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• W2033662995 abstract "CD34 glycoprotein in human hematopoiesis is expressed on a subset of progenitor cells capable of self-renewal, multilineage differentiation, and hematopoietic reconstitution. Nucleolin is an abundant multifunctional phosphoprotein of growing eukaryotic cells, involved in regulation of gene transcription, chromatin remodeling, and RNA metabolism, whose transcripts are enriched in murine hematopoietic stem cells, as opposed to differentiated tissue. Here we show that, in human CD34-positive hematopoietic cells, nucleolin activates endogenous CD34 and Bcl-2 gene expression, and cell surface CD34 protein expression is thereby enhanced by nucleolin. Nucleolin-mediated activation of CD34 gene transcription results from direct sequence-specific interactions with the CD34 promoter region. Nucleolin expression prevails in CD34-positive cells mobilized into peripheral blood (PB), as opposed to CD34-negative peripheral blood mononuclear cells (PBMCs). Therefore, in intact CD34-positive mobilized PB cells, a recruitment of nucleolin to the CD34 promoter region takes place, accompanied by nucleosomal determinants of gene activity, which are absent from the CD34 promoter region in CD34-negative PBMCs. Our data show that nucleolin acts as a component of the gene regulation program of CD34-positive hematopoietic cells and provide further insights into processes by which human CD34-positive hematopoietic stem/progenitor cells are maintained. CD34 glycoprotein in human hematopoiesis is expressed on a subset of progenitor cells capable of self-renewal, multilineage differentiation, and hematopoietic reconstitution. Nucleolin is an abundant multifunctional phosphoprotein of growing eukaryotic cells, involved in regulation of gene transcription, chromatin remodeling, and RNA metabolism, whose transcripts are enriched in murine hematopoietic stem cells, as opposed to differentiated tissue. Here we show that, in human CD34-positive hematopoietic cells, nucleolin activates endogenous CD34 and Bcl-2 gene expression, and cell surface CD34 protein expression is thereby enhanced by nucleolin. Nucleolin-mediated activation of CD34 gene transcription results from direct sequence-specific interactions with the CD34 promoter region. Nucleolin expression prevails in CD34-positive cells mobilized into peripheral blood (PB), as opposed to CD34-negative peripheral blood mononuclear cells (PBMCs). Therefore, in intact CD34-positive mobilized PB cells, a recruitment of nucleolin to the CD34 promoter region takes place, accompanied by nucleosomal determinants of gene activity, which are absent from the CD34 promoter region in CD34-negative PBMCs. Our data show that nucleolin acts as a component of the gene regulation program of CD34-positive hematopoietic cells and provide further insights into processes by which human CD34-positive hematopoietic stem/progenitor cells are maintained. CD34 glycoprotein in human hematopoiesis is expressed on a subset of progenitor cells capable of self-renewal, multilineage differentiation, and hematopoietic reconstitution (1Civin C.I. Strauss L.C. Brovall C. Fackler M.J. Schwartz J.F. Shaper J.H. J. Immunol. 1984; 133: 157-165PubMed Google Scholar, 2Dunbar C.E. Cottler-Fox M. O'Shaughnessy J.A. Doren S. Carter C. Berenson R. Brown S. Moen R.C. Greenblatt J. Stewart F.M. Leitman S.F. Wilson W.H. Cowan K. Young N.S. Nienhuis A.W. Blood. 1995; 85: 3048-3057Crossref PubMed Google Scholar), reviewed in Ref. 3Krause D.S. Fackler M.J. Civin C.I. May W.S. Blood. 1996; 87: 1-13Crossref PubMed Google Scholar. The expression of cell surface CD34 glycoprotein is broadly utilized for enumeration of stem/progenitor cells for clinical bone marrow (BM) 2The abbreviations used are: BM, bone marrow; Ab, antibody; AML, acute myelogenous leukemia; ChIP, chromatin immunoprecipitation; CT, threshold cycle; EMSA, electrophoretic mobility shift assay; GST, glutathioneS-transferase; FACS, fluorescence-activated cell sorter; H4-AcK8, histone H4 acetylated on lysine K8; H3-tri-methyl K4, histone H3 tri-methylated on lysine K4; H3-di-methyl K9, histone H3 di-methylated on lysine K9; HPV18, human papillomavirus type 18; mAb, monoclonal antibody; NK, natural killer; PB, peripheral blood; PBMCs, peripheral blood mononuclear cells; PCNA, proliferating cell nuclear antigen; PE, phycoerythrin; RT-PCR, reverse transcription PCR; siRNA, small interfering RNA; WT, wild-type. transplantation, and the number of 5-year survivors of hematopoietic stem cell transplantation is estimated to be 100,000 worldwide (3Krause D.S. Fackler M.J. Civin C.I. May W.S. Blood. 1996; 87: 1-13Crossref PubMed Google Scholar, 4Lee S.J. Joffe S. Kim H.T. Socie G. Gilman A.L. Wingard J.R. Horowitz M.M. Cella D. Syrjala K.L. Blood. 2004; 104: 2194-2200Crossref PubMed Scopus (59) Google Scholar). Experimental evidence for cellular function of CD34 and its involvement in hematopoietic reconstitution have been presented (5Drew E. Merzaban J.S. Seo W. Ziltener H.J. McNagny K.M. Immunity. 2005; 22: 43-57Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Regulation of the human CD34 gene has been investigated as a model of hematopoietic stem/progenitor cell-specific gene control (3Krause D.S. Fackler M.J. Civin C.I. May W.S. Blood. 1996; 87: 1-13Crossref PubMed Google Scholar, 6Burn T.C. Satterthwaite A.B. Tenen D.G. Blood. 1992; 80: 3051-3059Crossref PubMed Google Scholar, 7He X.Y. Antao V.P. Basila D. Marx J.C. Davis B.R. Blood. 1992; 79: 2296-2302Crossref PubMed Google Scholar, 8He X.Y. Cockerill P.N. Cen D. Davis B.R. Blood. 1994; 83: 1822-1830Crossref PubMed Google Scholar, 9Radomska H.S. Satterthwaite A.B. Burn T.C. Oliff I.A. Huettner C.S. Tenen D.G. Gene (Amst.). 1998; 222: 305-318Crossref PubMed Scopus (24) Google Scholar, 10Okuno Y. Huettner C.S. Radomska H.S. Petkova V. Iwasaki H. Akashi K. Tenen D.G. Blood. 2002; 100: 4420-4426Crossref PubMed Scopus (37) Google Scholar, 11Melotti P. Ku D. Calabretta B. J. Exp. Med. 1994; 179: 1023-1028Crossref PubMed Scopus (79) Google Scholar, 12Melotti P. Calabretta B. J. Biol. Chem. 1994; 269: 25303-25309Abstract Full Text PDF PubMed Google Scholar, 13Morris J.F. Rauscher F.J. Davis B.R. Klemsz M. Xu D. Tenen D.G. Hromas R. Blood. 1995; 86: 3640PubMed Google Scholar, 14Perrotti D. Melotti P. Skorski T. Casella I. Peschle C. Calabretta B. Mol. Cell. Biol. 1995; 15: 6075-6087Crossref PubMed Scopus (104) Google Scholar, 15Radomska H.S. Satterthwaite A.B. Taranenko N. Narravula S. Krause D.S. Tenen D.G. Blood. 1999; 94: 3772-3780Crossref PubMed Google Scholar). The promoter region of the CD34 gene as well as the 3′ enhancer have been identified (6Burn T.C. Satterthwaite A.B. Tenen D.G. Blood. 1992; 80: 3051-3059Crossref PubMed Google Scholar, 7He X.Y. Antao V.P. Basila D. Marx J.C. Davis B.R. Blood. 1992; 79: 2296-2302Crossref PubMed Google Scholar, 8He X.Y. Cockerill P.N. Cen D. Davis B.R. Blood. 1994; 83: 1822-1830Crossref PubMed Google Scholar), and it was shown that multiple regulatory elements, likely acting in the context of chromatin structure, are necessary to provide proper control of expression (8He X.Y. Cockerill P.N. Cen D. Davis B.R. Blood. 1994; 83: 1822-1830Crossref PubMed Google Scholar, 9Radomska H.S. Satterthwaite A.B. Burn T.C. Oliff I.A. Huettner C.S. Tenen D.G. Gene (Amst.). 1998; 222: 305-318Crossref PubMed Scopus (24) Google Scholar, 10Okuno Y. Huettner C.S. Radomska H.S. Petkova V. Iwasaki H. Akashi K. Tenen D.G. Blood. 2002; 100: 4420-4426Crossref PubMed Scopus (37) Google Scholar). Furthermore, transcription regulators including c-Myb, Ets-2, MZF-1, and nuclear factor Y have been shown to bind to their cognate sites and modulate human CD34 promoter region activity (11Melotti P. Ku D. Calabretta B. J. Exp. Med. 1994; 179: 1023-1028Crossref PubMed Scopus (79) Google Scholar, 12Melotti P. Calabretta B. J. Biol. Chem. 1994; 269: 25303-25309Abstract Full Text PDF PubMed Google Scholar, 13Morris J.F. Rauscher F.J. Davis B.R. Klemsz M. Xu D. Tenen D.G. Hromas R. Blood. 1995; 86: 3640PubMed Google Scholar, 14Perrotti D. Melotti P. Skorski T. Casella I. Peschle C. Calabretta B. Mol. Cell. Biol. 1995; 15: 6075-6087Crossref PubMed Scopus (104) Google Scholar, 15Radomska H.S. Satterthwaite A.B. Taranenko N. Narravula S. Krause D.S. Tenen D.G. Blood. 1999; 94: 3772-3780Crossref PubMed Google Scholar). It is essential to work out mechanisms controlling expression of the CD34 gene, as well as of other genes involved in the maintenance of CD34-positive human hematopoietic cells to understand the processes underlying homeostasis and reconstitution of the hematopoietic system at a molecular level. For instance, Bcl-2 is the founder member of a family of proteins that play a central role in the regulation of apoptosis, reviewed in Refs. 16Opferman J.T. Korsmeyer S.J. Nat. Immunol. 2003; 4: 410-415Crossref PubMed Scopus (415) Google Scholar and 17Cory S. Huang D.C. Adams J.M. Oncogene. 2003; 22: 8590-8607Crossref PubMed Scopus (1305) Google Scholar, and constitutive expression of Bcl-2 increases the frequency and in vivo repopulation potential of hematopoietic stem cells (18Domen J. Cheshier S.H. Weissman I.L. J. Exp. Med. 2000; 191: 253-264Crossref PubMed Scopus (270) Google Scholar). Nucleolin is a multifunctional DNA- and RNA-binding protein, which is abundant in growing and cancerous cells (19Lapeyre B. Bourbon H. Amalric F. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1472-1476Crossref PubMed Scopus (316) Google Scholar, 20Derenzini M. Sirri V. Trere D. Ochs R.L. Lab. Investig. 1995; 73: 497-502PubMed Google Scholar), found in the nucleolus, in the nucleus, in the cytoplasm and at the cell surface (19Lapeyre B. Bourbon H. Amalric F. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1472-1476Crossref PubMed Scopus (316) Google Scholar, 21Borer R.A. Lehner C.F. Eppenberger H.M. Nigg E.A. Cell. 1989; 56: 379-390Abstract Full Text PDF PubMed Scopus (923) Google Scholar, 22Hovanessian A.G. Puvion-Dutilleul F. Nisole S. Svab J. Perret E. Deng J.S. Krust B. Exp. Cell Res. 2000; 261: 312-328Crossref PubMed Scopus (189) Google Scholar). Interactions of nucleolin with p53 and the retinoblastoma protein have been described (23Daniely Y. Dimitrovy D.D. Borowiec J.A. Mol. Cell. Biol. 2002; 22: 6014-6022Crossref PubMed Scopus (145) Google Scholar, 24Grinstein E. Shan Y. Karawajew L. Snijders P.J. Meijer C.J. Royer H.D. Wernet P. J. Biol. Chem. 2006; 281: 22223-22235Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). A role of nucleolin in regulation of gene transcription, chromatin remodeling, and RNA metabolism has been shown (21Borer R.A. Lehner C.F. Eppenberger H.M. Nigg E.A. Cell. 1989; 56: 379-390Abstract Full Text PDF PubMed Scopus (923) Google Scholar, 25Hanakahi L.A. Dempsey L.A. Li M.J. Maizels N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3605-3610Crossref PubMed Scopus (116) Google Scholar, 26Grinstein E. Wernet P. Snijders P.J. Rösl F. Weinert I. Jia W. Kraft R. Ch Schewe Schwabe M. Hauptmann S. Dietel M. Meijer C.J.L.M. Royer H.D. J. Exp. Med. 2002; 196: 1067-1078Crossref PubMed Scopus (60) Google Scholar, 27Brys A. Maizels N. Proc. Natl. Acad. Sci. U. S. A. 1994; 9: 4915-4919Crossref Scopus (47) Google Scholar, 28Angelov D. Bondarenko V.A. Almagro S. Menoni H. Mongelard F. Hans F. Mietton F. Studitsky V.M. Hamiche A. Dimitrov S. Bouvet P. EMBO J. 2006; 25: 1669-1679Crossref PubMed Scopus (192) Google Scholar, 29Takagi M. Absalon M.J. McLure K.G. Kastan M.B. Cell. 2005; 123: 49-63Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar). For instance, nucleolin acts as a subunit of the transcription factor LR1, which activates expression of the c-myc gene in B-cell lymphomas (25Hanakahi L.A. Dempsey L.A. Li M.J. Maizels N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3605-3610Crossref PubMed Scopus (116) Google Scholar, 27Brys A. Maizels N. Proc. Natl. Acad. Sci. U. S. A. 1994; 9: 4915-4919Crossref Scopus (47) Google Scholar). Nucleolin is also directly involved in post-transcriptional inhibition of the p53 gene expression (29Takagi M. Absalon M.J. McLure K.G. Kastan M.B. Cell. 2005; 123: 49-63Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar). Enrichment of nucleolin gene transcripts in murine hematopoietic stem cells, as opposed to differentiated tissue, has been reported (30Phillips R.L. Ernst R.E. Brunk B. Ivanova N. Mahan M.A. Deanehan J.K. Moore K.A. Overton G.C. Lemischka I.R. Science. 2000; 288: 1635-1640Crossref PubMed Scopus (364) Google Scholar, 31Terskikh A.V. Easterday M.C. Li L. Hood L. Kornblum H.I. Geschwind D.H. Weissman I.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7934-7939Crossref PubMed Scopus (251) Google Scholar). Here we analyzed a possible involvement of nucleolin in gene regulation in human CD34-positive hematopoietic cells. We show that nucleolin activates endogenous CD34 and Bcl-2 gene expression, and cell surface CD34 protein expression is thereby enhanced by nucleolin. Nucleolin-mediated activation of CD34 gene transcription results from direct sequence-specific interactions with the CD34 promoter region. Nucleolin expression prevails in CD34-positive cells mobilized into PB, as opposed to CD34-negative PBMCs. Therefore, in intact CD34-positive mobilized PB cells, a recruitment of nucleolin to the CD34 promoter region takes place, accompanied by nucleosomal determinants of gene activity, which are absent from the CD34 promoter region in CD34-negative PBMCs. Our data show that nucleolin acts as a component of the gene regulation program of CD34-positive hematopoietic cells and provide further insights into processes by which human CD34-positive hematopoietic stem/progenitor cells are maintained. Cell Culture and Creation of Stable Transfected Cells—KG1 cells (32Koeffler H.P. Golde D.W. Science. 1978; 200: 1153-1154Crossref PubMed Scopus (361) Google Scholar) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, KG1a cells (33Koeffler H.P. Billing R. Lusis A.J. Sparkes R. Golde D.W. Blood. 1980; 56: 265-273Crossref PubMed Google Scholar) in RPMI 1640 medium supplemented with 20% fetal calf serum; NIH-3T3 cells, in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Stable KG1-Nuc transfectant cells were obtained using nucleolin expression vector containing the full-length human nucleolin cDNA in-frame with N-terminal FLAG tag, in the pCMV-Tag2B expression vector (Stratagene), and the selection was performed by addition of 200 μg/ml G418 to the culture medium. Stable KG1-mock transfectant cells were obtained using the same vector with no insert. Exponentially growing cells were used in all experiments. Cell Separation and Fluorescence-activated Cell Sorter (FACS) Analysis—Mobilized PB CD34-positive cells were obtained from apheresis collections from normal adult volunteers and enriched using CD34 MicroBeads (Miltenyi Biotech), after ammonium chloride red cell lysis, according to the supplier's instructions. For enrichment of natural killer (NK) cells, red cell lysis was followed by selection with the CD56 MultiSort Kit, and depletion of residual T cells using CD3 MicroBeads (Miltenyi Biotech). The purity of all preparations was monitored by FACS analysis with the following antibodies (Abs), directly coupled to phycoerythrin (PE) or fluorescein isothiocyanate: anti-CD34 (8G12), anti-CD56 and anti-CD3 (BD Pharmingen), and isotype-matched control monoclonal antibodies (mAbs). The purity of cell preparations was: CD34-positive cells, 96.5–97%; CD34-depleted cells 99–100%, NK cells >97%. Transient Transfection Assays—Reporter construct, containing CD34 promoter region (6Burn T.C. Satterthwaite A.B. Tenen D.G. Blood. 1992; 80: 3051-3059Crossref PubMed Google Scholar, 7He X.Y. Antao V.P. Basila D. Marx J.C. Davis B.R. Blood. 1992; 79: 2296-2302Crossref PubMed Google Scholar) nucleotides –666 to +175, cloned into the pGL3basic vector (Promega), is referred to as CD34WT-pGL3, and derivatives thereof, lacking nucleotide numbers –431 to –427, or –367 to –363 of the CD34 promoter region, are referred to as CD34mutA-pGL3 and CD34mutB-pGL3, respectively. Nucleolin expression vector contained the full-length human nucleolin cDNA downstream of the cytomegalovirus promoter. DNA was purified using a plasmid purification system (Qiagen). KG1 and KG1a cells were transiently transfected using the DMRIE-C transfection reagent (Invitrogen), and NIH-3T3 cells, using the Polyfect transfection reagent (Qiagen), according to the respective manufacturer's protocols. Measurements of luciferase were performed according to the recommended procedures by the producer of the luciferase kit (Roche Applied Science), and the results were normalized to protein concentration according to manufacturer's specifications. Results in figures represent at least three independent experiments. Nucleolin Down-modulation Experiments—Distinct small interfering RNAs (siRNAs) targeted to different regions of the mRNA of human nucleolin were used: a pool of four siRNA molecules targeting human nucleolin (Dharmacon, Lafayette, CO, M-003854-00), referred to as nucleolin siRNA-A, or the siRNA duplex corresponding to nucleolin mRNA nucleotide numbers 1755–1774 (nucleolin sequence from GenBank™ accession NM_005381), referred to as nucleolin siRNA-B. Validation of these siRNAs has been reported previously (34Huddleson J.P. Ahmad N. Lingrel J.B. J. Biol. Chem. 2006; 281: 15121-15128Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 35Masumi A. Fukazawa H. Shimazu T. Yoshida M. Ozato K. Komuro K. Yamaguchi K. Oncogene. 2006; 25: 5113-5124Crossref PubMed Scopus (27) Google Scholar). Non-targeting siRNAs (Santa Cruz, sc-37007, sc-44230) were used as a negative control. siRNA experiments were performed generally following the procedures recommended by Dharmacon. In brief, siRNA (100 nm) was applied to growing KG1 cells as described above, however, twice at 24-h intervals. Cells were harvested 2 days after transfection for analysis of RNA, and 2–4 days after transfection for immunoblotting. For antisense-mediated inhibition of nucleolin expression, phosphorothioate-modified nucleolin antisense oligonucleotide 5′-TCACCATGATGGCGGCGG-3′ was used, that is complementary to the 5′ end of the human nucleolin mRNA encompassing the translation initiation region. The antisense oligonucleotide has been described in detail and validated in our previous study (26Grinstein E. Wernet P. Snijders P.J. Rösl F. Weinert I. Jia W. Kraft R. Ch Schewe Schwabe M. Hauptmann S. Dietel M. Meijer C.J.L.M. Royer H.D. J. Exp. Med. 2002; 196: 1067-1078Crossref PubMed Scopus (60) Google Scholar). In brief, antisense or sense oligonucleotides (1 μm) were applied to exponentially growing cells as described (26Grinstein E. Wernet P. Snijders P.J. Rösl F. Weinert I. Jia W. Kraft R. Ch Schewe Schwabe M. Hauptmann S. Dietel M. Meijer C.J.L.M. Royer H.D. J. Exp. Med. 2002; 196: 1067-1078Crossref PubMed Scopus (60) Google Scholar), and the selectivity of antisense oligonucleotide was controlled in all experiments by Western blotting. Western Blotting and Electrophoretic Mobility Shift Assay (EMSA)—Immunoblot analysis followed standard procedures. The following are Abs and their dilutions: Ab specific for a N-terminal peptide of nucleolin, purified on the peptide column (26Grinstein E. Wernet P. Snijders P.J. Rösl F. Weinert I. Jia W. Kraft R. Ch Schewe Schwabe M. Hauptmann S. Dietel M. Meijer C.J.L.M. Royer H.D. J. Exp. Med. 2002; 196: 1067-1078Crossref PubMed Scopus (60) Google Scholar), 1:10,000; Ab specific for CD34 (Santa Cruz Biotechnology, H-140), 1:1500; mAb specific for Bcl-2 (Santa Cruz Biotechnology, C-2), 1:1500; mAb specific for β-actin (Sigma), 1:2000; mAb specific for proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, PC10), 1:2000; mAb specific for FLAG tag (Sigma) 1:1500. Prestained molecular weight marker proteins (Bio-Rad) were used. The conditions for DNA binding and EMSA were described (26Grinstein E. Wernet P. Snijders P.J. Rösl F. Weinert I. Jia W. Kraft R. Ch Schewe Schwabe M. Hauptmann S. Dietel M. Meijer C.J.L.M. Royer H.D. J. Exp. Med. 2002; 196: 1067-1078Crossref PubMed Scopus (60) Google Scholar). In brief, end-labeled double-stranded oligonucleotides from the CD34 gene promoter region (6Burn T.C. Satterthwaite A.B. Tenen D.G. Blood. 1992; 80: 3051-3059Crossref PubMed Google Scholar, 7He X.Y. Antao V.P. Basila D. Marx J.C. Davis B.R. Blood. 1992; 79: 2296-2302Crossref PubMed Google Scholar), corresponding to nucleotide numbers –446 to –412 (CD34A) or –379 to –347 (CD34B) were used, together with 5 μg of poly(dIdC)(dIdC) and 5 μg of nuclear extract, in the DNA binding buffer containing 10 mm Tris(hydroxymethyl)aminomethane, pH 7.5, 50 mm NaCl, 5% glycerol, 1 mm 1,4-dithio-dl-threitol, and protease inhibitors. Nucleolin-glutathione S-transferase (GST) fusion protein, comprising amino acid residues 289–709 of the human nucleolin cDNA, was generated and employed in EMSA as described (26Grinstein E. Wernet P. Snijders P.J. Rösl F. Weinert I. Jia W. Kraft R. Ch Schewe Schwabe M. Hauptmann S. Dietel M. Meijer C.J.L.M. Royer H.D. J. Exp. Med. 2002; 196: 1067-1078Crossref PubMed Scopus (60) Google Scholar). In brief, the nucleolin-GST fusion protein was generated by cloning the cDNA fragment corresponding to amino acid residues 289–709 of the nucleolin cDNA in-frame with GST cDNA into vector pGEX2T. The fusion protein was produced in Escherichia coli and purified by affinity chromatography using glutathione-Sepharose (Amersham Biosciences) according to the manufacturer's protocol. Chromatin Immunoprecipitation (ChIP) Assay—Formaldehyde cross-linking procedure (36Boyd K.E. Wells J. Gutman J. Bartley S.M. Farnham P.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13887-13892Crossref PubMed Scopus (246) Google Scholar) was employed, and ChIP kit (Upstate Biotechnology) was used, following the manufacturer's protocol. Average length of sonicated DNA was 400–1000 bp. Abs used were: Ab specific for N-terminal peptide of nucleolin, purified on the peptide column (26Grinstein E. Wernet P. Snijders P.J. Rösl F. Weinert I. Jia W. Kraft R. Ch Schewe Schwabe M. Hauptmann S. Dietel M. Meijer C.J.L.M. Royer H.D. J. Exp. Med. 2002; 196: 1067-1078Crossref PubMed Scopus (60) Google Scholar), Ab specific for histone H4 acetylated on residue Lys8 (H4-AcK8) (Upstate Biotechnology), Ab specific for histone H3 trimethylated on residue Lys4 (H3-tri-methyl K4) (Upstate Biotechnology), Ab specific for histone H3 dimethylated on residue Lys9 (H3-dimethyl K9) (a kind gift of Prof. T. Jenuwein), together with Ab specific for epidermal growth factor receptor (Santa Cruz Biotechnology, sc-03) or preimmune rabbit serum, as appropriate controls. Immunoprecipitated DNA was quantified by realtime PCR, using a QuantiTect SYBR Green PCR Kit (Qiagen) with an ABI 7700 sequence detector and normalized to input DNA, as described (37Litt M.D. Simpson M. Recillas-Targa F. Prioleau M.N. Felsenfeld G. EMBO J. 2001; 20: 2224-2235Crossref PubMed Scopus (323) Google Scholar). Specificity of PCR products was controlled by melting curve analysis and in Figs. 2C and 4B, additionally on agarose gels. Specific primers used are: CD34 promoter, 5′-GATGGTGATGGGGAACTAAATGG-3′ and 5′-GCCAGTAACAATCTTGCAAAAGG-3′ (size: 338 bp); KIR2DL3 promoter, 5′-TGTATGAGAGGTTGGATCTGAG-3′ and 5′-GCCCTTCCAGGACTCACC-3′ (size: 321 bp).FIGURE 4Prevalent expression and recruitment of nucleolin to CD34 promoter in CD34-positive mobilized PB cells. A, left, lysates from CD34-positive cells mobilized into PB (lane 1) and CD34-depleted PBMCs (lane 2) were analyzed by immunoblotting with Abs specific for nucleolin (top) and β-actin (bottom). Right, levels of nucleolin mRNA in CD34-positive cells mobilized into PB (lane 3), CD34-depleted PBMCs (lane 4) and purified NK cells (lane 5) were analyzed by real-time RT-PCR and normalized to β-actin (bottom). Top, representative real-time RT-PCR amplification plots from the same run of RNA samples. Delta CT, average differences of CT values. B, left, top, EMSA with labeled CD34B oligonucleotide and nuclear extract from CD34-positive cells mobilized into PB (lane 1), CD34-depleted PBMCs (lane 2), or with no extract added (lane 3). Retarded nucleolin/DNA complex is indicated by an arrow. Left, bottom, nuclear extracts were analyzed by immunoblotting with Abs specific for nucleolin and β-actin. Middle and right, ChIP performed with nucleolin peptide Ab (lanes 4, 8, 12, 16, 18, and 20), with nucleolin Ab presaturated with the immunizing nucleolin peptide (lanes 5, 9, 13, 17, 19, and 21), with control Ab (lanes 6, 10, and 14), or with no Ab (lanes 7, 11, and 15). Lanes 4–7, 16 and 17, CD34-positive cells mobilized into PB; lanes 8–11, 18, and 19, CD34-depleted PBMCs; lanes 12–15, 20, and 21, purified NK cells. Lanes 4–15, co-precipitated CD34 promoter was quantified by real-time PCR. The y-axis indicates the ratio between bound and input DNA, as arbitrary units. Lanes 16–21, top, co-precipitated CD34 promoter was detected after semiquantitative PCR on agarose gel; bottom, control showing DNA quantities before IP. Lanes 16–17, 18–19, and 20–21 are adjacent parts of the same exposure of the same gel. C, left, ChIP performed with Ab specific for H3-tri-methyl K4 (lanes 1 and 4), with Ab specific for H4-AcK8 (lanes 7 and 10), with control Ab (lanes 2, 5, 8, and 11), with no Ab (lanes 3, 6, 9 and 12). Lanes 1–3 and 7–9, CD34-positive cells mobilized into PB; lanes 4–6 and 10–12, CD34-depleted PBMCs. Lanes 1–12, co-precipitated CD34 promoter was quantified by real-time PCR. The y-axis indicates the ratio between bound and input DNA, as arbitrary units. Right, CD34 mRNA levels in CD34-positive cells mobilized into PB (lane 13) and CD34-depleted PBMCs (lane 14) were quantified by real-time RT-PCR and normalized to β-actin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Real-time Reverse Transcription PCR (RT-PCR)—2 μg of total RNA, isolated with RNeasy kit (Qiagen), were reverse-transcribed using oligo(dT) primers, and levels of gene expression were quantified by real-time PCR, using a QuantiTect SYBR Green PCR Kit (Qiagen) with an ABI 7700 sequence detector. Sequences of specific primers used are: nucleolin cDNA, 5′-GATCACCTAATGCCAGAAGCCAGCCATCC-3′ and 5′-CAAAGCCGCCTCTGCCTCCACCAC-3′ (size: 297 bp); CD34 cDNA, 5′-CATCACAGAAACGACAGTCAA-3′ and 5′-ACTCCGCACAGCTGGAGG-3′ (size: 354 bp); Bcl-2 cDNA, 5′-GCATCTTCTCCTCCCAGCC-3′ and 5′-TGGACATCTCGGCGAAGTC-3′ (size: 209 bp); β-actin cDNA 5′-GCACTCTTCCAGCCTTCC-3′ and 5′-CTAGAAGCATTTGCGGTG-3′ (size: 351 bp). Specificity of PCR products was controlled by melting curve analysis and on agarose gels. The comparative threshold cycle (CT) method and an internal control (β-actin) were used to normalize target gene expression. Activation of Endogenous CD34 and Bcl-2 Expression by Nucleolin in CD34-positive Cells—Human BM-derived CD34-positive myeloblast cell line KG1 (32Koeffler H.P. Golde D.W. Science. 1978; 200: 1153-1154Crossref PubMed Scopus (361) Google Scholar) was stably transfected with a nucleolin expression vector, containing nucleolin cDNA in-frame with FLAG tag under control of cytomegalovirus promoter, referred to as KG1-Nuc cells, or with the empty expression vector, referred to as KG1-mock cells. Nucleolin expression was 3.5-fold increased in KG1-Nuc versus KG1-mock cells (Fig. 1A, top, lanes 1 and 2), and expression of exogenous nucleolin in KG1-Nuc cells was verified by immunoblotting with an Ab directed at the FLAG tag (Fig. 1A, lane 1). Immunoblotting of cell extracts with Abs specific for CD34, Bcl-2, and β-actin demonstrated that the levels of CD34 and Bcl-2 proteins were significantly increased in KG1-Nuc cells, ∼7-fold and 5-fold, respectively (Fig. 1A, lanes 3 and 4), while β-actin levels were identical (Fig. 1A, bottom, lanes 1–4). Furthermore, FACS analysis revealed that the increased nucleolin expression resulted in a ∼3-fold up-regulation of the cell surface CD34 (Fig. 1A, right). The levels of CD34 and Bcl-2 transcripts were increased in KG1-Nuc versus KG1-mock cells, as was determined by quantitative real-time RT-PCR, using β-actin as internal control (Fig. 1B). To provide further evidence for nucleolin-dependent activation of CD34 and Bcl-2 expression in CD34-positive cells, siRNA approach was employed. Two previously validated siRNAs (34Huddleson J.P. Ahmad N. Lingrel J.B. J. Biol. Chem. 2006; 281: 15121-15128Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 35Masumi A. Fukazawa H. Shimazu T. Yoshida M. Ozato K. Komuro K. Yamaguchi K. Oncogene. 2006; 25: 5113-5124Crossref PubMed Scopus (27) Google Scholar) that are targeted to different regions of the mRNA of human nucleolin, referred to as nucleolin siRNA-A and nucleolin siRNA-B, were used in experiments with KG1 cells. As compared with control non-targeting siRNA, either nucleolin-targeting siRNA significantly reduced nucleolin protein levels (Fig. 1C, top, lanes 2 and 5). β-Actin expression was not changed (Fig. 1C, bottom, lanes 1–5), and no significant effect on PCNA protein was detected (data not shown). The level of nucleolin in untransfected cells did not differ detectably from that in cells treated with the control siRNA (Fig. 1C, top, lanes 3 and 1, respectively). The knockdown of nucleolin expression with either nucleolin-targeting siRNA was accompanied by a significant reduction of CD34 and Bcl-2 protein levels, as was demonstrated by immunoblotting of cell extracts with Abs specific for CD34 and Bcl-2 (Fig. 1C, lanes 2 and 5). The levels of CD34 and Bcl-2 transcripts were investigated by quantitative real-time RT-PCR (Fig. 1C, lanes 6–8 and 9–11, respectively). As internal control β-actin wa" @default.
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• W2033662995 title "Nucleolin Regulates Gene Expression in CD34-positive Hematopoietic Cells" @default.
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