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• W2022792299 abstract "We have cloned two novel, alternatively spliced messages of human cyclin D-binding Myb-like protein (hDMP1). The known, full-length protein has been named hDMP1α and the new isoforms, hDMP1β and hDMP1γ. The hDMP1α, -β, and -γ splice variants have unique expression patterns in normal hematopoietic cells; hDMP1β mRNA transcripts are strongly expressed in quiescent CD34+ cells and freshly isolated peripheral blood leukocytes, as compared with hDMP1α. In contrast, activated T-cells and developing myeloid cells, macrophages, and granulocytes express low levels of hDMP1β transcripts, and hDMP1γ is ubiquitously and weakly expressed. Mouse Dmp1 has been shown to activate CD13/aminopeptidase N (APN) and p19ARF gene expression via binding to canonical DNA recognition sites in the respective promoters. Assessment of CD13/APN promoter responsiveness demonstrated that hDMP1α but not hDMP1β and -γ, is a transcriptional activator. Furthermore, hDMP1β was found to inhibit the CD13/APN promoter transactivation ability of hDMP1α. Stable, ectopic expression of hDMP1β and, to a lesser extent hDMP1γ, reduced endogenous cell surface levels of CD13/APN in U937 cells. Moreover, stable, ectopic expression of hDMP1β altered phorbol 12-myristate 13-acetate-induced terminal differentiation of U937 cells to macrophages and resulted in maintenance of proliferation. These results demonstrate that hDMP1β antagonizes hDMP1α activity and suggest that cellular functions of hDMP1 may be regulated by cellular hDMP1 isoform levels. We have cloned two novel, alternatively spliced messages of human cyclin D-binding Myb-like protein (hDMP1). The known, full-length protein has been named hDMP1α and the new isoforms, hDMP1β and hDMP1γ. The hDMP1α, -β, and -γ splice variants have unique expression patterns in normal hematopoietic cells; hDMP1β mRNA transcripts are strongly expressed in quiescent CD34+ cells and freshly isolated peripheral blood leukocytes, as compared with hDMP1α. In contrast, activated T-cells and developing myeloid cells, macrophages, and granulocytes express low levels of hDMP1β transcripts, and hDMP1γ is ubiquitously and weakly expressed. Mouse Dmp1 has been shown to activate CD13/aminopeptidase N (APN) and p19ARF gene expression via binding to canonical DNA recognition sites in the respective promoters. Assessment of CD13/APN promoter responsiveness demonstrated that hDMP1α but not hDMP1β and -γ, is a transcriptional activator. Furthermore, hDMP1β was found to inhibit the CD13/APN promoter transactivation ability of hDMP1α. Stable, ectopic expression of hDMP1β and, to a lesser extent hDMP1γ, reduced endogenous cell surface levels of CD13/APN in U937 cells. Moreover, stable, ectopic expression of hDMP1β altered phorbol 12-myristate 13-acetate-induced terminal differentiation of U937 cells to macrophages and resulted in maintenance of proliferation. These results demonstrate that hDMP1β antagonizes hDMP1α activity and suggest that cellular functions of hDMP1 may be regulated by cellular hDMP1 isoform levels. The 761-amino acid mouse cyclin D-binding Myb-like protein (mDmp1) transcription factor was first identified using cyclin D2 as bait in a yeast two-hybrid screen, and human DMP1 was cloned shortly thereafter (1Hirai H. Sherr C.J. Mol. Cell Biol. 1996; 16: 6457-6467Crossref PubMed Scopus (115) Google Scholar, 2Bodner S.M. Naeve C.W. Rakestraw K.M. Jones B.G. Valentine V.A. Valentine M.B. Luthardt F.W. Willman C.L. Raimondi S.C. Downing J.R. Roussel M.F. Sherr C.J. Look A.T. Gene. 1999; 229: 223-228Crossref PubMed Scopus (32) Google Scholar). Both mouse and human DMP1 are composed of a central domain containing three Myb-like repeats flanked by an acidic transactivation, a cyclin binding domain on the N terminus, and an acidic transactivation domain at the C terminus (3Inoue K. Sherr C.J. Mol. Cell Biol. 1998; 18: 1590-1600Crossref PubMed Scopus (143) Google Scholar). In humans, hDMP1 mRNA is ubiquitously expressed at low levels in normal human tissues, with highest expression levels in testis, spleen, thymus, and peripheral blood leukocytes (2Bodner S.M. Naeve C.W. Rakestraw K.M. Jones B.G. Valentine V.A. Valentine M.B. Luthardt F.W. Willman C.L. Raimondi S.C. Downing J.R. Roussel M.F. Sherr C.J. Look A.T. Gene. 1999; 229: 223-228Crossref PubMed Scopus (32) Google Scholar). Interestingly the hDMP1 locus is localized on chromosome 7q21, a region frequently deleted as part of the 7q-minus and monosomy 7 abnormalities of acute myeloid leukemia and myelodysplastic syndrome. DMP1 binds to nonameric Ets consensus DNA sequences containing G(G/T)A cores, competes with Ets family proteins, and has been shown to be a transcriptional activator (1Hirai H. Sherr C.J. Mol. Cell Biol. 1996; 16: 6457-6467Crossref PubMed Scopus (115) Google Scholar). DMP1 binding sites are found in the promoters of two well known genes, CD13/aminopeptidase N (APN) 1The abbreviations used are: APN, aminopeptidase N; aa, amino acid; FITC, fluorescein; PE, phycoerythrin; PMA, phorbol 12-myristate 13-acetate; h, human; hr, human recombinant; CSF, colony-stimulating factor; tNGFR, truncated nerve growth factor receptor; PBMNC, peripheral blood mononuclear cell; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; HIV, human immunodeficiency virus. and the mouse tumor suppressor p19Arf (also referred to as ARF or p14ARF in humans), which is encoded from the Ink4a-Arf locus (4Sherr C.J. Weber J.D. Curr. Opin. Genet. Dev. 2000; 10: 94-99Crossref PubMed Scopus (573) Google Scholar). DMP1 has been shown to be present in non-dividing cells and has a role in cell differentiation in certain hematopoietic lineages (1Hirai H. Sherr C.J. Mol. Cell Biol. 1996; 16: 6457-6467Crossref PubMed Scopus (115) Google Scholar, 3Inoue K. Sherr C.J. Mol. Cell Biol. 1998; 18: 1590-1600Crossref PubMed Scopus (143) Google Scholar) and is critical for cell cycle control via regulation of ARF (5Inoue K. Roussel M.F. Sherr C.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3993-3998Crossref PubMed Scopus (125) Google Scholar). One such gene regulated by DMP1, the ectoenzyme CD13/APN, has been implicated in myeloid development (6Inoue K. Sherr C.J. Shapiro L.H. J. Biol. Chem. 1998; 273: 29188-29194Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). CD13/APN is a membrane-bound metalloproteinase that is expressed on normal human myeloid and lymphoid cells (7Drexler H.G. Leukemia (Baltimore). 1987; 1: 697-705PubMed Google Scholar, 8Pui C.H. Hancock M.L. Head D.R. Rivera G.K. Look A.T. Sandlund J.T. Behm F.G. Blood. 1993; 82: 889-894Crossref PubMed Google Scholar) as well as non-hematopoietic cells (9Shipp M.A. Look A.T. Blood. 1993; 82: 1052-1070Crossref PubMed Google Scholar). CD13/APN expression is developmentally regulated (10Riemann D. Kehlen A. Langner J. Immunol Today. 1999; 20: 83-88Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), functions in peptide degradation and cell-cell adhesion (11Menrad A. Speicher D. Wacker J. Herlyn M. Cancer Res. 1993; 53: 1450-1455PubMed Google Scholar, 12Bhagwat S.V. Petrovic N. Okamoto Y. Shapiro L.H. Blood. 2002; 101: 1818-1826Crossref PubMed Scopus (95) Google Scholar), and participates in growth and development of both hematopoietic and endothelial cells (reviewed in Riemann et al. (10Riemann D. Kehlen A. Langner J. Immunol Today. 1999; 20: 83-88Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar)). A direct role for CD13/APN has been proposed for human dendritic cell development in culture (13Rosenzwajg M. Tailleux L. Gluckman J.C. Blood. 2000; 95: 453-460Crossref PubMed Google Scholar). In myeloid cells it has been shown that c-Myb, Ets family members, and mDmp1 regulate CD13/APN transcription (6Inoue K. Sherr C.J. Shapiro L.H. J. Biol. Chem. 1998; 273: 29188-29194Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Activation of CD13/APN gene expression is enhanced by a cooperative interaction between c-Myb bound to its cognate site and mDmp1, bound to one of the three downstream GGA core sites (6Inoue K. Sherr C.J. Shapiro L.H. J. Biol. Chem. 1998; 273: 29188-29194Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Thus, CD13/APN promoter activity requires the intact DNA binding and transactivation domains of mDmp1, since mutants disrupted in either domain are biologically inert. Intriguingly, that c-Myb and mDmp1 show synergistic effects in activating the CD13/APN promoter implies that two different Myb family proteins collaborate in regulating CD13/APN gene expression and points to an important role for DMP1 in normal myeloid cell development (6Inoue K. Sherr C.J. Shapiro L.H. J. Biol. Chem. 1998; 273: 29188-29194Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Herein we describe two new splice variants of human DMP1 message generated by alternative splicing through the use of two different splice acceptor sites. We propose that the previously cloned hDMP1 message (2Bodner S.M. Naeve C.W. Rakestraw K.M. Jones B.G. Valentine V.A. Valentine M.B. Luthardt F.W. Willman C.L. Raimondi S.C. Downing J.R. Roussel M.F. Sherr C.J. Look A.T. Gene. 1999; 229: 223-228Crossref PubMed Scopus (32) Google Scholar) be termed hDMP1α and the new splice variants hDMP1β and -γ. Distinct patterns of hDMP1α, -β, and -γ mRNA transcript expression in primary myeloid and lymphoid cells during in vitro development led us to consider the possibility that alternative hDMP1β and -γ isoforms might have distinct cellular functions as compared with hDMP1α. We confirm a role for the original DMP1 isoform, hDMP1α, in activation of CD13/APN gene expression (6Inoue K. Sherr C.J. Shapiro L.H. J. Biol. Chem. 1998; 273: 29188-29194Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and document antagonistic functional properties for the hDMP1β isoform in CD13/APN regulation using CD13/APN promoter-reporters and through stable, ectopic hDMP1β and -γ isoform expression in the U937 myeloid cell line. Moreover, stable, ectopic expression of hDMP1β in U937 cells altered PMA-induced terminal differentiation to macrophages. Cell Isolations—Peripheral blood mononuclear cell (PBMNC) samples were obtained from healthy donors recruited by the General Clinical Research Center at Green Hospital, La Jolla, CA, and cord blood samples were from placentas acquired from mothers with normal, full term deliveries at Scripps Memorial Hospital, La Jolla, CA. Protocols and the use of all human samples were approved by the Human Subjects Institutional Review Board of The Scripps Research Institute. CD34+ cells from cord and peripheral blood, as well as PBMNCs and neutrophils from peripheral blood, were isolated as previously reported (14Miyoshi H. Smith K.A. Mosier D.E. Verma I.M. Torbett B.E. Science. 1999; 283: 682-686Crossref PubMed Scopus (557) Google Scholar, 15Tschan M.P. Vonlanthen S. Cajot J.F. Peters U.R. Oppliger E. Betticher D.C. Yarbrough W.G. Fey M.F. Tobler A. Leuk. Lymphoma. 2001; 42: 1077-1087Crossref PubMed Scopus (16) Google Scholar). RT-PCR Reactions—Total RNA was prepared using the RNeasy mini kit (Qiagen, Valencia, CA), and cDNA from 0.5–1 μg of sample RNA was generated (oligo(dT) primers, Roche Applied Science; Moloney murine leukemia virus reverse transcriptase was from Promega, Madison, WI). CD34+ RNA was prepared from 2 × 104 cells, and cDNA was generated using Superscript™II reverse transcriptase (Invitrogen). The relative expression of the hDMP1 splice variants β and γ as well as the previously cloned hDMP1α was determined by RT-PCR using primers encompassing the found insertions (Fig. 2A, forward primer, 5′-TACAGGACTATAGCATGGGGTC-3′; reverse primer, 5′-ACTTCCCTGTGTTGCAAGTATC-3′). Conditions for PCR amplification were: one cycle for 5 min at 94 °C, 35 cycles with 15 s at 94 °C, 30 s at 58 °C, 60 s at 72 °C, and a last cycle at 72 °C for 7 min. HDMP1β and -γ were specifically amplified using the same forward primer as above but with a β- and γ-specific reverse primer (5′-CCATTTGACTGGTTTGGAAGTTG-3′) in the unique DNA insertion of these splice variants. PCR conditions were the same as above but using 32 cycles. GAPDH PCR was performed as described (16Zwahlen D. Tschan M.P. Grob T.J. Peters U.R. Fink D. Haenggi W. Altermatt H.J. Cajot J.F. Tobler A. Fey M.F. Aebi S. Int. J. Cancer. 2000; 88: 66-70Crossref PubMed Scopus (37) Google Scholar). Cell Culture—Cord blood CD34+ cells were differentiated in culture to either neutrophils or monocytes during a 15-day period with combinations of human recombinant cytokines. Initially, CD34+ cells were cultured in 10% BIT 9500 (StemCell Technology, Vancouver, Canada) supplemented serum-free Iscove's modified Dulbecco's medium containing hr stem cell factor (300 ng/ml), hr granulocyte CSF (10 ng/ml), hr interleukin-6 (10 units/ml), hr granulocyte-macrophage CSF (50 ng/ml), and hr Flt-3 ligand (300 ng/ml) to expand primitive progenitors. Growth factors were purchased from PeproTech (Rocky Hill, NJ). All cultures were maintained in a 37 °C humidified incubator containing 5% CO2. After 4 days of culture the cells were removed and washed, and the medium was changed to Myelocult™ H5100 (StemCell Technology) containing hr CSF (300 ng/ml), hr granulocyte-macrophage CSF (10 ng/ml), hr interleukin-6 (10 units/ml), and hr interleukin-3 (10 ng/ml) to promote expansion of primitive myeloid progenitors. On day 10 of the cultures, cells were removed, washed, and resuspended in either Myelocult™ hr granulocyte CSF (10 ng/ml) for neutrophil development or Myelocult™ and hr macrophage CSF (5000 units/ml) for monocyte/macrophage development. Stage of development was assessed on cellular morphology and stage-specific cell surface markers using flow cytometry and appropriate antibody reagents (14Miyoshi H. Smith K.A. Mosier D.E. Verma I.M. Torbett B.E. Science. 1999; 283: 682-686Crossref PubMed Scopus (557) Google Scholar, 17Berliner N. Hsing A. Graubert T. Sigurdsson F. Zain M. Bruno E. Hoffman R. Blood. 1995; 85: 799-803Crossref PubMed Google Scholar, 18Anderson K.L. Smith K.A. Conners K. McKercher S.R. Maki R.A. Torbett B.E. Blood. 1998; 91: 3702-3710Crossref PubMed Google Scholar). To obtain peripheral blood-derived macrophages, 2 × 106 PBMNCs in 1 ml of RPMI containing 10% fetal bovine serum (Hyclone, Ogden, UT), 2 mm l-glutamine, and 100 units/ml penicillin and streptomycin (C-RPMI) were added to each well of a 24-well tissue culture plate and maintained overnight in a 37 °C humidified incubator containing 5% CO2. The next day non-adherent cells were removed by gentle washing, and cells were then cultured in 1 ml of C-RPMI and 5000 units/ml of hr macrophage CSF. The human U937 monocytic cell line was maintained in DMEM containing 10% fetal bovine serum (Hyclone), 2 mm l-glutamine, and 100 units/ml penicillin and streptomycin (C-DMEM). U937 cells were differentiated using PMA (Sigma-Aldrich). Briefly, PMA first was dissolved in Me2SO at a concentration of 1.0 × 10–4m. For macrophage differentiation, PMA was diluted to concentrations as indicated in C-DMEM, and cells were cultured at a density of 0.3 × 106 cells/ml for 1–4 days. All cultures were maintained in a 37 °C humidified incubator containing 10% CO2. Non-adherent PBMNC were cultured in C-RPMI on 12-well plates pre-coated with anti-CD3 (1 μg/ml; BD Biosciences, San Jose, CA) and anti-CD28 (1 μg/ml; BD Biosciences) antibody for T-cell activation. Activation of T-cells was measured by fluorescence-activated cell sorter analysis of the CD69 surface marker (peridinin chlorophyll protein-conjugated CD69 antibody, BD Biosciences) after 6 days of incubation in a 37 °C humidified incubator containing 5% CO2. Expression Plasmid and HIV Vector Construction and Preparation of Vector Stocks—hDMP1α and its two variants were cloned by RT-PCR. Briefly, total RNA from KG-1 cells was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Promega). The open reading frame of hDMP1α was amplified with the Expand High Fidelity PCR System (Roche Applied Science) and the following primers: forward, 5′-ATGAGCACAGTGGAAGAGGATTC-3′, and reverse, 5′-ATGACAGTTTACCAAATCTTC-3′. Blunt-ended fragments were 3′ A-tailed with Taq DNA polymerase, purified, and ligated into the pcDNA3.1/V5-His-TOPO vector (Invitrogen). Plasmid pCR-XL-CSPre was constructed by cloning the MluI-ApaI fragment from the self-inactivating (SIN) HIV vector pHIV-SINPre (19Miyoshi H. Blomer U. Takahashi M. Gage F.H. Verma I.M. J. Virol. 1998; 72: 8150-8157Crossref PubMed Google Scholar) into the pCR-XL Topo backbone (Invitrogen). The cytomegalovirus promoter from pcDNA3.1/V5-His-TOPO (BglII-BamHI fragment) was cloned into the BamHI site of pCR-XL-CSPre. The HIV vector VIPER was constructed by cloning a fragment containing the central polypurine tract (20Follenzi A. Ailles L.E. Bakovic S. Geuna M. Naldini L. Nat. Genet. 2000; 25: 217-222Crossref PubMed Scopus (786) Google Scholar), the human elongation factor five internal ribosomal entry site (21Gan W. Celle M.L. Rhoads R.E. J. Biol. Chem. 1998; 273: 5006-5012Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), the truncated nerve growth factor receptor (tNGFR), and scaffold attachment region sequence (22Agarwal M. Austin T.W. Morel F. Chen J. Bohnlein E. Plavec I. J. Virol. 1998; 72: 3720-3728Crossref PubMed Google Scholar) between the BamHI and SacII of pCR-XL-CSPre. HDMP1β and -γ were cloned into the unique BamHI site of VIPER. The VSV-G-pseudotyped HIV vectors were generated by transient transfection using the selected transgene plasmid and the following third-generation packaging plasmids: pMD.G (VSV-G), pMDLg/p.RRE (gag and pol), and pRSV-Rev (rev) (23Galimi F. Noll M. Kanazawa Y. Lax T. Chen C. Grompe M. Verma I.M. Blood. 2002; 100: 2732-2736Crossref PubMed Scopus (78) Google Scholar). The VIPER vector-containing supernatant was harvested after 24 h, concentrated by ultracentrifugation, and resuspended in serum-free medium. HIV vector titers, defined as transducing units/ml were determined by transduction of 293T-cells and flow cytometry analysis to determine the percentage of cells expressing tNGFR (14Miyoshi H. Smith K.A. Mosier D.E. Verma I.M. Torbett B.E. Science. 1999; 283: 682-686Crossref PubMed Scopus (557) Google Scholar). In Vitro Transcription and Translation of hDMP1 Isoforms—Cell-free transcription and translation of hDMP1α, -β, and -γ plasmid DNA was performed using the TnT T7-coupled reticulocyte lysate system (TnT® system) and the Transcend™ non-radioactive translation detection system according to the manufacturer's instructions (Promega). A luciferase plasmid encoding a 61-kDa protein was used as positive control. The empty pcDNA3.1/V5-His-TOPO vector served as a negative control. To detect reaction products 15 μl were separated by SDS-PAGE, blotted, and detected by chemiluminescence. Dual Luciferase Reporter Assays—Firefly luciferase reporter plasmids containing a contiguous stretch of eight concatamerized DMP1 binding sites, pGL2-BS2, kindly provided by Charles J. Sherr, St. Jude Children's Hospital, Memphis, TN, or the myeloid CD13/APN promoter (pMyo), kindly provided by Jørgen Olson, University of Copenhagen, Denmark, were used for functional analysis of the different hDMP1 isoforms. For transfection experiments, 293T-cells were grown to 70–80% confluence in 60-mm dishes and transfected using a modified calcium phosphate method (24Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4826) Google Scholar); 6 μg of the reporter plasmids were co-transfected with 4 μg of the different hDMP1 expression vectors or the empty pcDNA3.1/V5-His-TOPO vector. Twenty ng of the pRL-TK Renilla luciferase vector were co-transfected in each experiment as an internal control for transfection efficiency. For mixing experiments, 4 μg of the reporter plasmids and 1.5 μg of pcDNA3.1-hDMP1α were co-transfected with varying amounts of pcDNA3.1-hDMP1β and -γ. The total amount of transfected DNA was adjusted to 11.5 μg with empty pcDNA3.1/V5-His-TOPO vector. Cell lysates from transfected cells were prepared 48 h after transfection, and luciferase activity was measured according to the manufacturer's Dual-Luciferase™ reporter assay protocol (Promega). The ratio of firefly to Renilla luciferase was calculated to obtain the relative luciferase activity. Each experiment was performed in triplicate, and data are presented as the mean ± S.E. HIV VIPER Transduction and Cell Sorting—For VIPER vector transduction 2 × 105 U937 cells were added to round bottom 96-well plates in 0.1 ml of C-DMEM. Cells were transduced with VIPER control (tNGFR gene, no hDMP1 gene), VIPERβ (hDMP1β) and -γ (hDMP1γ) vectors at a multiplicity of infection of 20 in the presence of 8 μg/ml Polybrene. The plates were spun for 45 min at 1250 rpm at room temperature using a Sorvall RT 6000D centrifuge and then incubated for 4 h in 10% CO2 at 37 °C. Cells were washed once and resuspended in fresh medium. The transduction was repeated the next day, and cells were cultured for 10 days. After 10 days the upper 20% tNGFR-expressing U937 cells were isolated on a fluorescence-activated cell sorter Vantage SE II cell sorter (BD Biosciences) using FITC-conjugated tNGFR antibodies. Analysis of Cell Surface Proteins—Flow cytometry was used to analyze expression of CD13/APN, CD11c, and tNGFR on transduced U937 cells. In brief, 2 × 105 U937 cells in 100 μl of ice-cold fluorescence-activated cell sorter buffer were incubated with 1 μl of the FITC-conjugated CD13/APN (10M13, AMAC, Westbrook, ME), the FITC-conjugated CD11c (Leu-M5, BD Biosciences), or the PE-conjugated NGFR antibody (C40–1457, BD Biosciences), washed, and analyzed for flow cytometry or sorted. For antibody specificity, irrelevant FITC- and PE-conjugated mouse isotype controls (Beckman Coulter, Miami, FL) were used. For flow cytometry analysis, 104 cell events per sample were acquired on a FACSCalibur machine (BD Biosciences) running the Cell Quest acquisition software, and analysis was accomplished using Cell Quest analysis software, Version 3.4. Proliferation Assays—Proliferation was assessed by labeling cells with [3H]thymidine. Differentiating U937 VIPER and VIPERβ transfectants were resuspended in C-DMEM containing PMA at concentrations as indicated and seeded in 96-well plates. [3H]Thymidine was added for the last 5 h of cell treatment (1 μCi/well). Cells were harvested at time points indicated using a Mach III cell harvester (Tomtec, Orange, CT), and β-radiation was detected on a Wallac 1450 MicroBeta Liquid Scintillation Counter (PerkinElmer Life Sciences). Morphological Evaluation—Morphological features of differentiated U937 VIPER and VIPERβ cells were reviewed on cytospin slide preparations stained with May-Gruenwald-Giemsa stain. Classically defined morphological features were used as indicators of macrophage differentiation including cell shape, adherence, and ratio of cytoplasmic region to nuclei and cytoplasmic granulation. Genomic Organization and Translation of the Human DMP1 Gene and Its Splice Variants—During routine RT-PCR cloning for full-length hDMP1 message from the myeloid leukemic cell line KG-1, we obtained three distinct hDMP1 cDNA clones (“Experimental Procedures”). Sequence comparison of each unique clone to the hDMP1 genomic clone (accession number RG227L24) allowed identification of the genomic organization of hDMP1. The analysis revealed a perfect match for each of the 18 exons with corresponding splice donor and acceptor sites found in the genomic BAC clone (Table I). hDMP1 contains a large 11-kilobase first intron with the translational start site located in exon 3 (Fig. 1A). Further comparison of the two unique hDMP1 message sequences with the hDMP1 genomic sequence revealed inserts of 172 and 211 nucleotides, respectively, at nucleotide 985 (accession number AF084530). These sequences are identical to the genomic sequence of intron 9 and were generated by alternative splicing using two putative splicing acceptor sites as seen in Table I. Based on our findings we now propose to refer to hDMP1 as hDMP1α and the new splice variants as hDMP1β and -γ, respectively (accession numbers AF202144 and AF202145). The open reading frames of hDMP1β and -γ encode identical initial amino acid (aa) sequences to hDMP1α up to the splice site at aa 237. However, after aa 237, hDMP1β and -γ show novel stretches of 35 and 48 aa, respectively, followed by a premature TAA stop signal occurring in the alternatively spliced intronic sequence (Fig. 1B). The newly defined hDMP1 isoforms still contain the complete acidic N-terminal transactivation domain, the cyclin D binding domain (CBS), and an Myb-homology remnant but no C-terminal transactivation domain. The hDMP1β and -γ Myb-homology remnant is composed of first the 14/169 aa of the N-terminal sequence of the Myb-homology domain followed by sequences derived from intron 9 and concluding with 35 aa showing homology to the C terminus of the Myb-homology domain (Fig. 1B). HDMP1γ contains additional 13 aa as compared with -β, bridging the 14- and 35-aa remnants of the N- and C-terminal Myb-homology region. The predicted length of proteins encoded by hDMP1α,-β, and -γ would be 760, 272, and 285 aa, respectively.Table IExon-intron boundaries of hDMP1αExon no.ExonExon sizeSequence of exon-intron junction3′ splice acceptor5′ splice donorIntron sizebpkilobases11-144145ggggc GCGGCAGGAA gtaag10.942145-267123ttcag AGTGTTCTAG gtaag1.323268-384109tacag ATTTGGAATG gtagg1.434385-507123attag AAGCGTCCAC gtaag4.395508-60295tgaag TTTCATACAG gtata2.446603-717115tcaag ATTTTTAAAG gtaag0.947718-79477taaag GACATTTAAG gtatc4.878795-952158tttag GCACGGGAAA gtatg1.619953-98533tctag ATATAAAGGA gtaag0.8910986-1095110tttag GCTCCCACAG gtact2.0610β+172282cacag ACAACCACAG gtact1.8910γ+211321cccag GAAAACACAG gtact1.85111096-1324229ttcag GGAAGCTCAG gtttg1.20121325-1476152tgtag GATAGCCCTG gtaat2.11131477-1686210ttcag TCTTATGTTT gtaag2.64141687-176983cacag CTTCATTCCC gtgag2.17151770-1925156tttag TCTTTTATCC gtatg0.37161926-2303378tatag CCAGACTGAG gtaag0.58172304-2448145tttag GGTTTAACAG gtact0.20182449-37671319tacag ATCCC Open table in a new tab We next performed cell-free transcription and translation assays to verify the existence of protein translation from the hDMP1β and -γ message cDNA and to determine the approximate molecular weight of the translated proteins. HDMP1 expression plasmids produced all protein isoforms and displayed the molecular masses of 125, 31, and 32 kDa for hDMP1α, -β, and -γ, respectively (Fig. 1C). These data provide evidence that two new human DMP1 splice variants can be expressed from cloned hDMP1β and hDMP1γ message, and both encode shorter proteins than found for full-length hDMP1α. Expression Patterns of hDMP1 mRNA Splice Variants in Primary Hematopoietic Cells—hDMP1 is expressed in cells obtained from spleen, peripheral blood, and thymus (2Bodner S.M. Naeve C.W. Rakestraw K.M. Jones B.G. Valentine V.A. Valentine M.B. Luthardt F.W. Willman C.L. Raimondi S.C. Downing J.R. Roussel M.F. Sherr C.J. Look A.T. Gene. 1999; 229: 223-228Crossref PubMed Scopus (32) Google Scholar). To investigate the expression patterns of the two novel hDMP1β and -γ mRNA splice variants and hDMP1α in hematopoietic cells, we designed primers flanking the intronic insertions of hDMP1β and -γ, allowing us to amplify all three variants in the same PCR reaction and thereby allowing intra-transcript comparisons (Fig. 2A). A number of distinct hematopoietic cell lineages were used as poly-A mRNA sources for evaluation of hDMP1 transcript expression. As can be seen in a representative panel of RT-PCR results presented in Fig. 2B, expression of hDMP1α, -β, and -γ was seen in RNA samples obtained from freshly isolated CD34+ hematopoietic cells (7/7 samples) derived from mobilized peripheral blood, PBMNCs (6/6 samples), and cultured macrophages derived from peripheral blood (3/3 samples). In CD34+ cells the ratio of hDMP1α to hDMP1β transcripts was decreased (0.6 ± 0.1; n = 5), indicating increased relative amounts of hDMP1β transcripts as compared with hDMP1α. PBMNCs, a heterogeneous population of T-cells, B-cells, and monocytes expressed equivalent amounts of hDMP1α and -β mRNA transcripts (hDMP1α:hDMP1β ratio 0.9 ± 0.1; n = 6). In contrast to CD34+ cells, macrophages derived in tissue culture from peripheral blood monocytes predominantly and consistently demonstrated an increased hDMP1α: hDMP1β ratio (3.3 ± 1.0; n = 3), implying increased relative hDMP1α transcript amounts as compared with hDMP1β.Inall cell populations examined from peripheral blood, hDMP1γ transcripts were weakly expressed. To provide further insight into hDMP1 transcript expression in developing myeloid cells, cord blood CD34+ cells were differentiated in tissue culture using conditions that favored myeloid development. The presence of hDMP1 splice variants was assessed at various times early in differentiation and in culture-derived neutrophils (Fig. 2C , top right panel) and monocytes (Fig. 2C , bottom right panel). Myeloid stages of cells grown in culture were confirmed by phenotypic and morphologic criteria. After 1 day of differentiation a marked down-regulation of hDMP1β was observed (Fig. 2C). During the entire differentiation period relative levels of hDMP1β stayed low, whereas hDMP1α was the predominant splice variant identified. There were no substantial changes in hDMP1γ transcript expression. In contrast, terminally differentiated neutrophils expressed relatively lower amounts of all hDMP1 splice variants but increased amounts of hDMP1α versus hDMP1β, whereas monocytes expressed relatively higher amounts of hDMP1α transcripts. Assessment of hDMP1 transcripts from freshly isolated neutrophils obtained from blood confirmed (data n" @default.
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• W2022792299 title "Alternative Splicing of the Human Cyclin D-binding Myb-like Protein (hDMP1) Yields a Truncated Protein Isoform That Alters Macrophage Differentiation Patterns" @default.
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