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Hemogen is a novel nuclear factor specifically expressed in mouse hematopoietic development and its human homologue EDAG maps to chromosome 9q22, a region containing breakpoints of hematological neoplasms
Mechanisms of Development 104 (2001) 105±111
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Hemogen is a novel nuclear factor speci®cally expressed in mouse hematopoietic development and its human homologue EDAG maps to chromosome 9q22, a region containing breakpoints of hematological neoplasms Li V. Yang a, Rhonda H. Nicholson a, Joseph Kaplan b, Anne Galy c, Li Li a,d,* a
Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI 48201, USA b Department of Pediatrics, Wayne State University, Detroit, MI 48201, USA c Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA d Department of Internal Medicine, Wayne State University, 421 E. Can®eld Ave. # 1107 Detroit, MI 48201, USA Received 1 February 2001; received in revised form 30 March 2001; accepted 5 April 2001
Abstract We cloned a novel murine gene, designated Hemogen (hemopoietic gene), which was sequentially expressed in active hematopoietic sites and downregulated in the process of blood cell differentiation. Hemogen transcripts were speci®cally detected in blood islands, primitive blood cells and fetal liver during embryogenesis, and then remained in bone marrow and spleen in adult mice. Immunostaining demonstrated that Hemogen was a nuclear protein. We also identi®ed a human homologue of Hemogen, named EDAG, which was mapped to chromosome 9q22, a leukemia breakpoint. Like Hemogen, EDAG exhibited speci®c expression in hematopoietic tissues and cells. Taken together, these data are consistent with Hemogen and EDAG playing an important role in hematopoietic development and neoplasms. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hematopoiesis; Stem cell; Blood island; Bone marrow; Gene expression; Hemogen; EDAG; RP59; Development; Leukemia; Embryogenesis; In situ hybridization; Chromosome 9q22; Breakpoints
1. Introduction Hematopoiesis is a dynamic process with sequential shifting of primary hematopoietic sites from yolk sac to fetal liver and ®nally bone marrow. During embryonic development, hematopoietic tissues are derived from the ventral mesoderm. The ®rst blood cells, primitive erythrocytes, appear in blood islands in extraembryonic yolk sac at about embryonic day 7.5 (E7.5) in mice. Yolk sac also serves as the ®rst source of de®nitive hematopoietic progenitors (Palis et al., 1999). By E12, fetal liver becomes the predominant site of blood cell formation in the embryo (Dzierzak and Medvinsky, 1995). Just prior to birth and thereafter, the hematopoiesis in fetal liver gradually decreases, and bone marrow becomes the major hematopoietic site. The mouse spleen, different from the human * Corresponding author. Department of Internal Medicine, Wayne State University, 421 E. Can®eld Ave. # 1107 Detroit, MI 48201, USA. Tel.: 11313-577-8749; fax: 11-313-577-8615. E-mail address: [email protected] (L. Li).
spleen, is still very active in hematopoiesis, particularly erythropoiesis, in the red pulp at the adult stage (Seifert and Marks, 1985). Mature blood cells of three main hematopoietic lineages, erythroid, myeloid and lymphoid, are derived from common pluripotent hematopoietic stem cells (Spangrude et al., 1988). Research on the molecular basis of hematopoiesis has been facilitated by the identi®cation of a variety of hematopoietic regulating genes that are important for hematopoietic induction, lineage selection and blood cell differentiation (Engel and Murre, 1999; Orkin, 1995; Sieweke and Graf, 1998). Mutations or translocations of many of these genes have proven to play a role in hematopoietic malignancies (Rowley, 1998; Sawyers, 1998). We now report the identi®cation of a new nuclear protein, Hemogen, which shows a spatial-temporal expression pattern corresponding to the ontogeny of mouse hematopoiesis. We have also identi®ed a human homologue of Hemogen, EDAG, which is also speci®cally expressed in hematopoietic cells. Interestingly, EDAG maps to chromosome 9q22, a region containing the breakpoints of several hematopoietic neoplasms.
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00376-8
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2. Results and discussion 2.1. Cloning and sequence analysis of a novel murine gene Hemogen We initially performed a PCR-based cDNA subtraction, aiming to identify developmentally regulated genes in mouse E10.5 and E16.5 heart tissues. A number of differentially expressed genes were identi®ed in this screening. Perhaps due to the blood cells trapped in the heart tissues, some hematopoietic genes, such as embryonic 1 and bH1 globins, were also cloned (data not shown). One of the clones, 6B2, showed the differential expression at E10.5. Searching against GenBank, this clone showed homology with several mouse ESTs, e.g. GenBank accession nos. AA051237, AI121196 and AI006512. These three independent clones were sequenced to obtain the full-length cDNA sequence from which the amino acid sequence was deduced (Fig. 1A). This novel gene, now designated Hemogen (hemopoietic gene, accession no. AF269248), encodes 503 amino acids with a calculated molecular weight of 55 043 Da and a pI of 4.84. Sequence analysis suggested that the ®rst ATG is the translation initiation codon and the surrounding sequence AAGATGG is consistent with the Kozak consensus sequence with a purine at position 23 and a G at position 14 (Kozak, 1997). The polyadenylation signal sequence is ATTAAA (2288±2293 nt), a most common variant of the canonical sequence AATAAA (Graber et al., 1999). The putative Hemogen protein has a basic domain (34±78 residues with a net charge of 115) at the N-terminus and an acidic domain (450±480 residues with a net charge of 211) at the C-terminus. In the basic domain, the region from 34 to 50 amino acids is predicted (at the window size of 14) to be a coiled-coil domain (Lupas, 1996), which is implicated in protein polymerization. The 61±78 amino acids contain a bipartite nuclear localization signal suggesting that Hemogen may be a nuclear protein (Dingwall and Laskey, 1991). The analysis of amino acid composition shows that the usage of proline (10.3%), glutamate (8.7%) and glutamic acid (11.9%) is higher than the average level (Brendel et al., 1992). 2.2. Hemogen encodes a nuclear protein To con®rm that the nuclear localization signal in Hemogen protein was functional, we transfected the mammalian expression vector containing the Hemogen-FLAG fusion gene into COS-7 cells, and used anti-FLAG antibody to detect the subcellular localization of this protein. The results showed that Hemogen was located in cell nuclei but not in nucleoli or cytoplasm (Fig. 1B). 2.3. Hemogen shows alternate locations of expression coincident with the shifting sites of hematopoiesis during ontogeny To study the expression pattern of Hemogen during
mouse embryogenesis, in situ hybridization was used to detect mRNA transcripts. Hemogen was expressed during early embryogenesis at E8.5 in the blood islands of the yolk sac and in the circulating primitive blood cells (Fig. 2A±C). The expression in the circulating blood cells was detected strongly at E9.5 and E10.5 (Fig. 2D±G). As the organogenesis of liver emerged at E10.5, Hemogen expression began to be detectable in the developing hepatic primordia (Fig. 2F,H). From E11.5, Hemogen was exclusively expressed in the fetal liver (Fig. 2I,K), while the expression in circulating blood cells was downregulated dramatically to undetectable levels (Fig. 2J). The same expression patterns were observed in E12.5 and E14.5 mouse embryos (Fig. 2L±Q). We examined the tissue distribution of Hemogen in adult mice by Northern blot. A 2.4 kb transcript was speci®cally expressed in the bone marrow and spleen (Fig. 3A). When the same ®lter was overexposed, weak signals were detected in the peripheral blood at a much lower level. Two additional transcripts at 1.1 and 3.7 kb were also identi®ed and the 1.1 kb band gave the strongest signal in peripheral blood (Fig. 3A). This indicated that multiple isoforms exist in peripheral blood cells. No expression was detected in the thymus or non-hematopoietic tissues, including brain, heart, kidney, liver, lung, skeletal muscle and stomach. In adult mouse spleen, the red pulp is active in erythropoiesis (Seifert and Marks, 1985) and the white pulp is a lymphoid tissue that contains mature B and T lymphocytes (van Ewijk and Nieuwenhuis, 1985). In situ hybridization showed that Hemogen was expressed in the red pulp, but not in the white pulp (Fig. 3C,D). 2.4. Hemogen is primarily expressed in immature hematopoietic cells To further investigate which hematopoietic cells express Hemogen, we puri®ed adult mouse bone marrow cells using antibodies and ¯uorescence activated cell sorting (FACS), and assessed Hemogen expression by RT-PCR. As shown in Fig. 3E, Hemogen was primarily expressed in Lineage 2 blast cells, Lin lockit 1Sca-1 1 pluripotent stem cells and CD34 1 stem cells. Previous studies have shown that Lineage 2 blast cells (Spangrude et al., 1988), Lin lockit 1Sca-1 1 cells (Li and Johnson, 1995) and CD34 1 cells (Krause et al., 1996) are enriched with early multipotential stem cells. A low level of expression was found in cultured macrophages and natural killer cells. However, no expression was detected in freshly isolated CD3 1 T cells, B220 1 B cells, Terr-119 1 erythrocytes and GR-1 1 granulocytes, which are differentiated blood cells. Hence, the data suggest that Hemogen is differentially expressed in the hematopoietic precursor cells and downregulated in mature blood cells. This notion is consistent with the observation that Hemogen is expressed in the active hematopoietic sites known to harbor hematopoietic progenitor cells (Figs. 2 and 3A). However, the expression is diminished in the peripheral blood that mainly contains mature blood cells (Fig. 3A).
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2.5. A human homologous gene of Hemogen, EDAG, maps to chromosome 9q22, a location associated with blood disease breakpoints To search for human homologues of Hemogen in the GenBank, we identi®ed two human ESTs (accession nos. T52254 and AA393302). During the process of this study, three human homologous sequences, including a hypothetical protein EDAG-1 (accession no. AF228713) and two draft human genomic sequences (accession nos. AC015928 and AL354726), were deposited to GenBank. When we
Fig. 1. Sequence analysis and nuclear localization of mouse Hemogen. (A) The ®rst ATG (191±193 nt) represents the putative start codon with multiple upstream stop codons (upper case and bold) in all three reading frames. The Kozak consensus sequence is underlined. A polyadenylation signal (bold and underlined) is 16 nucleotides upstream of the poly(A) tail. The deduced protein contains a basic domain at the N-terminus (underlined) and an acidic domain at the C-terminus (italic and underlined). The region from 34 to 50 amino acids (in brackets) is predicted as a coiled-coil domain. This protein also contains a bipartite nuclear localization signal (61±78 amino acids, highlighted and underlined) that is a basic amino acid cluster. (B) The Hemogen-FLAG fusion gene was transfected into COS-7 cells and detected with anti-FLAG antibody. The signal was localized in the cell nuclei (nu) but not in the nucleoli (no) or cytoplasm (cy).
Fig. 2. Expression of Hemogen during mouse embryogenesis by in situ hybridization. Digoxigenin-labeled antisense RNA probes were hybridized with mouse frontal section at E8.5 (A) and sagittal sections at 9.5, 10.5, 11.5, 12.5 and 14.5 (D,F,I,L,O, respectively). The Hemogen transcripts are shown as purple staining. (B,E,G,J,M,P) Circulating blood cells in (A,D,F,I,L,O) at high magni®cation (1000£). The high magni®cation (1000£) of the blood island is shown in (C). The high magni®cation (1000£) of fetal livers is shown in (H,K,N,Q). Scale bars, 1 mm. ao, aorta; bc, blood cell; bi, blood island; lv, liver.
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Fig. 3. Expression analysis of Hemogen in adult mouse tissues and cells. (A) Total RNA (,15 mg) from each indicated tissue was hybridized with 32P-labeled antisense RNA probe derived from Hemogen. A ,2.4 kb message was detected in the spleen and bone marrow by 5 h of exposure. When the same ®lter was overexposed for 5 days, besides the signals in bone marrow and spleen, three very weak bands (arrow) were detected in the peripheral blood, but not in other tissues. The same agarose gel was stained with ethidium bromide before transfer to monitor the RNA loading. (B) The tissue section of adult mouse spleen was hybridized with the Hemogen sense RNA probes as a negative control. (C) By hybridization with antisense RNA probe, Hemogen transcripts were localized in the red pulp (rp) but not in the white pulp (wp) in adult mouse spleen. (D) The high magni®cation (1000£) of the positive-staining cells in the red pulp. Scale bars, 1 mm. (E) Expression analysis of Hemogen by RT-PCR. The PCR products were ampli®ed from the templates as indicated. Lanes containing RT-PCR reactions without reverse transcriptase are labeled RT(2). Histone H3 was used as the internal control.
were preparing this manuscript, a rat homologous gene RP59 (accession no. AJ302650) was also deposited. From the GenBank information, EDAG-1 encodes a 309 amino acid protein with unknown function. However, when compared with mouse Hemogen and rat RP59 by sequence alignment, the deduced EDAG-1 protein obviously lacks the part of N-terminal sequence (Fig. 4). Based on the genomic sequence AC015928, we used RT-PCR to clone a further 5 0 upstream cDNA fragment of EDAG-1 from K562 cells (data not shown). From the sequence information of this cDNA fragment and genomic clone AC015928, an upstream ATG start codon encodes a 484 amino acid open reading frame (ORF) that shows a similar size to Hemogen and RP59, suggesting that the deposited 309 amino acid EDAG-1 protein may be incomplete. After we sequenced two human EST clones (accession nos. T52254 and AA393302), we obtained the full-length cDNA that also encodes a 484 amino acid ORF and appears to be an isoform of EDAG-1 cDNA. This cDNA with a 484 amino acid ORF was named EDAG (accession no. AF322875) to distinguish it from the previous EDAG-1 that lacks the N-terminal 175
amino acids. Protein sequence alignment showed overall 43% identity between mouse Hemogen and human EDAG (Fig. 4). Moreover, the nuclear localization signal and coiled-coil domain are highly conserved with 94 and 76% similarity, respectively, which suggests that EDAG may also be a nuclear protein. Hemogen shares 70% identity with rat RP59. The nuclear localization signal and coiledcoil domain are almost identical (Fig. 4). We searched the human gene-mapping database to determine the chromosome localization of EDAG. By searching the Map Viewer at NCBI, the genomic sequence AC015928 that contains EDAG was located in the region 85.1±85.3 Mb of chromosome 9 in the GenBank map. This ,160 kb BAC clone was also found to contain the forkhead box E1 (FOXE1/FKHL15) gene that has been mapped to chromosome 9q22 (Chadwick et al., 1997). The results indicate that the genomic clone AC015928, containing FOXE1/FKHL15 and EDAG, is located at the same position 9q22. Furthermore, by electronic PCR at NCBI (http://www.ncbi.nlm.nih.gov/genome/sts/epcr.cgi), EDAG was found to contain the STS marker SHGC-33415. Searching GeneMap'99
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(http://www.ncbi.nlm.nih.gov/genemap/) and the GDB database (http://gdbwww.gdb.org/gdb/advancedSearch.html) revealed that the position of the marker SHGC-33415 is on the long arm of chromosome 9 between the markers D9S287 and D9S176, which correspond to the 9q22 region on the cytogenetic ideogram. Therefore, the human gene EDAG, homologous with Hemogen, maps to chromosome 9q22. This region contains the breakpoints in several hematological neoplasms, for example acute myeloid leukemia with deletions del(9)(q22), del(9)(q12q22), del(9)(q13q22) or translocation t(9;10)(q22;q22) (Kao et al., 1986; Mitelman, 1991; Mitelman et al., 1997; Sreekantaiah et al., 1989; Yunis et al., 1984). A genetic disease, the familial hemophagocytic lymphohistiocytosis (HPLH1), is also mapped to 9q21.3-q22 (Ohadi et al., 1999). It has been previously suggested that the chromosome region 9q21-q22 contains a cluster of leukemia breakpoints and genes important for leukemogenesis may reside in this region (Sreekantaiah et al., 1989). 2.6. EDAG is also speci®cally expressed in human hematopoietic tissues and cells
Fig. 4. Amino acid sequence alignment of Hemogen, RP59, EDAG and EDAG-1. The sequences were aligned by the ClustalW program (http:// www2.ebi.ac.uk/clustalw/). The mouse gene Hemogen (accession no. AF269248) shares 70 and 43% identity with a rat gene RP59 (accession no. AJ302650) and a human gene EDAG (accession no. AF322875), respectively, at the amino acid level. The nuclear localization signal (61± 78 residues) and coiled-coil domain (34±50 residues) are highly conserved. The conserved residues between Hemogen, RP59 and EDAG are highlighted. The hypothetical protein EDAG-1 (accession no. AF228713) lacks the N-terminal 175 amino acids of EDAG.
The Northern hybridization of the EDAG cDNA probe with human tissue RNA blots revealed that EDAG was expressed in the active hematopoietic organs bone marrow and fetal liver (Fig. 5A). Two isoforms, a 2.4 kb major isoform and a 1.8 kb minor isoform, were detected. No expression was found in the spleen, lymph node, thymus or peripheral blood leukocytes (Fig. 5A). In human adults, spleen and thymus are inactive in hematopoiesis under normal physiological conditions. By RT-PCR, a high level of transcripts was detected in myelogenous leukemia cell line K562, K562 stimulated with phorbol myristate acetate (PMA), adult bone marrow and CD34 1 progenitor cells. A low level of expression appeared in the child thymus and histiocytic lymphoma cell line U-937. No expression was
Fig. 5. Expression analysis of EDAG in human tissues, culture cells and cell lines. (A) Northern analysis of human tissue RNA blots with 32P-labeled EDAG cDNA probes. Two isoforms, a 2.4 kb major isoform and a 1.8 kb minor isoform, were detected (arrow). (B) The expression of EDAG in hematopoietic tissues, culture cells, cell lines and non-hematopoietic cell lines by RT-PCR. Histone H3 was used as the internal control.
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detected in cultured blood T cells, monocytes or other nonhematopoietic cell lines including SV-40 transformed thymus epithelial cell line 24SV48, endothelial cell line HUVEC and breast epithelial cell line SKBR3 (Fig. 5B). The results revealed that EDAG was speci®cally expressed in hematopoietic cells and the expression was developmentally regulated. 3. Experimental procedures 3.1. Plasmid constructs Mammalian expression vector pcDNA3.1 (Invitrogen) was modi®ed by inserting a FLAG tag into the multiple cloning sites to express the FLAG-fusion protein. This plasmid was re-named as pcDNA3.1-FLAG (a gift from Dr Maozhou Yang in the lab). To generate a mammalian expression vector of Hemogen, the ORF (191±1699 bp) was cloned into pcDNA3.1-FLAG. The resulting construct, named pcDNA3.1-Hemogen, was sequenced to con®rm that Hemogen ORF was in frame fused with the FLAG tag at the C-terminus. 3.2. Immunostaining COS-7 cells were cultured in Dulbecco's modi®ed Eagle's medium with 10% fetal bovine serum (Gibco BRL). Using LIPOFECTAMINE PLUSe reagent (Gibco BRL), 1 mg plasmid DNA of pcDNA3.1-Hemogen was transfected into COS-7 cells to express the HemogenFLAG fusion protein. In parallel, the same amount of pcDNA3.1-FLAG vector was transfected as a negative control. For immunostaining, 24 h after transfection, cells were ®xed in 4% paraformaldehyde for 4 min. The endogenous peroxidase was quenched with 0.3% H2O2 in methanol. After the treatment with 2% blocking serum, the cells were incubated with the mouse anti-FLAG M2 monoclonal antibody (Sigma), and then, after washing, with the antimouse IgG antibody conjugated with peroxidase (Vector). The signals were detected through the reaction of peroxidase with the substrate DAB (Roche). 3.3. In situ hybridization A 224 bp EcoRI-ApaI fragment of Hemogen cDNA was cloned into pBluescript-SKII vector to produce riboprobe labeled with digoxigenin (Roche) by in vitro transcription. Mouse embryos and tissues were ®xed in 4% paraformaldehyde overnight at 48C, embed in paraf®n and then sectioned. In situ hybridization was carried out using a modi®cation of a previously described method (Wilkinson, 1992). Tissue sections were pretreated with 0.2 N HCl for 20 min, 10 mg/ml proteinase K for 15 min at 378C, 4% formaldehyde for 20 min, and 0.5% acetic anhydride in 0.1 M TEA (pH 8.0) for 10 min. The prehybridization was performed in the hybridization buffer (50% formamide, 5 £ SSC (pH 4.5),
2% blocking reagent (Roche), 0.1% Tween-20, 0.5% CHAPS, 50 mg/ml yeast RNA, 5 mM EDTA, and 50 mg/ ml heparin) at 60±658C for 1 h. The hybridization was done with 1 mg/ml digoxigenin-labeled RNA probes at 60±658C overnight. Non-speci®c reactants were removed by three 15 min washes in 50% formamide, 2 £ SSC and 0.1% CHAPS at 60±658C. The samples were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche) overnight at 48C, and then washed to remove the non-speci®c binding. Signals were developed with the substrate NBT/ BCIP (Roche). 3.4. RNA isolation and Northern analysis RNAs were extracted from tissues or cells with TRIzol reagent (Gibco BRL), and fractionated in 1.2% agaroseformaldehyde gel. The Northern blots were performed following standard methods (Sambrook et al., 1989). Human tissue blot was purchased from Clontech. 3.5. Expression analysis of Hemogen and EDAG by RT-PCR Total RNA was extracted from a variety of freshly isolated tissues, ¯ow sorted bone marrow cells, cultured cells and transformed cell lines, and gene expression was analyzed by RT-PCR. The cells were isolated as previously described (Nicholson et al., 2000). In brief, adult mouse bone marrow cells were sorted using different antibodies by FACS (FACS Vantage, Becton Dickinson). Natural killer cells were generated as previously described (Hirayama et al., 1998) by culturing mouse newborn liver cells for 21 days with 500 units/ml recombinant human IL2. Bone marrow-derived macrophages were obtained as previously described (Li and Chen, 1995). All human tissues were obtained with approval from the Institutional Review Board of Wayne State University. Adult bone marrow cells were isolated from fragments of ribs that were removed from patients undergoing thoracic surgery. Child thymus was obtained from children undergoing cardiac surgery. CD34 1 cells and monocytes were obtained from the blood of cancer patients undergoing peripheral mobilization for autologous transplant. T cells were obtained from blood, maintained with IL-2 for several weeks and contained .99% CD3 1 T cells. Hematopoietic cell lines K562 and U-937 and non-hematopoietic cell lines 24SV48, HUVEC and SKBR3 were cultured in RPMI 1640 medium with 10% calf serum. Primer pairs for RT-PCR were: Hemogen, 5 0 -AAACACACCTCTCTCCTACCAC3 0 and 5 0 -CCTACTTTCTGGGCTCCTTCTG-3 0 ; EDAG, 5 0 -AAGCACCATCAGACACCTGACC-3 0 and 5 0 -TGCTTGAAGAGAGCATCCTGCC-3 0 ; Histone H3, 5 0 -CCACTGAACTTCTGATTCGC-3 0 and 5 0 -GGGTGCTAGCTGGATGTCTT-3 0 . The PCR products of Hemogen, EDAG and Histone are 881, 751 and 214 bp, respectively.
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3.6. GenBank accession nos. The Hemogen and EDAG sequences have been assigned GenBank accession nos. AF269248 and AF322875, respectively. Editor in proof After this paper was submitted, International Human Genome Sequencing Consortium (2001) also reported the location of EDAG-1 to chromosome 9q22.33. Acknowledgements This work was supported by NHLBI/NIH grant HL58916-01A1 (to L.L.). L.V.Y. is ®nancially supported by the graduate research assistantship from the Center for Molecular Medicine and Genetics at Wayne State University. We would like to thank members in Dr Li Li's lab for helpful discussion. References Brendel, V., Bucher, P., Nourbakhsh, I.R., Blaisdell, B.E., Karlin, S., 1992. Methods and algorithms for statistical analysis of protein sequences. Proc. Natl. Acad. Sci. USA 89, 2002±2006. Chadwick, B.P., Obermayr, F., Frischauf, A.M., 1997. FKHL15, a new human member of the forkhead gene family located on chromosome 9q22. Genomics 41, 390±396. Dingwall, C., Laskey, R.A., 1991. Nuclear targeting sequences ± a consensus? Trends Biochem. Sci. 16, 478±481. Dzierzak, E., Medvinsky, A., 1995. Mouse embryonic hematopoiesis. Trends Genet. 11, 359±366. Engel, I., Murre, C., 1999. Transcription factors in hematopoiesis. Curr. Opin. Genet. Dev. 9, 575±579. Graber, J.H., Cantor, C.R., Mohr, S.C., Smith, T.F., 1999. In silico detection of control signals: mRNA 3 0 -end-processing sequences in diverse species. Proc. Natl. Acad. Sci. USA 96, 14055±14060. Hirayama, M., Genyea, C., Brownell, A., Kaplan, J., 1998. IL-2-activated murine newborn liver NK cells enhance engraftment of hematopoietic stem cells in MHC-mismatched recipients. Bone Marrow Transpl. 21, 1245±1252. International Human Genome Sequencing Consortium, 2001. Initial sequencing and analysis of the human genome. Nature 409, 860±921. Kao, Y.S., Sartin, B.W., Van Brunt, J., Hew, A.Y., 1986. Interstitial 9q deletion (q12q22) in two cases of acute myeloblastic leukemia. Cancer Genet. Cytogenet. 19, 365±366. Kozak, M., 1997. Recognition of AUG and alternative initiator codons is augmented by G in position 14 but is not generally affected by the nucleotides in positions 15 and 16. EMBO J. 16, 2482±2492.
111
Krause, D.S., Fackler, M.J., Civin, C.I., May, W.S., 1996. CD34: structure, biology, and clinical utility. Blood 87, 1±13. Li, C.L., Johnson, G.R., 1995. Murine hematopoietic stem and progenitor cells: I. Enrichment and biologic characterization. Blood 85, 1472± 1479. Li, Y., Chen, B., 1995. Differential regulation of fyn-associated protein tyrosine kinase activity by macrophage colony-stimulating factor (MCSF) and granulocyte-macrophage colony-stimulating factor (GMCSF). J. Leukocyte Biol. 57, 484±490. Lupas, A., 1996. Prediction and analysis of coiled-coil structures. Methods Enzymol. 266, 513±525. Mitelman, F., 1991. Catalog of Chromosome Aberrations in Cancer, WileyLiss, New York. Mitelman, F., Mertens, F., Johansson, B., 1997. A breakpoint map of recurrent chromosomal rearrangements in human neoplasia. Nat. Genet. 15, 417±474. Nicholson, R.H., Pantano, S., Eliason, J.F., Galy, A., Weiler, S., Kaplan, J., Hughes, M.R., Ko, M.S., 2000. Phemx, a novel mouse gene expressed in hematopoietic cells maps to the imprinted cluster on distal chromosome 7. Genomics 68, 13±21. Ohadi, M., Lalloz, M.R., Sham, P., Zhao, J., Dearlove, A.M., Shiach, C., Kinsey, S., Rhodes, M., Layton, D.M., 1999. Localization of a gene for familial hemophagocytic lymphohistiocytosis at chromosome 9q21.3-22 by homozygosity mapping. Am. J. Hum. Genet. 64, 165± 171. Orkin, S.H., 1995. Transcription factors and hematopoietic development. J. Biol. Chem. 270, 4955±4958. Palis, J., Robertson, S., Kennedy, M., Wall, C., Keller, G., 1999. Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 126, 5073±5084. Rowley, J.D., 1998. The critical role of chromosome translocations in human leukemias. Annu. Rev. Genet. 32, 495±519. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sawyers, C.L., 1998. Molecular abnormalities in myeloid leukemias and myelodysplastic syndromes. Leukemia Res. 22, 1113±1122. Seifert, M.F., Marks Jr., S.C., 1985. The regulation of hemopoiesis in the spleen. Experientia 41, 192±199. Sieweke, M.H., Graf, T., 1998. A transcription factor party during blood cell differentiation. Curr. Opin. Genet. Dev. 8, 545±551. Spangrude, G.J., Heimfeld, S., Weissman, I.L., 1988. Puri®cation and characterization of mouse hematopoietic stem cells. Science 241, 58±62. Sreekantaiah, C., Baer, M.R., Preisler, H.D., Sandberg, A.A., 1989. Involvement of bands 9q21-q22 in ®ve cases of acute nonlymphocytic leukemia. Cancer Genet. Cytogenet. 39, 55±64. van Ewijk, W., Nieuwenhuis, P., 1985. Compartments, domains and migration pathways of lymphoid cells in the splenic pulp. Experientia 41, 199±208. Wilkinson, D.G., 1992. In Situ Hybridization: A Practical Approach, The Practical Approach Series, IRL Press at Oxford University Press, New York. Yunis, J.J., Brunning, R.D., Howe, R.B., Lobell, M., 1984. High-resolution chromosomes as an independent prognostic indicator in adult acute nonlymphocytic leukemia. N. Engl. J. Med. 311, 812±818.
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Short communication
Hemogen is a novel nuclear factor speci®cally expressed in mouse hematopoietic development and its human homologue EDAG maps to chromosome 9q22, a region containing breakpoints of hematological neoplasms Li V. Yang a, Rhonda H. Nicholson a, Joseph Kaplan b, Anne Galy c, Li Li a,d,* a
Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI 48201, USA b Department of Pediatrics, Wayne State University, Detroit, MI 48201, USA c Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA d Department of Internal Medicine, Wayne State University, 421 E. Can®eld Ave. # 1107 Detroit, MI 48201, USA Received 1 February 2001; received in revised form 30 March 2001; accepted 5 April 2001
Abstract We cloned a novel murine gene, designated Hemogen (hemopoietic gene), which was sequentially expressed in active hematopoietic sites and downregulated in the process of blood cell differentiation. Hemogen transcripts were speci®cally detected in blood islands, primitive blood cells and fetal liver during embryogenesis, and then remained in bone marrow and spleen in adult mice. Immunostaining demonstrated that Hemogen was a nuclear protein. We also identi®ed a human homologue of Hemogen, named EDAG, which was mapped to chromosome 9q22, a leukemia breakpoint. Like Hemogen, EDAG exhibited speci®c expression in hematopoietic tissues and cells. Taken together, these data are consistent with Hemogen and EDAG playing an important role in hematopoietic development and neoplasms. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hematopoiesis; Stem cell; Blood island; Bone marrow; Gene expression; Hemogen; EDAG; RP59; Development; Leukemia; Embryogenesis; In situ hybridization; Chromosome 9q22; Breakpoints
1. Introduction Hematopoiesis is a dynamic process with sequential shifting of primary hematopoietic sites from yolk sac to fetal liver and ®nally bone marrow. During embryonic development, hematopoietic tissues are derived from the ventral mesoderm. The ®rst blood cells, primitive erythrocytes, appear in blood islands in extraembryonic yolk sac at about embryonic day 7.5 (E7.5) in mice. Yolk sac also serves as the ®rst source of de®nitive hematopoietic progenitors (Palis et al., 1999). By E12, fetal liver becomes the predominant site of blood cell formation in the embryo (Dzierzak and Medvinsky, 1995). Just prior to birth and thereafter, the hematopoiesis in fetal liver gradually decreases, and bone marrow becomes the major hematopoietic site. The mouse spleen, different from the human * Corresponding author. Department of Internal Medicine, Wayne State University, 421 E. Can®eld Ave. # 1107 Detroit, MI 48201, USA. Tel.: 11313-577-8749; fax: 11-313-577-8615. E-mail address: [email protected] (L. Li).
spleen, is still very active in hematopoiesis, particularly erythropoiesis, in the red pulp at the adult stage (Seifert and Marks, 1985). Mature blood cells of three main hematopoietic lineages, erythroid, myeloid and lymphoid, are derived from common pluripotent hematopoietic stem cells (Spangrude et al., 1988). Research on the molecular basis of hematopoiesis has been facilitated by the identi®cation of a variety of hematopoietic regulating genes that are important for hematopoietic induction, lineage selection and blood cell differentiation (Engel and Murre, 1999; Orkin, 1995; Sieweke and Graf, 1998). Mutations or translocations of many of these genes have proven to play a role in hematopoietic malignancies (Rowley, 1998; Sawyers, 1998). We now report the identi®cation of a new nuclear protein, Hemogen, which shows a spatial-temporal expression pattern corresponding to the ontogeny of mouse hematopoiesis. We have also identi®ed a human homologue of Hemogen, EDAG, which is also speci®cally expressed in hematopoietic cells. Interestingly, EDAG maps to chromosome 9q22, a region containing the breakpoints of several hematopoietic neoplasms.
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00376-8
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2. Results and discussion 2.1. Cloning and sequence analysis of a novel murine gene Hemogen We initially performed a PCR-based cDNA subtraction, aiming to identify developmentally regulated genes in mouse E10.5 and E16.5 heart tissues. A number of differentially expressed genes were identi®ed in this screening. Perhaps due to the blood cells trapped in the heart tissues, some hematopoietic genes, such as embryonic 1 and bH1 globins, were also cloned (data not shown). One of the clones, 6B2, showed the differential expression at E10.5. Searching against GenBank, this clone showed homology with several mouse ESTs, e.g. GenBank accession nos. AA051237, AI121196 and AI006512. These three independent clones were sequenced to obtain the full-length cDNA sequence from which the amino acid sequence was deduced (Fig. 1A). This novel gene, now designated Hemogen (hemopoietic gene, accession no. AF269248), encodes 503 amino acids with a calculated molecular weight of 55 043 Da and a pI of 4.84. Sequence analysis suggested that the ®rst ATG is the translation initiation codon and the surrounding sequence AAGATGG is consistent with the Kozak consensus sequence with a purine at position 23 and a G at position 14 (Kozak, 1997). The polyadenylation signal sequence is ATTAAA (2288±2293 nt), a most common variant of the canonical sequence AATAAA (Graber et al., 1999). The putative Hemogen protein has a basic domain (34±78 residues with a net charge of 115) at the N-terminus and an acidic domain (450±480 residues with a net charge of 211) at the C-terminus. In the basic domain, the region from 34 to 50 amino acids is predicted (at the window size of 14) to be a coiled-coil domain (Lupas, 1996), which is implicated in protein polymerization. The 61±78 amino acids contain a bipartite nuclear localization signal suggesting that Hemogen may be a nuclear protein (Dingwall and Laskey, 1991). The analysis of amino acid composition shows that the usage of proline (10.3%), glutamate (8.7%) and glutamic acid (11.9%) is higher than the average level (Brendel et al., 1992). 2.2. Hemogen encodes a nuclear protein To con®rm that the nuclear localization signal in Hemogen protein was functional, we transfected the mammalian expression vector containing the Hemogen-FLAG fusion gene into COS-7 cells, and used anti-FLAG antibody to detect the subcellular localization of this protein. The results showed that Hemogen was located in cell nuclei but not in nucleoli or cytoplasm (Fig. 1B). 2.3. Hemogen shows alternate locations of expression coincident with the shifting sites of hematopoiesis during ontogeny To study the expression pattern of Hemogen during
mouse embryogenesis, in situ hybridization was used to detect mRNA transcripts. Hemogen was expressed during early embryogenesis at E8.5 in the blood islands of the yolk sac and in the circulating primitive blood cells (Fig. 2A±C). The expression in the circulating blood cells was detected strongly at E9.5 and E10.5 (Fig. 2D±G). As the organogenesis of liver emerged at E10.5, Hemogen expression began to be detectable in the developing hepatic primordia (Fig. 2F,H). From E11.5, Hemogen was exclusively expressed in the fetal liver (Fig. 2I,K), while the expression in circulating blood cells was downregulated dramatically to undetectable levels (Fig. 2J). The same expression patterns were observed in E12.5 and E14.5 mouse embryos (Fig. 2L±Q). We examined the tissue distribution of Hemogen in adult mice by Northern blot. A 2.4 kb transcript was speci®cally expressed in the bone marrow and spleen (Fig. 3A). When the same ®lter was overexposed, weak signals were detected in the peripheral blood at a much lower level. Two additional transcripts at 1.1 and 3.7 kb were also identi®ed and the 1.1 kb band gave the strongest signal in peripheral blood (Fig. 3A). This indicated that multiple isoforms exist in peripheral blood cells. No expression was detected in the thymus or non-hematopoietic tissues, including brain, heart, kidney, liver, lung, skeletal muscle and stomach. In adult mouse spleen, the red pulp is active in erythropoiesis (Seifert and Marks, 1985) and the white pulp is a lymphoid tissue that contains mature B and T lymphocytes (van Ewijk and Nieuwenhuis, 1985). In situ hybridization showed that Hemogen was expressed in the red pulp, but not in the white pulp (Fig. 3C,D). 2.4. Hemogen is primarily expressed in immature hematopoietic cells To further investigate which hematopoietic cells express Hemogen, we puri®ed adult mouse bone marrow cells using antibodies and ¯uorescence activated cell sorting (FACS), and assessed Hemogen expression by RT-PCR. As shown in Fig. 3E, Hemogen was primarily expressed in Lineage 2 blast cells, Lin lockit 1Sca-1 1 pluripotent stem cells and CD34 1 stem cells. Previous studies have shown that Lineage 2 blast cells (Spangrude et al., 1988), Lin lockit 1Sca-1 1 cells (Li and Johnson, 1995) and CD34 1 cells (Krause et al., 1996) are enriched with early multipotential stem cells. A low level of expression was found in cultured macrophages and natural killer cells. However, no expression was detected in freshly isolated CD3 1 T cells, B220 1 B cells, Terr-119 1 erythrocytes and GR-1 1 granulocytes, which are differentiated blood cells. Hence, the data suggest that Hemogen is differentially expressed in the hematopoietic precursor cells and downregulated in mature blood cells. This notion is consistent with the observation that Hemogen is expressed in the active hematopoietic sites known to harbor hematopoietic progenitor cells (Figs. 2 and 3A). However, the expression is diminished in the peripheral blood that mainly contains mature blood cells (Fig. 3A).
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2.5. A human homologous gene of Hemogen, EDAG, maps to chromosome 9q22, a location associated with blood disease breakpoints To search for human homologues of Hemogen in the GenBank, we identi®ed two human ESTs (accession nos. T52254 and AA393302). During the process of this study, three human homologous sequences, including a hypothetical protein EDAG-1 (accession no. AF228713) and two draft human genomic sequences (accession nos. AC015928 and AL354726), were deposited to GenBank. When we
Fig. 1. Sequence analysis and nuclear localization of mouse Hemogen. (A) The ®rst ATG (191±193 nt) represents the putative start codon with multiple upstream stop codons (upper case and bold) in all three reading frames. The Kozak consensus sequence is underlined. A polyadenylation signal (bold and underlined) is 16 nucleotides upstream of the poly(A) tail. The deduced protein contains a basic domain at the N-terminus (underlined) and an acidic domain at the C-terminus (italic and underlined). The region from 34 to 50 amino acids (in brackets) is predicted as a coiled-coil domain. This protein also contains a bipartite nuclear localization signal (61±78 amino acids, highlighted and underlined) that is a basic amino acid cluster. (B) The Hemogen-FLAG fusion gene was transfected into COS-7 cells and detected with anti-FLAG antibody. The signal was localized in the cell nuclei (nu) but not in the nucleoli (no) or cytoplasm (cy).
Fig. 2. Expression of Hemogen during mouse embryogenesis by in situ hybridization. Digoxigenin-labeled antisense RNA probes were hybridized with mouse frontal section at E8.5 (A) and sagittal sections at 9.5, 10.5, 11.5, 12.5 and 14.5 (D,F,I,L,O, respectively). The Hemogen transcripts are shown as purple staining. (B,E,G,J,M,P) Circulating blood cells in (A,D,F,I,L,O) at high magni®cation (1000£). The high magni®cation (1000£) of the blood island is shown in (C). The high magni®cation (1000£) of fetal livers is shown in (H,K,N,Q). Scale bars, 1 mm. ao, aorta; bc, blood cell; bi, blood island; lv, liver.
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Fig. 3. Expression analysis of Hemogen in adult mouse tissues and cells. (A) Total RNA (,15 mg) from each indicated tissue was hybridized with 32P-labeled antisense RNA probe derived from Hemogen. A ,2.4 kb message was detected in the spleen and bone marrow by 5 h of exposure. When the same ®lter was overexposed for 5 days, besides the signals in bone marrow and spleen, three very weak bands (arrow) were detected in the peripheral blood, but not in other tissues. The same agarose gel was stained with ethidium bromide before transfer to monitor the RNA loading. (B) The tissue section of adult mouse spleen was hybridized with the Hemogen sense RNA probes as a negative control. (C) By hybridization with antisense RNA probe, Hemogen transcripts were localized in the red pulp (rp) but not in the white pulp (wp) in adult mouse spleen. (D) The high magni®cation (1000£) of the positive-staining cells in the red pulp. Scale bars, 1 mm. (E) Expression analysis of Hemogen by RT-PCR. The PCR products were ampli®ed from the templates as indicated. Lanes containing RT-PCR reactions without reverse transcriptase are labeled RT(2). Histone H3 was used as the internal control.
were preparing this manuscript, a rat homologous gene RP59 (accession no. AJ302650) was also deposited. From the GenBank information, EDAG-1 encodes a 309 amino acid protein with unknown function. However, when compared with mouse Hemogen and rat RP59 by sequence alignment, the deduced EDAG-1 protein obviously lacks the part of N-terminal sequence (Fig. 4). Based on the genomic sequence AC015928, we used RT-PCR to clone a further 5 0 upstream cDNA fragment of EDAG-1 from K562 cells (data not shown). From the sequence information of this cDNA fragment and genomic clone AC015928, an upstream ATG start codon encodes a 484 amino acid open reading frame (ORF) that shows a similar size to Hemogen and RP59, suggesting that the deposited 309 amino acid EDAG-1 protein may be incomplete. After we sequenced two human EST clones (accession nos. T52254 and AA393302), we obtained the full-length cDNA that also encodes a 484 amino acid ORF and appears to be an isoform of EDAG-1 cDNA. This cDNA with a 484 amino acid ORF was named EDAG (accession no. AF322875) to distinguish it from the previous EDAG-1 that lacks the N-terminal 175
amino acids. Protein sequence alignment showed overall 43% identity between mouse Hemogen and human EDAG (Fig. 4). Moreover, the nuclear localization signal and coiled-coil domain are highly conserved with 94 and 76% similarity, respectively, which suggests that EDAG may also be a nuclear protein. Hemogen shares 70% identity with rat RP59. The nuclear localization signal and coiledcoil domain are almost identical (Fig. 4). We searched the human gene-mapping database to determine the chromosome localization of EDAG. By searching the Map Viewer at NCBI, the genomic sequence AC015928 that contains EDAG was located in the region 85.1±85.3 Mb of chromosome 9 in the GenBank map. This ,160 kb BAC clone was also found to contain the forkhead box E1 (FOXE1/FKHL15) gene that has been mapped to chromosome 9q22 (Chadwick et al., 1997). The results indicate that the genomic clone AC015928, containing FOXE1/FKHL15 and EDAG, is located at the same position 9q22. Furthermore, by electronic PCR at NCBI (http://www.ncbi.nlm.nih.gov/genome/sts/epcr.cgi), EDAG was found to contain the STS marker SHGC-33415. Searching GeneMap'99
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(http://www.ncbi.nlm.nih.gov/genemap/) and the GDB database (http://gdbwww.gdb.org/gdb/advancedSearch.html) revealed that the position of the marker SHGC-33415 is on the long arm of chromosome 9 between the markers D9S287 and D9S176, which correspond to the 9q22 region on the cytogenetic ideogram. Therefore, the human gene EDAG, homologous with Hemogen, maps to chromosome 9q22. This region contains the breakpoints in several hematological neoplasms, for example acute myeloid leukemia with deletions del(9)(q22), del(9)(q12q22), del(9)(q13q22) or translocation t(9;10)(q22;q22) (Kao et al., 1986; Mitelman, 1991; Mitelman et al., 1997; Sreekantaiah et al., 1989; Yunis et al., 1984). A genetic disease, the familial hemophagocytic lymphohistiocytosis (HPLH1), is also mapped to 9q21.3-q22 (Ohadi et al., 1999). It has been previously suggested that the chromosome region 9q21-q22 contains a cluster of leukemia breakpoints and genes important for leukemogenesis may reside in this region (Sreekantaiah et al., 1989). 2.6. EDAG is also speci®cally expressed in human hematopoietic tissues and cells
Fig. 4. Amino acid sequence alignment of Hemogen, RP59, EDAG and EDAG-1. The sequences were aligned by the ClustalW program (http:// www2.ebi.ac.uk/clustalw/). The mouse gene Hemogen (accession no. AF269248) shares 70 and 43% identity with a rat gene RP59 (accession no. AJ302650) and a human gene EDAG (accession no. AF322875), respectively, at the amino acid level. The nuclear localization signal (61± 78 residues) and coiled-coil domain (34±50 residues) are highly conserved. The conserved residues between Hemogen, RP59 and EDAG are highlighted. The hypothetical protein EDAG-1 (accession no. AF228713) lacks the N-terminal 175 amino acids of EDAG.
The Northern hybridization of the EDAG cDNA probe with human tissue RNA blots revealed that EDAG was expressed in the active hematopoietic organs bone marrow and fetal liver (Fig. 5A). Two isoforms, a 2.4 kb major isoform and a 1.8 kb minor isoform, were detected. No expression was found in the spleen, lymph node, thymus or peripheral blood leukocytes (Fig. 5A). In human adults, spleen and thymus are inactive in hematopoiesis under normal physiological conditions. By RT-PCR, a high level of transcripts was detected in myelogenous leukemia cell line K562, K562 stimulated with phorbol myristate acetate (PMA), adult bone marrow and CD34 1 progenitor cells. A low level of expression appeared in the child thymus and histiocytic lymphoma cell line U-937. No expression was
Fig. 5. Expression analysis of EDAG in human tissues, culture cells and cell lines. (A) Northern analysis of human tissue RNA blots with 32P-labeled EDAG cDNA probes. Two isoforms, a 2.4 kb major isoform and a 1.8 kb minor isoform, were detected (arrow). (B) The expression of EDAG in hematopoietic tissues, culture cells, cell lines and non-hematopoietic cell lines by RT-PCR. Histone H3 was used as the internal control.
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detected in cultured blood T cells, monocytes or other nonhematopoietic cell lines including SV-40 transformed thymus epithelial cell line 24SV48, endothelial cell line HUVEC and breast epithelial cell line SKBR3 (Fig. 5B). The results revealed that EDAG was speci®cally expressed in hematopoietic cells and the expression was developmentally regulated. 3. Experimental procedures 3.1. Plasmid constructs Mammalian expression vector pcDNA3.1 (Invitrogen) was modi®ed by inserting a FLAG tag into the multiple cloning sites to express the FLAG-fusion protein. This plasmid was re-named as pcDNA3.1-FLAG (a gift from Dr Maozhou Yang in the lab). To generate a mammalian expression vector of Hemogen, the ORF (191±1699 bp) was cloned into pcDNA3.1-FLAG. The resulting construct, named pcDNA3.1-Hemogen, was sequenced to con®rm that Hemogen ORF was in frame fused with the FLAG tag at the C-terminus. 3.2. Immunostaining COS-7 cells were cultured in Dulbecco's modi®ed Eagle's medium with 10% fetal bovine serum (Gibco BRL). Using LIPOFECTAMINE PLUSe reagent (Gibco BRL), 1 mg plasmid DNA of pcDNA3.1-Hemogen was transfected into COS-7 cells to express the HemogenFLAG fusion protein. In parallel, the same amount of pcDNA3.1-FLAG vector was transfected as a negative control. For immunostaining, 24 h after transfection, cells were ®xed in 4% paraformaldehyde for 4 min. The endogenous peroxidase was quenched with 0.3% H2O2 in methanol. After the treatment with 2% blocking serum, the cells were incubated with the mouse anti-FLAG M2 monoclonal antibody (Sigma), and then, after washing, with the antimouse IgG antibody conjugated with peroxidase (Vector). The signals were detected through the reaction of peroxidase with the substrate DAB (Roche). 3.3. In situ hybridization A 224 bp EcoRI-ApaI fragment of Hemogen cDNA was cloned into pBluescript-SKII vector to produce riboprobe labeled with digoxigenin (Roche) by in vitro transcription. Mouse embryos and tissues were ®xed in 4% paraformaldehyde overnight at 48C, embed in paraf®n and then sectioned. In situ hybridization was carried out using a modi®cation of a previously described method (Wilkinson, 1992). Tissue sections were pretreated with 0.2 N HCl for 20 min, 10 mg/ml proteinase K for 15 min at 378C, 4% formaldehyde for 20 min, and 0.5% acetic anhydride in 0.1 M TEA (pH 8.0) for 10 min. The prehybridization was performed in the hybridization buffer (50% formamide, 5 £ SSC (pH 4.5),
2% blocking reagent (Roche), 0.1% Tween-20, 0.5% CHAPS, 50 mg/ml yeast RNA, 5 mM EDTA, and 50 mg/ ml heparin) at 60±658C for 1 h. The hybridization was done with 1 mg/ml digoxigenin-labeled RNA probes at 60±658C overnight. Non-speci®c reactants were removed by three 15 min washes in 50% formamide, 2 £ SSC and 0.1% CHAPS at 60±658C. The samples were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche) overnight at 48C, and then washed to remove the non-speci®c binding. Signals were developed with the substrate NBT/ BCIP (Roche). 3.4. RNA isolation and Northern analysis RNAs were extracted from tissues or cells with TRIzol reagent (Gibco BRL), and fractionated in 1.2% agaroseformaldehyde gel. The Northern blots were performed following standard methods (Sambrook et al., 1989). Human tissue blot was purchased from Clontech. 3.5. Expression analysis of Hemogen and EDAG by RT-PCR Total RNA was extracted from a variety of freshly isolated tissues, ¯ow sorted bone marrow cells, cultured cells and transformed cell lines, and gene expression was analyzed by RT-PCR. The cells were isolated as previously described (Nicholson et al., 2000). In brief, adult mouse bone marrow cells were sorted using different antibodies by FACS (FACS Vantage, Becton Dickinson). Natural killer cells were generated as previously described (Hirayama et al., 1998) by culturing mouse newborn liver cells for 21 days with 500 units/ml recombinant human IL2. Bone marrow-derived macrophages were obtained as previously described (Li and Chen, 1995). All human tissues were obtained with approval from the Institutional Review Board of Wayne State University. Adult bone marrow cells were isolated from fragments of ribs that were removed from patients undergoing thoracic surgery. Child thymus was obtained from children undergoing cardiac surgery. CD34 1 cells and monocytes were obtained from the blood of cancer patients undergoing peripheral mobilization for autologous transplant. T cells were obtained from blood, maintained with IL-2 for several weeks and contained .99% CD3 1 T cells. Hematopoietic cell lines K562 and U-937 and non-hematopoietic cell lines 24SV48, HUVEC and SKBR3 were cultured in RPMI 1640 medium with 10% calf serum. Primer pairs for RT-PCR were: Hemogen, 5 0 -AAACACACCTCTCTCCTACCAC3 0 and 5 0 -CCTACTTTCTGGGCTCCTTCTG-3 0 ; EDAG, 5 0 -AAGCACCATCAGACACCTGACC-3 0 and 5 0 -TGCTTGAAGAGAGCATCCTGCC-3 0 ; Histone H3, 5 0 -CCACTGAACTTCTGATTCGC-3 0 and 5 0 -GGGTGCTAGCTGGATGTCTT-3 0 . The PCR products of Hemogen, EDAG and Histone are 881, 751 and 214 bp, respectively.
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3.6. GenBank accession nos. The Hemogen and EDAG sequences have been assigned GenBank accession nos. AF269248 and AF322875, respectively. Editor in proof After this paper was submitted, International Human Genome Sequencing Consortium (2001) also reported the location of EDAG-1 to chromosome 9q22.33. Acknowledgements This work was supported by NHLBI/NIH grant HL58916-01A1 (to L.L.). L.V.Y. is ®nancially supported by the graduate research assistantship from the Center for Molecular Medicine and Genetics at Wayne State University. We would like to thank members in Dr Li Li's lab for helpful discussion. References Brendel, V., Bucher, P., Nourbakhsh, I.R., Blaisdell, B.E., Karlin, S., 1992. Methods and algorithms for statistical analysis of protein sequences. Proc. Natl. Acad. Sci. USA 89, 2002±2006. Chadwick, B.P., Obermayr, F., Frischauf, A.M., 1997. FKHL15, a new human member of the forkhead gene family located on chromosome 9q22. Genomics 41, 390±396. Dingwall, C., Laskey, R.A., 1991. Nuclear targeting sequences ± a consensus? Trends Biochem. Sci. 16, 478±481. Dzierzak, E., Medvinsky, A., 1995. Mouse embryonic hematopoiesis. Trends Genet. 11, 359±366. Engel, I., Murre, C., 1999. Transcription factors in hematopoiesis. Curr. Opin. Genet. Dev. 9, 575±579. Graber, J.H., Cantor, C.R., Mohr, S.C., Smith, T.F., 1999. In silico detection of control signals: mRNA 3 0 -end-processing sequences in diverse species. Proc. Natl. Acad. Sci. USA 96, 14055±14060. Hirayama, M., Genyea, C., Brownell, A., Kaplan, J., 1998. IL-2-activated murine newborn liver NK cells enhance engraftment of hematopoietic stem cells in MHC-mismatched recipients. Bone Marrow Transpl. 21, 1245±1252. International Human Genome Sequencing Consortium, 2001. Initial sequencing and analysis of the human genome. Nature 409, 860±921. Kao, Y.S., Sartin, B.W., Van Brunt, J., Hew, A.Y., 1986. Interstitial 9q deletion (q12q22) in two cases of acute myeloblastic leukemia. Cancer Genet. Cytogenet. 19, 365±366. Kozak, M., 1997. Recognition of AUG and alternative initiator codons is augmented by G in position 14 but is not generally affected by the nucleotides in positions 15 and 16. EMBO J. 16, 2482±2492.
111
Krause, D.S., Fackler, M.J., Civin, C.I., May, W.S., 1996. CD34: structure, biology, and clinical utility. Blood 87, 1±13. Li, C.L., Johnson, G.R., 1995. Murine hematopoietic stem and progenitor cells: I. Enrichment and biologic characterization. Blood 85, 1472± 1479. Li, Y., Chen, B., 1995. Differential regulation of fyn-associated protein tyrosine kinase activity by macrophage colony-stimulating factor (MCSF) and granulocyte-macrophage colony-stimulating factor (GMCSF). J. Leukocyte Biol. 57, 484±490. Lupas, A., 1996. Prediction and analysis of coiled-coil structures. Methods Enzymol. 266, 513±525. Mitelman, F., 1991. Catalog of Chromosome Aberrations in Cancer, WileyLiss, New York. Mitelman, F., Mertens, F., Johansson, B., 1997. A breakpoint map of recurrent chromosomal rearrangements in human neoplasia. Nat. Genet. 15, 417±474. Nicholson, R.H., Pantano, S., Eliason, J.F., Galy, A., Weiler, S., Kaplan, J., Hughes, M.R., Ko, M.S., 2000. Phemx, a novel mouse gene expressed in hematopoietic cells maps to the imprinted cluster on distal chromosome 7. Genomics 68, 13±21. Ohadi, M., Lalloz, M.R., Sham, P., Zhao, J., Dearlove, A.M., Shiach, C., Kinsey, S., Rhodes, M., Layton, D.M., 1999. Localization of a gene for familial hemophagocytic lymphohistiocytosis at chromosome 9q21.3-22 by homozygosity mapping. Am. J. Hum. Genet. 64, 165± 171. Orkin, S.H., 1995. Transcription factors and hematopoietic development. J. Biol. Chem. 270, 4955±4958. Palis, J., Robertson, S., Kennedy, M., Wall, C., Keller, G., 1999. Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 126, 5073±5084. Rowley, J.D., 1998. The critical role of chromosome translocations in human leukemias. Annu. Rev. Genet. 32, 495±519. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sawyers, C.L., 1998. Molecular abnormalities in myeloid leukemias and myelodysplastic syndromes. Leukemia Res. 22, 1113±1122. Seifert, M.F., Marks Jr., S.C., 1985. The regulation of hemopoiesis in the spleen. Experientia 41, 192±199. Sieweke, M.H., Graf, T., 1998. A transcription factor party during blood cell differentiation. Curr. Opin. Genet. Dev. 8, 545±551. Spangrude, G.J., Heimfeld, S., Weissman, I.L., 1988. Puri®cation and characterization of mouse hematopoietic stem cells. Science 241, 58±62. Sreekantaiah, C., Baer, M.R., Preisler, H.D., Sandberg, A.A., 1989. Involvement of bands 9q21-q22 in ®ve cases of acute nonlymphocytic leukemia. Cancer Genet. Cytogenet. 39, 55±64. van Ewijk, W., Nieuwenhuis, P., 1985. Compartments, domains and migration pathways of lymphoid cells in the splenic pulp. Experientia 41, 199±208. Wilkinson, D.G., 1992. In Situ Hybridization: A Practical Approach, The Practical Approach Series, IRL Press at Oxford University Press, New York. Yunis, J.J., Brunning, R.D., Howe, R.B., Lobell, M., 1984. High-resolution chromosomes as an independent prognostic indicator in adult acute nonlymphocytic leukemia. N. Engl. J. Med. 311, 812±818.