" rowspan="1" colspan="1">Symbol</th><th align="left" rowspan="1" colspan="1">Fold change</th><th align="left" rowspan="1" colspan="1">SE</th><th align="left" rowspan="1" colspan="1">p-value</th><th align="left" rowspan="1" colspan="1">Plate No.</th><th align="left" rowspan="1" colspan="1">Symbol</th><th align="left" rowspan="1" colspan="1">Fold change</th><th align="left" rowspan="1" colspan="1">SE</th><th align="left" rowspan="1" colspan="1">p-value</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">MDM2</td><td align="right" rowspan="1" colspan="1">0.73</td><td align="right" rowspan="1" colspan="1">0.14</td><td align="right" rowspan="1" colspan="1">0.006</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">CITED2</td><td align="right" rowspan="1" colspan="1">0.39</td><td align="right" rowspan="1" colspan="1">0.03</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">DDX54</td><td align="right" rowspan="1" colspan="1">0.74</td><td align="right" rowspan="1" colspan="1">0.11</td><td align="right" rowspan="1" colspan="1">0.004</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">CNOT7</td><td align="right" rowspan="1" colspan="1">0.40</td><td align="right" rowspan="1" colspan="1">0.03</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="right" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">HEXIM1</td><td align="right" rowspan="1" colspan="1">0.77</td><td align="right" rowspan="1" colspan="1">0.14</td><td align="right" rowspan="1" colspan="1">0.013</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BRDT</td><td align="right" rowspan="1" colspan="1">0.41</td><td align="right" rowspan="1" colspan="1">0.01</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">CXXC1</td><td align="right" rowspan="1" colspan="1">0.79</td><td align="right" rowspan="1" colspan="1">0.10</td><td align="right" rowspan="1" colspan="1">0.009</td><td align="right" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">CNOT2</td><td align="right" rowspan="1" colspan="1">0.42</td><td align="right" rowspan="1" colspan="1">0.09</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">MED12</td><td align="right" rowspan="1" colspan="1">0.79</td><td align="right" rowspan="1" colspan="1">0.11</td><td align="right" rowspan="1" colspan="1">0.012</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">CITED1</td><td align="right" rowspan="1" colspan="1">0.42</td><td align="right" rowspan="1" colspan="1">0.01</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">MIXL1</td><td align="right" rowspan="1" colspan="1">0.74</td><td align="right" rowspan="1" colspan="1">0.05</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BRCA1</td><td align="right" rowspan="1" colspan="1">0.43</td><td align="right" rowspan="1" colspan="1">0.04</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">ESRRB</td><td align="right" rowspan="1" colspan="1">0.78</td><td align="right" rowspan="1" colspan="1">0.11</td><td align="right" rowspan="1" colspan="1">0.007</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">EGR1</td><td align="right" rowspan="1" colspan="1">0.43</td><td align="right" rowspan="1" colspan="1">0.04</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">MED31</td><td align="right" rowspan="1" colspan="1">0.78</td><td align="right" rowspan="1" colspan="1">0.07</td><td align="right" rowspan="1" colspan="1">0.002</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">DLX3</td><td align="right" rowspan="1" colspan="1">0.43</td><td align="right" rowspan="1" colspan="1">0.06</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="right" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">HOP</td><td align="right" rowspan="1" colspan="1">0.78</td><td align="right" rowspan="1" colspan="1">0.12</td><td align="right" rowspan="1" colspan="1">0.009</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">FOXD1</td><td align="right" rowspan="1" colspan="1">0.69</td><td align="right" rowspan="1" colspan="1">0.09</td><td align="right" rowspan="1" colspan="1">0.014</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">EN1</td><td align="right" rowspan="1" colspan="1">0.80</td><td align="right" rowspan="1" colspan="1">0.03</td><td align="right" rowspan="1" colspan="1">0.001</td><td align="right" rowspan="1" colspan="1">8</td><td align="left" rowspan="1" colspan="1">GATA1</td><td align="right" rowspan="1" colspan="1">0.71</td><td align="right" rowspan="1" colspan="1">0.13</td><td align="right" rowspan="1" colspan="1">0.025</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">ERG</td><td align="right" rowspan="1" colspan="1">0.82</td><td align="right" rowspan="1" colspan="1">0.04</td><td align="right" rowspan="1" colspan="1">0.004</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">GFI1B</td><td align="right" rowspan="1" colspan="1">0.75</td><td align="right" rowspan="1" colspan="1">0.06</td><td align="right" rowspan="1" colspan="1">0.035</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">HLX1</td><td align="right" rowspan="1" colspan="1">0.83</td><td align="right" rowspan="1" colspan="1">0.09</td><td align="right" rowspan="1" colspan="1">0.018</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">KLF11</td><td align="right" rowspan="1" colspan="1">0.57</td><td align="right" rowspan="1" colspan="1">0.09</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">LEF1</td><td align="right" rowspan="1" colspan="1">0.83</td><td align="right" rowspan="1" colspan="1">0.06</td><td align="right" rowspan="1" colspan="1">0.009</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">MYOD1</td><td align="right" rowspan="1" colspan="1">0.60</td><td align="right" rowspan="1" colspan="1">0.05</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">MYT1L</td><td align="right" rowspan="1" colspan="1">0.61</td><td align="right" rowspan="1" colspan="1">0.11</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">KLF5</td><td align="right" rowspan="1" colspan="1">0.63</td><td align="right" rowspan="1" colspan="1">0.13</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">RARG</td><td align="right" rowspan="1" colspan="1">0.71</td><td align="right" rowspan="1" colspan="1">0.02</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="right" rowspan="1" colspan="1">9</td><td align="left" rowspan="1" colspan="1">MLL</td><td align="right" rowspan="1" colspan="1">0.64</td><td align="right" rowspan="1" colspan="1">0.04</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">MYST2</td><td align="right" rowspan="1" colspan="1">0.71</td><td align="right" rowspan="1" colspan="1">0.18</td><td align="right" rowspan="1" colspan="1">0.006</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">ISGF3G</td><td align="right" rowspan="1" colspan="1">0.64</td><td align="right" rowspan="1" colspan="1">0.04</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="right" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">NKX2-2</td><td align="right" rowspan="1" colspan="1">0.71</td><td align="right" rowspan="1" colspan="1">0.03</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">NAB2</td><td align="right" rowspan="1" colspan="1">0.65</td><td align="right" rowspan="1" colspan="1">0.06</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">NPAS3</td><td align="right" rowspan="1" colspan="1">0.73</td><td align="right" rowspan="1" colspan="1">0.04</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">NEUROG1</td><td align="right" rowspan="1" colspan="1">0.65</td><td align="right" rowspan="1" colspan="1">0.06</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">NR1H2</td><td align="right" rowspan="1" colspan="1">0.74</td><td align="right" rowspan="1" colspan="1">0.10</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">FTHL8</td><td align="right" rowspan="1" colspan="1">0.68</td><td align="right" rowspan="1" colspan="1">0.09</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">PKNOX2</td><td align="right" rowspan="1" colspan="1">0.75</td><td align="right" rowspan="1" colspan="1">0.07</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">PLAG1</td><td align="right" rowspan="1" colspan="1">0.59</td><td align="right" rowspan="1" colspan="1">0.06</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">MTA3</td><td align="right" rowspan="1" colspan="1">0.75</td><td align="right" rowspan="1" colspan="1">0.21</td><td align="right" rowspan="1" colspan="1">0.025</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">PTTG1</td><td align="right" rowspan="1" colspan="1">0.60</td><td align="right" rowspan="1" colspan="1">0.05</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">NFY</td><td align="right" rowspan="1" colspan="1">0.57</td><td align="right" rowspan="1" colspan="1">0.08</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">NFRKB</td><td align="right" rowspan="1" colspan="1">0.61</td><td align="right" rowspan="1" colspan="1">0.01</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">VDR</td><td align="right" rowspan="1" colspan="1">0.59</td><td align="right" rowspan="1" colspan="1">0.04</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="right" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1">POUF2</td><td align="right" rowspan="1" colspan="1">0.63</td><td align="right" rowspan="1" colspan="1">0.08</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">PPARD</td><td align="right" rowspan="1" colspan="1">0.59</td><td align="right" rowspan="1" colspan="1">0.05</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">PIAS1</td><td align="right" rowspan="1" colspan="1">0.64</td><td align="right" rowspan="1" colspan="1">0.06</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="right" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">ZFHX1B</td><td align="right" rowspan="1" colspan="1">0.60</td><td align="right" rowspan="1" colspan="1">0.09</td><td align="right" rowspan="1" colspan="1">0.002</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">NR3C1</td><td align="right" rowspan="1" colspan="1">0.64</td><td align="right" rowspan="1" colspan="1">0.06</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">CBFA2T2</td><td align="right" rowspan="1" colspan="1">0.60</td><td align="right" rowspan="1" colspan="1">0.08</td><td align="right" rowspan="1" colspan="1">0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">POU2F1</td><td align="right" rowspan="1" colspan="1">0.64</td><td align="right" rowspan="1" colspan="1">0.09</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">ZNF694</td><td align="right" rowspan="1" colspan="1">0.60</td><td align="right" rowspan="1" colspan="1">0.05</td><td align="right" rowspan="1" colspan="1">0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">POU6F1</td><td align="right" rowspan="1" colspan="1">0.64</td><td align="right" rowspan="1" colspan="1">0.05</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">NKX2-5</td><td align="right" rowspan="1" colspan="1">0.61</td><td align="right" rowspan="1" colspan="1">0.04</td><td align="right" rowspan="1" colspan="1">0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">TBX4</td><td align="right" rowspan="1" colspan="1">0.62</td><td align="right" rowspan="1" colspan="1">0.04</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">ZFHX4</td><td align="right" rowspan="1" colspan="1">0.61</td><td align="right" rowspan="1" colspan="1">0.02</td><td align="right" rowspan="1" colspan="1">0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">TCEA1</td><td align="right" rowspan="1" colspan="1">0.71</td><td align="right" rowspan="1" colspan="1">0.09</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">FOXF2</td><td align="right" rowspan="1" colspan="1">0.55</td><td align="right" rowspan="1" colspan="1">0.03</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">TBX22</td><td align="right" rowspan="1" colspan="1">0.71</td><td align="right" rowspan="1" colspan="1">0.02</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">HEYL</td><td align="right" rowspan="1" colspan="1">0.63</td><td align="right" rowspan="1" colspan="1">0.13</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="right" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">TSFM</td><td align="right" rowspan="1" colspan="1">0.71</td><td align="right" rowspan="1" colspan="1">0.01</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">GTF2IRD1</td><td align="right" rowspan="1" colspan="1">0.67</td><td align="right" rowspan="1" colspan="1">0.16</td><td align="right" rowspan="1" colspan="1">0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">TCF8</td><td align="right" rowspan="1" colspan="1">0.73</td><td align="right" rowspan="1" colspan="1">0.18</td><td align="right" rowspan="1" colspan="1">0.016</td></tr><tr><td align="right" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">ELF4</td><td align="right" rowspan="1" colspan="1">0.73</td><td align="right" rowspan="1" colspan="1">0.08</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">TCEB3B</td><td align="right" rowspan="1" colspan="1">0.75</td><td align="right" rowspan="1" colspan="1">0.02</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">FOXF1</td><td align="right" rowspan="1" colspan="1">0.76</td><td align="right" rowspan="1" colspan="1">0.09</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">TBX3</td><td align="right" rowspan="1" colspan="1">0.76</td><td align="right" rowspan="1" colspan="1">0.04</td><td align="right" rowspan="1" colspan="1">0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">FOXA2</td><td align="right" rowspan="1" colspan="1">0.77</td><td align="right" rowspan="1" colspan="1">0.07</td><td align="right" rowspan="1" colspan="1">< 0.001</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">TCFL5</td><td align="right" rowspan="1" colspan="1">0.80</td><td align="right" rowspan="1" colspan="1">0.03</td><td align="right" rowspan="1" colspan="1">0.003</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">HCFC1</td><td align="right" rowspan="1" colspan="1">0.77</td><td align="right" rowspan="1" colspan="1">0.17</td><td align="right" rowspan="1" colspan="1">0.018</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">ZNF140</td><td align="right" rowspan="1" colspan="1">0.64</td><td align="right" rowspan="1" colspan="1">0.04</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">MEF2D</td><td align="right" rowspan="1" colspan="1">0.78</td><td align="right" rowspan="1" colspan="1">0.13</td><td align="right" rowspan="1" colspan="1">0.007</td><td align="right" rowspan="1" colspan="1">13</td><td align="left" rowspan="1" colspan="1">NZF161</td><td align="right" rowspan="1" colspan="1">0.67</td><td align="right" rowspan="1" colspan="1">0.13</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">TFE3</td><td align="right" rowspan="1" colspan="1">0.74</td><td align="right" rowspan="1" colspan="1">0.16</td><td align="right" rowspan="1" colspan="1">0.018</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">ZNF35</td><td align="right" rowspan="1" colspan="1">0.67</td><td align="right" rowspan="1" colspan="1">0.02</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">T</td><td align="right" rowspan="1" colspan="1">0.76</td><td align="right" rowspan="1" colspan="1">0.00</td><td align="right" rowspan="1" colspan="1">0.002</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">ZIC1</td><td align="right" rowspan="1" colspan="1">0.67</td><td align="right" rowspan="1" colspan="1">0.11</td><td align="right" rowspan="1" colspan="1">< 0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">TCEB3</td><td align="right" rowspan="1" colspan="1">0.76</td><td align="right" rowspan="1" colspan="1">0.08</td><td align="right" rowspan="1" colspan="1">0.005</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="right" rowspan="1" colspan="1">6</td><td align="left" rowspan="1" colspan="1">ZNF398</td><td align="right" rowspan="1" colspan="1">0.77</td><td align="right" rowspan="1" colspan="1">0.07</td><td align="right" rowspan="1" colspan="1">0.005</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">VSX1</td><td align="right" rowspan="1" colspan="1">0.77</td><td align="right" rowspan="1" colspan="1">0.11</td><td align="right" rowspan="1" colspan="1">0.012</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">ARID4A</td><td align="right" rowspan="1" colspan="1">0.78</td><td align="right" rowspan="1" colspan="1">0.06</td><td align="right" rowspan="1" colspan="1">0.006</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">WT1</td><td align="right" rowspan="1" colspan="1">0.85</td><td align="right" rowspan="1" colspan="1">0.04</td><td align="right" rowspan="1" colspan="1">0.035</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr></tbody></table></alternatives><table-wrap-foot><fn id="t001fn001"><p>Fold change represents mean luciferase activity of each sample relative to that of negative control (n = 3).</p></fn></table-wrap-foot></table-wrap><p>We next knocked down <italic>CITED2</italic> using a pool of four synthetic siRNAs based on YN-1 cells expressing +9.9 kb/1S-Luc2. As shown in <xref rid="pone.0137079.g005" ref-type="fig">Fig 5A</xref>, the expression of <italic>CITED2</italic> genes was significantly repressed (by 12%) after siRNA transfection, a result that was confirmed by western blot analysis. Accordingly, we confirmed that <italic>GATA-2</italic> mRNA expression, GATA-2 1S transcript, and luciferase activity significantly decreased (by 63%, 55%, and 74%, respectively), under these conditions (<xref rid="pone.0137079.g005" ref-type="fig">Fig 5B</xref>). We further established two CITED2-knockout YN-1 cell clones based on CRISPR/Cas9 technique and confirmed these clones by western blotting (<xref rid="pone.0137079.g005" ref-type="fig">Fig 5C</xref>). Quantitative RT-PCR analysis confirmed significant downregulation of <italic>GATA-2</italic> mRNA and 1S transcript (<xref rid="pone.0137079.g005" ref-type="fig">Fig 5D</xref>). To reveal how CITED2 contributes to the GATA-2 expression in general, we also performed siRNA-mediated CITED2 knockdown based on K562 cells, which exhibited relatively lower 1S transcript level than YN-1 cells (<xref rid="pone.0137079.g001" ref-type="fig">Fig 1C</xref>). As shown in <xref rid="pone.0137079.g006" ref-type="fig">Fig 6</xref>, CITED2 knockdown in K562 cells resulted in significant downregulation of total GATA-2 mRNA as well as both 1S and 1G transcript levels, suggesting that CITED2 might not specifically activate +9.9 kb/1S promoter. Furthermore, to test if CITED2 would specifically affect +9.9 kb enhancer activity, luciferase analysis was conducted based on YN-1 cells and CITED2-knockout YN-1 cell clone (#1), demonstrating that CITED2 depletion significantly reduced both 1S alone and +9.9kb/1S-luciferase activity (<xref rid="pone.0137079.s002" ref-type="supplementary-material">S2 Fig</xref>). Thus, CITED2 might also regulate GATA-2 expression by impacting upon promoter region. Whereas our results indicated that CITED2 is an upstream regulator of GATA-2 in hematopoietic cells, further analysis would be required to reveal the molecular mechanism by which CITED2 contributes to the GATA-2 expression.</p><fig id="pone.0137079.g005" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0137079.g005</object-id><label>Fig 5</label><caption><title>Identification of CBP/p300-Interacting Transactivator with Glu/Asp-Rich C-Terminal Domain 2 (CITED2) as an activator of GATA-2 expression activators.</title><p>(A, B) <italic>CITED2</italic> was knocked down with a pool of four synthetic small interfering RNAs (siRNAs) (Dharmacon); this gene was targeted in YN-1 cells expressing +9.9 kb/1S-Luc2 to validate the screening analysis. The knockdown process was repeated twice at 24-h intervals. The cells were harvested and assayed 48 h after the first transfection. (A) <italic>CITED2</italic> mRNA was significantly repressed, by 12%, after siRNA transfection (mean ± standard error [SE], n = 3, p < 0.001), and this result was confirmed by western blotting. Alpha-tubulin was used as a loading control. (B) <italic>GATA-2</italic> mRNA expression, <italic>GATA-2</italic> 1S transcript, and luciferase activity decreased by 63% (mean ± SE, n = 3, p < 0.001), 55% (mean ± SE, n = 3, p < 0.001), and 74% (mean ± SE, n = 3, p < 0.001), respectively. (C, D) CITED2 knockout clones (#1, #2), established from YN-1 cells, and confirmed by western blotting (C). In these clones, <italic>GATA-2</italic> mRNA expression and <italic>GATA-2</italic> 1S transcript were significantly decreased (mean ± SE, n = 3, p < 0.001) (D). * p<0.05.</p></caption><graphic xlink:href="pone.0137079.g005"></graphic></fig><fig id="pone.0137079.g006" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0137079.g006</object-id><label>Fig 6</label><caption><title><italic>CBP/p300-Interacting Transactivator with Glu/Asp-Rich C-Terminal Domain 2</italic> (CITED2) regulates GATA-2 expression in K562 cells.</title><p>(A) <italic>CITED2</italic> was knocked down with a pool of four synthetic small interfering RNAs (siRNAs) (Dharmacon) in K562 cells. The knockdown process was repeated twice at 24-h intervals. The cells were harvested and assayed 48 h after the first transfection. (A) <italic>CITED2</italic> mRNA was significantly repressed after siRNA transfection (mean ± standard error [SE], n = 3, p < 0.001), and this result was confirmed by western blotting. Actin was used as a loading control. (B,C) <italic>GATA-2</italic> mRNA expression and <italic>GATA-2</italic> 1S and 1G transcripts were significantly decreased (mean ± SE, n = 3, p < 0.001). * p<0.05.</p></caption><graphic xlink:href="pone.0137079.g006"></graphic></fig><p>CITED2 is known as CBP/p300-dependent transcriptional co-activator [<xref rid="pone.0137079.ref036" ref-type="bibr">36</xref>–<xref rid="pone.0137079.ref038" ref-type="bibr">38</xref>]. In a previous study, conditional deletion of <italic>Cited2</italic> in adult mice resulted a loss of HSCs, leading to bone marrow failure and increased lethality [<xref rid="pone.0137079.ref036" ref-type="bibr">36</xref>]. Noticeably, in <italic>Cited2</italic><sup>−/−</sup> Lin<sup>−</sup>c-Kit<sup>+</sup> cells exhibited reduced <italic>Bmi1</italic>, <italic>Notch1</italic>, <italic>Lef1</italic>, <italic>Mcl1</italic>, and <italic>Gata2</italic> expression [<xref rid="pone.0137079.ref037" ref-type="bibr">37</xref>]. Furthermore, a previous report demonstrated that BMI1 could induce <italic>Gata2</italic> expression [<xref rid="pone.0137079.ref039" ref-type="bibr">39</xref>]. Based on an analysis of clinical samples from patients with AML, Korthuis <italic>et al</italic>. recently demonstrated that CITED2 might play an important role in maintenance of this disease [<xref rid="pone.0137079.ref040" ref-type="bibr">40</xref>]. These data support our finding that <italic>CITED2</italic> is an upstream regulator of GATA-2 in humans.</p><p>Aplastic anemia (AA) is characterized by a decrease in HSCs and fatty marrow replacement. We, along with other research groups, have demonstrated reduced GATA-2 expression in CD34-positive cells from patients with AA [<xref rid="pone.0137079.ref024" ref-type="bibr">24</xref>, <xref rid="pone.0137079.ref025" ref-type="bibr">25</xref>]. On the other hand, decreased GATA-2 expression in mesenchymal stem cells has been shown to lead to accelerated adipocyte differentiation and an impaired ability to support hematopoiesis [<xref rid="pone.0137079.ref041" ref-type="bibr">41</xref>]. Therefore, in the bone marrow, GATA-2 not only generates and/or maintains HSCs, but also plays a role in the regulating the hematopoietic microenvironment. Thus, our system potentially represents a powerful tool with which to identify the regulatory mechanisms of GATA-2 and might lead to the development of novel therapeutic approaches for bone marrow failure syndrome.</p></sec></sec><sec sec-type="supplementary-material" id="sec019"><title>Supporting Information</title><supplementary-material content-type="local-data" id="pone.0137079.s001"><label>S1 Fig</label><caption><title>Identification of GATA-2 by western blot.</title><p>Western blot analysis to detect GATA-2 in K562 cells. To specify GATA-2 band, both overexpression and knockdown of GATA-2 were conducted in K562 cells. Alpha-tubulin was used as a loading control. The asterisk demotes cross-reactive band.</p><p>(EPS)</p></caption><media xlink:href="pone.0137079.s001.eps"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0137079.s002"><label>S2 Fig</label><caption><title><italic>CBP/p300-Interacting Transactivator with Glu/Asp-Rich C-Terminal Domain 2</italic> (CITED2) affects both 1S and +9.9 kb/1S luciferase activity.</title><p>Luciferase analysis was performed with empty or contained the <italic>GATA-2 1S</italic> promoter and/or +9.9 kb site in both YN-1 cells and CITED2-knockout YN-1 clone (<xref rid="pone.0137079.g005" ref-type="fig">Fig 5</xref>). Luciferase activity was measured 24 h after the first transfection. Results are presented as ratios of luciferase activities (means of Firefly/Renilla ± standard error, n = 3).* p<0.05.</p><p>(EPS)</p></caption><media xlink:href="pone.0137079.s002.eps"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0137079.s003"><label>S1 Table</label><caption><title>Oligonucleotide primers.</title><p>(EPS)</p></caption><media xlink:href="pone.0137079.s003.eps"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0137079.s004"><label>S2 Table</label><caption><title>Results of <italic>small interfering RNA (siRNA) screening</italic>.</title><p>(XLSX)</p></caption><media xlink:href="pone.0137079.s004.xlsx"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack><p>We thank the members of the Hematology and Rheumatology department of Tohoku University Graduate School of Medicine for providing helpful comments. We also acknowledge the technical support from the Biomedical Research Core of Tohoku University School of Medicine.</p></ack><ref-list><title>References</title><ref id="pone.0137079.ref001"><label>1</label><mixed-citation publication-type="journal"><name><surname>Orkin</surname><given-names>SH</given-names></name>, <name><surname>Zon</surname><given-names>LI</given-names></name>. (<year>2008</year>) <article-title>SnapShot: hematopoiesis</article-title>. <source>Cell</source>. <volume>20</volume>: <fpage>712</fpage>.<pub-id pub-id-type="pmid">18295585</pub-id></mixed-citation></ref><ref id="pone.0137079.ref002"><label>2</label><mixed-citation publication-type="journal"><name><surname>Evans</surname><given-names>T</given-names></name>, <name><surname>Reitman</surname><given-names>M</given-names></name>, <name><surname>Felsenfeld</surname><given-names>G</given-names></name>. (<year>1988</year>) <article-title>An erythrocyte-specific DNA-binding factor recognizes a regulatory sequence common to all chicken globin genes</article-title>. <source>Proc Natl Acad Sci U S A</source>. <volume>85</volume>: <fpage>5976</fpage>–<lpage>5980</lpage>. <pub-id pub-id-type="pmid">3413070</pub-id></mixed-citation></ref><ref id="pone.0137079.ref003"><label>3</label><mixed-citation publication-type="journal"><name><surname>Yamamoto</surname><given-names>M</given-names></name>, <name><surname>Ko</surname><given-names>LJ</given-names></name>, <name><surname>Leonard</surname><given-names>MW</given-names></name>, <name><surname>Beug</surname><given-names>H</given-names></name>, <name><surname>Orkin</surname><given-names>SH</given-names></name>, <name><surname>Engel</surname><given-names>JD</given-names></name>. (<year>1990</year>) <article-title>Activity and tissue-specific expression of the transcription factor NF-E1 multigene family</article-title>. <source>Genes Dev</source>. <volume>4</volume>: <fpage>1650</fpage>–<lpage>1662</lpage>. <pub-id pub-id-type="pmid">2249770</pub-id></mixed-citation></ref><ref id="pone.0137079.ref004"><label>4</label><mixed-citation publication-type="journal"><name><surname>Harigae</surname><given-names>H</given-names></name>. (<year>2006</year>) <article-title>GATA transcription factors and hematological diseases</article-title>. <source>Tohoku J Exp Med</source>. <volume>210</volume>: <fpage>1</fpage>–<lpage>9</lpage>. <pub-id pub-id-type="pmid">16960339</pub-id></mixed-citation></ref><ref id="pone.0137079.ref005"><label>5</label><mixed-citation publication-type="journal"><name><surname>Bresnick</surname><given-names>EH</given-names></name>, <name><surname>Lee</surname><given-names>HY</given-names></name>, <name><surname>Fujiwara</surname><given-names>T</given-names></name>, <name><surname>Johnson</surname><given-names>KD</given-names></name> and <name><surname>Keles</surname><given-names>S</given-names></name>. (<year>2010</year>) <article-title>GATA switches as developmental drivers</article-title>. <source>J Biol Chem</source>. <volume>285</volume>: <fpage>31087</fpage>–<lpage>31093</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1074/jbc.R110.159079">10.1074/jbc.R110.159079</ext-link></comment><pub-id pub-id-type="pmid">20670937</pub-id></mixed-citation></ref><ref id="pone.0137079.ref006"><label>6</label><mixed-citation publication-type="journal"><name><surname>Martin</surname><given-names>DI</given-names></name>, <name><surname>Zon</surname><given-names>LI</given-names></name>, <name><surname>Mutter</surname><given-names>G</given-names></name>, <name><surname>Orkin</surname><given-names>SH</given-names></name>. (<year>1990</year>) <article-title>Expression of an erythroid transcription factor in megakaryocytic and mast cell lineages</article-title>. <source>Nature</source>. <volume>344</volume>: <fpage>444</fpage>–<lpage>447</lpage>. <pub-id pub-id-type="pmid">2320112</pub-id></mixed-citation></ref><ref id="pone.0137079.ref007"><label>7</label><mixed-citation publication-type="journal"><name><surname>Lim</surname><given-names>KC</given-names></name>, <name><surname>Lakshmanan</surname><given-names>G</given-names></name>, <name><surname>Crawford</surname><given-names>SE</given-names></name>, <name><surname>Gu</surname><given-names>Y</given-names></name>, <name><surname>Grosveld</surname><given-names>F</given-names></name>, <name><surname>Engel</surname><given-names>JD</given-names></name>. (<year>2000</year>) <article-title>Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system</article-title>. <source>Nat Genet</source>. <volume>25</volume>: <fpage>209</fpage>–<lpage>212</lpage>. <pub-id pub-id-type="pmid">10835639</pub-id></mixed-citation></ref><ref id="pone.0137079.ref008"><label>8</label><mixed-citation publication-type="journal"><name><surname>Tsai</surname><given-names>FY</given-names></name>, <name><surname>Keller</surname><given-names>G</given-names></name>, <name><surname>Kuo</surname><given-names>FC</given-names></name>, <name><surname>Weiss</surname><given-names>M</given-names></name>, <name><surname>Chen</surname><given-names>J</given-names></name>, <name><surname>Rosenblatt</surname><given-names>M</given-names></name>, <etal>et al</etal> (<year>1994</year>) <article-title>An early haematopoietic defect in mice lacking the transcription factor GATA-2</article-title>. <source>Nature</source>. <volume>371</volume>: <fpage>221</fpage>–<lpage>226</lpage>. <pub-id pub-id-type="pmid">8078582</pub-id></mixed-citation></ref><ref id="pone.0137079.ref009"><label>9</label><mixed-citation publication-type="journal"><name><surname>Tsai</surname><given-names>FY</given-names></name>, <name><surname>Orkin</surname><given-names>SH</given-names></name>. (<year>1997</year>) <article-title>Transcription factor GATA-2 is required for proliferation/survival of early hematopoietic cells and mast cell formation, but not for erythroid and myeloid terminal differentiation</article-title>. <source>Blood</source>. <volume>89</volume>: <fpage>3636</fpage>–<lpage>3643</lpage>. <pub-id pub-id-type="pmid">9160668</pub-id></mixed-citation></ref><ref id="pone.0137079.ref010"><label>10</label><mixed-citation publication-type="journal"><name><surname>Ezoe</surname><given-names>S</given-names></name>, <name><surname>Matsumura</surname><given-names>I</given-names></name>, <name><surname>Nakata</surname><given-names>S</given-names></name>, <name><surname>Gale</surname><given-names>K</given-names></name>, <name><surname>Ishihara</surname><given-names>K</given-names></name>, <name><surname>Minegishi</surname><given-names>N</given-names></name>, <etal>et al</etal> (<year>2002</year>) <article-title>GATA-2/estrogen receptor chimera regulates cytokine-dependent growth of hematopoietic cells through accumulation of p21(WAF1) and p27(Kip1) proteins</article-title>. <source>Blood</source>. <volume>100</volume>: <fpage>3512</fpage>–<lpage>3520</lpage>. <pub-id pub-id-type="pmid">12393444</pub-id></mixed-citation></ref><ref id="pone.0137079.ref011"><label>11</label><mixed-citation publication-type="journal"><name><surname>Rodrigues</surname><given-names>NP</given-names></name>, <name><surname>Janzen</surname><given-names>V</given-names></name>, <name><surname>Forkert</surname><given-names>R</given-names></name>, <name><surname>Dombkowski</surname><given-names>DM</given-names></name>, <name><surname>Boyd</surname><given-names>AS</given-names></name>, <name><surname>Orkin</surname><given-names>SH</given-names></name>, <etal>et al</etal> (<year>2005</year>) <article-title>Haploinsufficiency of GATA-2 perturbs adult hematopoietic stem-cell homeostasis</article-title>. <source>Blood</source>. <volume>106</volume>: <fpage>477</fpage>–<lpage>484</lpage>. <pub-id pub-id-type="pmid">15811962</pub-id></mixed-citation></ref><ref id="pone.0137079.ref012"><label>12</label><mixed-citation publication-type="journal"><name><surname>Minegishi</surname><given-names>N</given-names></name>, <name><surname>Ohta</surname><given-names>J</given-names></name>, <name><surname>Suwabe</surname><given-names>N</given-names></name>, <name><surname>Nakauchi</surname><given-names>H</given-names></name>, <name><surname>Ishihara</surname><given-names>H</given-names></name>, <name><surname>Hayashi</surname><given-names>N</given-names></name>, <etal>et al</etal> (<year>1998</year>) <article-title>Alternative promoters regulate transcription of the mouse GATA-2 gene</article-title>. <source>J Biol Chem</source>. <volume>273</volume>: <fpage>3625</fpage>–<lpage>3634</lpage>. <pub-id pub-id-type="pmid">9452491</pub-id></mixed-citation></ref><ref id="pone.0137079.ref013"><label>13</label><mixed-citation publication-type="journal"><name><surname>Pan</surname><given-names>X</given-names></name>, <name><surname>Minegishi</surname><given-names>N</given-names></name>, <name><surname>Harigae</surname><given-names>H</given-names></name>, <name><surname>Yamagiwa</surname><given-names>H</given-names></name>, <name><surname>Minegishi</surname><given-names>M</given-names></name>, <name><surname>Akine</surname><given-names>Y</given-names></name>, <etal>et al</etal> (<year>2000</year>) <article-title>Identification of human GATA-2 gene distal IS exon and its expression in hematopoietic stem cell fractions</article-title>. <source>J Biochem</source>. <volume>127</volume>: <fpage>105</fpage>–<lpage>112</lpage>. <pub-id pub-id-type="pmid">10731672</pub-id></mixed-citation></ref><ref id="pone.0137079.ref014"><label>14</label><mixed-citation publication-type="journal"><name><surname>Moriguchi</surname><given-names>T</given-names></name>, <name><surname>Yamamoto</surname><given-names>M</given-names></name>. (<year>2014</year>) <article-title>A regulatory network governing Gata1 and Gata2 gene transcription orchestrates erythroid lineage differentiation</article-title>. <source>Int J Hematol</source>, <volume>100</volume>: <fpage>417</fpage>–<lpage>424</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1007/s12185-014-1568-0">10.1007/s12185-014-1568-0</ext-link></comment><pub-id pub-id-type="pmid">24638828</pub-id></mixed-citation></ref><ref id="pone.0137079.ref015"><label>15</label><mixed-citation publication-type="journal"><name><surname>Sanalkumar</surname><given-names>R</given-names></name>, <name><surname>Johnson</surname><given-names>KD</given-names></name>, <name><surname>Gao</surname><given-names>X</given-names></name>, <name><surname>Boyer</surname><given-names>ME</given-names></name>, <name><surname>Chang</surname><given-names>YI</given-names></name>, <name><surname>Hewitt</surname><given-names>KJ</given-names></name>, <etal>et al</etal> (<year>2014</year>) <article-title>Mechanism governing a stem cell-generating cis-regulatory element</article-title>. <source>Proc Natl Acad Sci U S A</source>. <volume>111</volume>: <fpage>E1091</fpage>–<lpage>E1100</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1073/pnas.1400065111">10.1073/pnas.1400065111</ext-link></comment><pub-id pub-id-type="pmid">24616499</pub-id></mixed-citation></ref><ref id="pone.0137079.ref016"><label>16</label><mixed-citation publication-type="journal"><name><surname>Snow</surname><given-names>JW</given-names></name>, <name><surname>Trowbridge</surname><given-names>JJ</given-names></name>, <name><surname>Johnson</surname><given-names>KD</given-names></name>, <name><surname>Fujiwara</surname><given-names>T</given-names></name>, <name><surname>Emambokus</surname><given-names>NE</given-names></name>, <name><surname>Grass</surname><given-names>JA</given-names></name>, <etal>et al</etal> (<year>2011</year>) <article-title>Context-dependent function of "GATA switch" sites in vivo</article-title>. <source>Blood</source>. <volume>117</volume>: <fpage>4769</fpage>–<lpage>4772</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1182/blood-2010-10-313031">10.1182/blood-2010-10-313031</ext-link></comment><pub-id pub-id-type="pmid">21398579</pub-id></mixed-citation></ref><ref id="pone.0137079.ref017"><label>17</label><mixed-citation publication-type="journal"><name><surname>Snow</surname><given-names>JW</given-names></name>, <name><surname>Trowbridge</surname><given-names>JJ</given-names></name>, <name><surname>Fujiwara</surname><given-names>T</given-names></name>, <name><surname>Emambokus</surname><given-names>NE</given-names></name>, <name><surname>Grass</surname><given-names>JA</given-names></name>, <name><surname>Orkin</surname><given-names>SH</given-names></name>, <etal>et al</etal> (<year>2010</year>) <article-title>A single cis element maintains repression of the key developmental regulator Gata2</article-title>. <source>PLoS Genet</source>. <volume>6</volume>: <fpage>e1001103</fpage><comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1371/journal.pgen.1001103">10.1371/journal.pgen.1001103</ext-link></comment><pub-id pub-id-type="pmid">20838598</pub-id></mixed-citation></ref><ref id="pone.0137079.ref018"><label>18</label><mixed-citation publication-type="journal"><name><surname>Johnson</surname><given-names>KD</given-names></name>, <name><surname>Hsu</surname><given-names>AP</given-names></name>, <name><surname>Ryu</surname><given-names>MJ</given-names></name>, <name><surname>Wang</surname><given-names>J</given-names></name>, <name><surname>Gao</surname><given-names>X</given-names></name>, <name><surname>Boyer</surname><given-names>ME</given-names></name>, <etal>et al</etal> (<year>2012</year>) <article-title>Cis-element mutated in GATA2-dependent immunodeficiency governs hematopoiesis and vascular integrity</article-title>. <source>J Clin Invest</source>. <volume>122</volume>: <fpage>3692</fpage>–<lpage>3704</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1172/JCI61623">10.1172/JCI61623</ext-link></comment><pub-id pub-id-type="pmid">22996659</pub-id></mixed-citation></ref><ref id="pone.0137079.ref019"><label>19</label><mixed-citation publication-type="journal"><name><surname>Dickinson</surname><given-names>RE</given-names></name>, <name><surname>Griffin</surname><given-names>H</given-names></name>, <name><surname>Bigley</surname><given-names>V</given-names></name>, <name><surname>Reynard</surname><given-names>LN</given-names></name>, <name><surname>Hussain</surname><given-names>R</given-names></name>, <name><surname>Haniffa</surname><given-names>M</given-names></name>, <etal>et al</etal> (<year>2011</year>) <article-title>Exome sequencing identifies GATA-2 mutation as the cause of dendritic cell, monocyte, B and NK lymphoid deficiency</article-title>. <source>Blood</source>. <volume>118</volume>: <fpage>2656</fpage>–<lpage>2658</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1182/blood-2011-06-360313">10.1182/blood-2011-06-360313</ext-link></comment><pub-id pub-id-type="pmid">21765025</pub-id></mixed-citation></ref><ref id="pone.0137079.ref020"><label>20</label><mixed-citation publication-type="journal"><name><surname>Hsu</surname><given-names>AP</given-names></name>, <name><surname>Sampaio</surname><given-names>EP</given-names></name>, <name><surname>Khan</surname><given-names>J</given-names></name>, <name><surname>Calvo</surname><given-names>KR</given-names></name>, <name><surname>Lemieux</surname><given-names>JE</given-names></name>, <name><surname>Patel</surname><given-names>SY</given-names></name>, <etal>et al</etal> (<year>2011</year>) <article-title>Mutations in GATA2 are associated with the autosomal dominant and sporadic monocytopenia and mycobacterial infection (MonoMAC) syndrome</article-title>. <source>Blood</source>. <volume>118</volume>: <fpage>2653</fpage>–<lpage>2655</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1182/blood-2011-05-356352">10.1182/blood-2011-05-356352</ext-link></comment><pub-id pub-id-type="pmid">21670465</pub-id></mixed-citation></ref><ref id="pone.0137079.ref021"><label>21</label><mixed-citation publication-type="journal"><name><surname>Ostergaard</surname><given-names>P</given-names></name>, <name><surname>Simpson</surname><given-names>MA</given-names></name>, <name><surname>Connell</surname><given-names>FC</given-names></name>, <name><surname>Steward</surname><given-names>CG</given-names></name>, <name><surname>Brice</surname><given-names>G</given-names></name>, <name><surname>Woollard</surname><given-names>WJ</given-names></name>, <etal>et al</etal> (<year>2011</year>) <article-title>Mutations in GATA2 cause primary lymphedema associated with a predisposition to acute myeloid leukemia (Emberger syndrome)</article-title>. <source>Nat Genet</source>. <volume>43</volume>: <fpage>929</fpage>–<lpage>931</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1038/ng.923">10.1038/ng.923</ext-link></comment><pub-id pub-id-type="pmid">21892158</pub-id></mixed-citation></ref><ref id="pone.0137079.ref022"><label>22</label><mixed-citation publication-type="journal"><name><surname>Guiu</surname><given-names>J</given-names></name>, <name><surname>Shimizu</surname><given-names>R</given-names></name>, <name><surname>D'Altri</surname><given-names>T</given-names></name>, <name><surname>Fraser</surname><given-names>ST</given-names></name>, <name><surname>Hatakeyama</surname><given-names>J</given-names></name>, <name><surname>Bresnick</surname><given-names>EH</given-names></name>, <etal>et al</etal> (<year>2013</year>) <article-title>Hes repressors are essential regulators of hematopoietic stem cell development downstream of Notch signaling</article-title>. <source>J Exp Med</source>. <volume>210</volume>: <fpage>71</fpage>–<lpage>84</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1084/jem.20120993">10.1084/jem.20120993</ext-link></comment><pub-id pub-id-type="pmid">23267012</pub-id></mixed-citation></ref><ref id="pone.0137079.ref023"><label>23</label><mixed-citation publication-type="journal"><name><surname>Trompouki</surname><given-names>E</given-names></name>, <name><surname>Bowman</surname><given-names>TV</given-names></name>, <name><surname>Lawton</surname><given-names>LN</given-names></name>, <name><surname>Fan</surname><given-names>ZP</given-names></name>, <name><surname>Wu</surname><given-names>DC</given-names></name>, <name><surname>DiBiase</surname><given-names>A</given-names></name>, <etal>et al</etal> (<year>2011</year>) <article-title>Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration</article-title>. <source>Cell</source>. <volume>147</volume>: <fpage>577</fpage>–<lpage>589</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1016/j.cell.2011.09.044">10.1016/j.cell.2011.09.044</ext-link></comment><pub-id pub-id-type="pmid">22036566</pub-id></mixed-citation></ref><ref id="pone.0137079.ref024"><label>24</label><mixed-citation publication-type="journal"><name><surname>Fujimaki</surname><given-names>S</given-names></name>, <name><surname>Harigae</surname><given-names>H</given-names></name>, <name><surname>Sugawara</surname><given-names>T</given-names></name>, <name><surname>Takasawa</surname><given-names>N</given-names></name>, <name><surname>Sasaki</surname><given-names>T</given-names></name>, <name><surname>Kaku</surname><given-names>M</given-names></name>. (<year>2001</year>) <article-title>Decreased expression of transcription factor GATA-2 in haematopoietic stem cells in patients with aplastic anaemia</article-title>. <source>Br J Haematol</source>. <volume>113</volume>: <fpage>52</fpage>–<lpage>57</lpage>. <pub-id pub-id-type="pmid">11328281</pub-id></mixed-citation></ref><ref id="pone.0137079.ref025"><label>25</label><mixed-citation publication-type="journal"><name><surname>Zeng</surname><given-names>W</given-names></name>, <name><surname>Chen</surname><given-names>G</given-names></name>, <name><surname>Kajigaya</surname><given-names>S</given-names></name>, <name><surname>Nunez</surname><given-names>O</given-names></name>, <name><surname>Charrow</surname><given-names>A</given-names></name>, <name><surname>Billings</surname><given-names>EM</given-names></name>, <etal>et al</etal> (<year>2004</year>) <article-title>Gene expression profiling in CD34 cells to identify differences between aplastic anemia patients and healthy volunteers</article-title>. <source>Blood</source>. <volume>103</volume>: <fpage>325</fpage>–<lpage>332</lpage>. <pub-id pub-id-type="pmid">14504100</pub-id></mixed-citation></ref><ref id="pone.0137079.ref026"><label>26</label><mixed-citation publication-type="journal"><name><surname>Kumar</surname><given-names>MS</given-names></name>, <name><surname>Hancock</surname><given-names>DC</given-names></name>, <name><surname>Molina-Arcas</surname><given-names>M</given-names></name>, <name><surname>Steckel</surname><given-names>M</given-names></name>, <name><surname>East</surname><given-names>P</given-names></name>, <name><surname>Diefenbacher</surname><given-names>M</given-names></name>, <etal>et al</etal> (<year>2012</year>) <article-title>The GATA2 transcriptional network is requisite for RAS oncogene-driven non-small cell lung cancer</article-title>. <source>Cell</source>. <volume>149</volume>, <fpage>642</fpage>–<lpage>655</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1016/j.cell.2012.02.059">10.1016/j.cell.2012.02.059</ext-link></comment><pub-id pub-id-type="pmid">22541434</pub-id></mixed-citation></ref><ref id="pone.0137079.ref027"><label>27</label><mixed-citation publication-type="journal"><name><surname>Endo</surname><given-names>K</given-names></name>, <name><surname>Harigae</surname><given-names>H</given-names></name>, <name><surname>Nagai</surname><given-names>T</given-names></name>, <name><surname>Fujie</surname><given-names>H</given-names></name>, <name><surname>Meguro</surname><given-names>K</given-names></name>, <name><surname>Watanabe</surname><given-names>N</given-names></name>, <etal>et al</etal> (<year>1993</year>) <article-title>Two chronic myelogenous leukaemia cell lines which represent different stages of erythroid differentiation</article-title>. <source>Br J Haematol</source>. <volume>85</volume>: <fpage>653</fpage>–<lpage>662</lpage>. <pub-id pub-id-type="pmid">7918028</pub-id></mixed-citation></ref><ref id="pone.0137079.ref028"><label>28</label><mixed-citation publication-type="journal"><name><surname>Dorfman</surname><given-names>DM</given-names></name>, <name><surname>Wilson</surname><given-names>DB</given-names></name>, <name><surname>Bruns</surname><given-names>GA</given-names></name>, <name><surname>Orkin</surname><given-names>SH</given-names></name>. (<year>1992</year>) <article-title>Human transcription factor GATA-2. Evidence for regulation of preproendothelin-1 gene expression in endothelial cells</article-title>. <source>J Biol Chem</source>. <volume>267</volume>: <fpage>1279</fpage>–<lpage>1285</lpage>. <pub-id pub-id-type="pmid">1370462</pub-id></mixed-citation></ref><ref id="pone.0137079.ref029"><label>29</label><mixed-citation publication-type="journal"><name><surname>Koeffler</surname><given-names>HP</given-names></name>, <name><surname>Billing</surname><given-names>R</given-names></name>, <name><surname>Lusis</surname><given-names>AJ</given-names></name>, <name><surname>Sparkes</surname><given-names>R</given-names></name>, <name><surname>Golde</surname><given-names>DW</given-names></name>. (<year>1980</year>) <article-title>An undifferentiated variant derived from the human acute myelogenous leukemia cell line (KG-1)</article-title>. <source>Blood</source>. <volume>56</volume>: <fpage>265</fpage>–<lpage>273</lpage>. <pub-id pub-id-type="pmid">6967340</pub-id></mixed-citation></ref><ref id="pone.0137079.ref030"><label>30</label><mixed-citation publication-type="journal"><name><surname>Fujiwara</surname><given-names>T</given-names></name>, <name><surname>Fukuhara</surname><given-names>N</given-names></name>, <name><surname>Funayama</surname><given-names>R</given-names></name>, <name><surname>Nariai</surname><given-names>N</given-names></name>, <name><surname>Kamata</surname><given-names>M</given-names></name>, <name><surname>Nagashima</surname><given-names>T</given-names></name>, <etal>et al</etal> (<year>2014</year>) <article-title>Identification of acquired mutations by whole-genome sequencing in GATA-2 deficiency evolving into myelodysplasia and acute leukemia</article-title>. <source>Ann Hematol</source>. <volume>93</volume>:<fpage>1515</fpage>–<lpage>1522</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1007/s00277-014-2090-4">10.1007/s00277-014-2090-4</ext-link></comment><pub-id pub-id-type="pmid">24782121</pub-id></mixed-citation></ref><ref id="pone.0137079.ref031"><label>31</label><mixed-citation publication-type="journal"><name><surname>Fujiwara</surname><given-names>T</given-names></name>, <name><surname>Yokoyama</surname><given-names>H</given-names></name>, <name><surname>Okitsu</surname><given-names>Y</given-names></name>, <name><surname>Kamata</surname><given-names>M</given-names></name>, <name><surname>Fukuhara</surname><given-names>N</given-names></name>, <name><surname>Onishi</surname><given-names>Y</given-names></name>, <etal>et al</etal> (<year>2012</year>) <article-title>Gene expression profiling identifies HOXB4 as a direct downstream target of GATA-2 in human CD34+ hematopoietic cells</article-title>. <source>PLoS One</source>. <volume>7</volume>: <fpage>e40959</fpage><comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1371/journal.pone.0040959">10.1371/journal.pone.0040959</ext-link></comment><pub-id pub-id-type="pmid">23028422</pub-id></mixed-citation></ref><ref id="pone.0137079.ref032"><label>32</label><mixed-citation publication-type="journal"><name><surname>Fujiwara</surname><given-names>T</given-names></name>, <name><surname>O'Geen</surname><given-names>H</given-names></name>, <name><surname>Keles</surname><given-names>S</given-names></name>, <name><surname>Blahnik</surname><given-names>K</given-names></name>, <name><surname>Linnemann</surname><given-names>AK</given-names></name>, <name><surname>Kang</surname><given-names>YA</given-names></name>, <etal>et al</etal> (<year>2009</year>) <article-title>Discovering hematopoietic mechanisms through genome-wide analysis of GATA factor chromatin occupancy</article-title>. <source>Mol Cell</source>. <volume>36</volume>: <fpage>667</fpage>–<lpage>681</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1016/j.molcel.2009.11.001">10.1016/j.molcel.2009.11.001</ext-link></comment><pub-id pub-id-type="pmid">19941826</pub-id></mixed-citation></ref><ref id="pone.0137079.ref033"><label>33</label><mixed-citation publication-type="journal"><name><surname>Cortes-Lavaud</surname><given-names>X</given-names></name>, <name><surname>Maicas</surname><given-names>M</given-names></name>, <name><surname>Vazquez</surname><given-names>I</given-names></name>, <name><surname>Vicente</surname><given-names>C</given-names></name>, <name><surname>Urquiza</surname><given-names>L</given-names></name>, <name><surname>Garcia-Sanchez</surname><given-names>MA</given-names></name>, <etal>et al</etal> (<year>2012</year>) <article-title>Functional analysis of the GATA2 promoter shows that mutations of GATA2 impair its own transcriptional regulation</article-title>. <source>Blood</source>. <volume>120</volume>: <fpage>1233</fpage>.</mixed-citation></ref><ref id="pone.0137079.ref034"><label>34</label><mixed-citation publication-type="journal"><name><surname>Wozniak</surname><given-names>RJ</given-names></name>, <name><surname>Boyer</surname><given-names>ME</given-names></name>, <name><surname>Grass</surname><given-names>JA</given-names></name>, <name><surname>Lee</surname><given-names>Y</given-names></name>, <name><surname>Bresnick</surname><given-names>EH</given-names></name>. (<year>2007</year>) <article-title>Context-dependent GATA factor function: combinatorial requirements for transcriptional control in hematopoietic and endothelial cells</article-title>. <source>J Biol Chem</source>. <volume>282</volume>: <fpage>14665</fpage>–<lpage>14674</lpage>. <pub-id pub-id-type="pmid">17347142</pub-id></mixed-citation></ref><ref id="pone.0137079.ref035"><label>35</label><mixed-citation publication-type="journal"><name><surname>Khandekar</surname><given-names>M</given-names></name>, <name><surname>Brandt</surname><given-names>W</given-names></name>, <name><surname>Zhou</surname><given-names>Y</given-names></name>, <name><surname>Dagenais</surname><given-names>S</given-names></name>, <name><surname>Glover</surname><given-names>TW</given-names></name>, <name><surname>Suzuki</surname><given-names>N</given-names></name>, <etal>et al</etal> (<year>2007</year>) <article-title>A Gata2 intronic enhancer confers its pan-endothelia-specific regulation</article-title>. <source>Development</source>. <volume>134</volume>: <fpage>1703</fpage>–<lpage>1712</lpage>. <pub-id pub-id-type="pmid">17395646</pub-id></mixed-citation></ref><ref id="pone.0137079.ref036"><label>36</label><mixed-citation publication-type="journal"><name><surname>Kranc</surname><given-names>KR</given-names></name>, <name><surname>Schepers</surname><given-names>H</given-names></name>, <name><surname>Rodrigues</surname><given-names>NP</given-names></name>, <name><surname>Bamforth</surname><given-names>S</given-names></name>, <name><surname>Villadsen</surname><given-names>E</given-names></name>, <name><surname>Ferry</surname><given-names>H</given-names></name>, <etal>et al</etal> (<year>2009</year>) <article-title>Cited2 is an essential regulator of adult hematopoietic stem cells</article-title>. <source>Cell Stem Cell</source>. <volume>5</volume>: <fpage>659</fpage>–<lpage>665</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1016/j.stem.2009.11.001">10.1016/j.stem.2009.11.001</ext-link></comment><pub-id pub-id-type="pmid">19951693</pub-id></mixed-citation></ref><ref id="pone.0137079.ref037"><label>37</label><mixed-citation publication-type="journal"><name><surname>Chen</surname><given-names>Y</given-names></name>, <name><surname>Haviernik</surname><given-names>P</given-names></name>, <name><surname>Bunting</surname><given-names>KD</given-names></name>, <name><surname>Yang</surname><given-names>YC</given-names></name>. (<year>2007</year>) <article-title>Cited2 is required for normal hematopoiesis in the murine fetal liver</article-title>. <source>Blood</source>. <volume>110</volume>: <fpage>2889</fpage>–<lpage>2898</lpage>. <pub-id pub-id-type="pmid">17644732</pub-id></mixed-citation></ref><ref id="pone.0137079.ref038"><label>38</label><mixed-citation publication-type="journal"><name><surname>Kranc</surname><given-names>KR</given-names></name>, <name><surname>Bamforth</surname><given-names>SD</given-names></name>, <name><surname>Bragança</surname><given-names>J</given-names></name>, <name><surname>Norbury</surname><given-names>C</given-names></name>, <name><surname>van Lohuizen</surname><given-names>M</given-names></name>, <name><surname>Bhattacharya</surname><given-names>S</given-names></name>. (<year>2003</year>) <article-title>Transcriptional coactivator Cited2 induces Bmi1 and Mel18 and controls fibroblast proliferation via Ink4a/ARF</article-title>. <source>Mol Cell Biol</source>. <volume>23</volume>: <fpage>7658</fpage>–<lpage>7666</lpage>. <pub-id pub-id-type="pmid">14560011</pub-id></mixed-citation></ref><ref id="pone.0137079.ref039"><label>39</label><mixed-citation publication-type="journal"><name><surname>Ding</surname><given-names>X</given-names></name>, <name><surname>Lin</surname><given-names>Q</given-names></name>, <name><surname>Ensenat-Waser</surname><given-names>R</given-names></name>, <name><surname>Rose-John</surname><given-names>S</given-names></name>, <name><surname>Zenke</surname><given-names>M</given-names></name>. (<year>2012</year>) <article-title>Polycomb group protein Bmi1 promotes hematopoietic cell development from embryonic stem cells</article-title>. <source>Stem Cells Dev</source>. <volume>21</volume>: <fpage>121</fpage>–<lpage>132</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1089/scd.2010.0539">10.1089/scd.2010.0539</ext-link></comment><pub-id pub-id-type="pmid">21545235</pub-id></mixed-citation></ref><ref id="pone.0137079.ref040"><label>40</label><mixed-citation publication-type="journal"><name><surname>Korthuis</surname><given-names>PM</given-names></name>, <name><surname>Berger</surname><given-names>G</given-names></name>, <name><surname>Bakker</surname><given-names>B</given-names></name>, <name><surname>Rozenveld-Geugien</surname><given-names>M</given-names></name>, <name><surname>Jaques</surname><given-names>J</given-names></name>, <name><surname>de Haan</surname><given-names>G</given-names></name>, <etal>et al</etal> (<year>2015</year>) <article-title>CITED2-mediated human hematopoietic stem cell maintenance is critical for acute myeloid leukemia</article-title>. <source>Leukemia</source>. <volume>29</volume>: <fpage>625</fpage>–<lpage>635</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1038/leu.2014.259">10.1038/leu.2014.259</ext-link></comment><pub-id pub-id-type="pmid">25184385</pub-id></mixed-citation></ref><ref id="pone.0137079.ref041"><label>41</label><mixed-citation publication-type="journal"><name><surname>Kamata</surname><given-names>M</given-names></name>, <name><surname>Okitsu</surname><given-names>Y</given-names></name>, <name><surname>Fujiwara</surname><given-names>T</given-names></name>, <name><surname>Kanehira</surname><given-names>M</given-names></name>, <name><surname>Nakajima</surname><given-names>S</given-names></name>, <name><surname>Takahashi</surname><given-names>T</given-names></name>, <etal>et al</etal> (<year>2014</year>) <article-title>GATA2 regulates differentiation of bone marrow-derived mesenchymal stem cells</article-title>. <source>Haematologica</source>. <volume>99</volume>: <fpage>1686</fpage>–<lpage>1696</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.3324/haematol.2014.105692">10.3324/haematol.2014.105692</ext-link></comment><pub-id pub-id-type="pmid">25150255</pub-id></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Sante/explor/LymphedemaV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000433 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000433 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Sante
|area= LymphedemaV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:4556642
|texte= High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:26325290" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a LymphedemaV1
| This area was generated with Dilib version V0.6.31. |  |