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Originally published In Press as doi:10.1074/jbc.M606314200 on August 1, 2006

J. Biol. Chem., Vol. 281, Issue 40, 29625-29632, October 6, 2006
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Expression of Mouse Abcg2 mRNA during Hematopoiesis Is Regulated by Alternative Use of Multiple Leader Exons and Promoters*

Yang Zong{ddagger}§1, Sheng Zhou{ddagger}, Soghra Fatima{ddagger}, and Brian P. Sorrentino{ddagger}§2

From the {ddagger}Division of Experimental Hematology, Department of Hematology/Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 and §The Interdisciplinary Program, College of Graduate Health Sciences, the University of Tennessee Health Science Center, Memphis, Tennessee 38163

Received for publication, July 3, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
ABCG2 encodes a transmembrane transporter associated with multidrug resistance in various cancer cells. ABCG2 is also highly expressed in hematopoietic stem cells (HSCs) and is down-regulated in most committed progenitors, whereas expression is sharply up-regulated during erythroid differentiation. The mechanisms for regulation of ABCG2 expression in hematopoietic cells are poorly understood. We have recently identified three novel leader exons (termed E1A, E1B, and E1C) located in the 5'-untranslated region of mouse Abcg2 mRNA by data base searches and reverse transcription-PCR. In a mouse erythroid cell line, reverse transcription-PCR analysis showed that the transcript containing E1B exon was the only isoform detected. Consistently, the E1B-containing transcript was the predominant isoform of Abcg2 mRNA in primary Ter119+ erythroid cells from mouse bone marrow as well as in mouse fetal liver cells. In contrast, the E1A-containing transcript was highly expressed in c-Kit+, Sca-1+, Lin– (KSL) bone marrow cells, especially in CD34– KSL fraction, which is highly enriched for repopulating HSCs. The differential expression pattern of Abcg2 mRNA isoforms in mouse HSCs and erythroid cells was confirmed by 5'-rapid amplification of cDNA ends, indicating that at least two different promoters control mouse Abcg2 transcription during hematopoiesis. Promoter functional assays using EGFP as reporter gene demonstrated that the E1A 5'-flanking region had promoter activity, which contains multiple putative hematopoietic transcription factor binding sites. In summary, our data show that the expression of Abcg2 during hematopoiesis is transcriptionally regulated by alternative use of multiple leader exons and promoters in a developmental stage-specific manner.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
ABCG23 (also known as BCRP, ABC-P, or MXR), a member of the G subfamily of ATP binding cassette (ABC) transport proteins, is associated with resistance to mitoxantrone, doxorubicin, bisantrene, topotecan, and methotrexate in various cancer cells (1). Expression of ABCG2 is mainly detected in epithelial tissues such as placenta, intestine, kidney, and hepatic canalicular membrane, reflecting its physiologic roles in the absorption, distribution, metabolism, and excretion of xenobiotics. High expression of ABCG2 has also been described in a variety of stem cell populations from several tissues, including hematopoietic stem cells (HSCs), neural stem cells, side population cells from skeletal muscle, and pancreatic islets as well as embryonic stem cells (2, 3).

Previous studies on mouse and human hematopoietic cells demonstrated that ABCG2 is highly expressed in HSCs and is down-regulated in most committed progenitors (3, 4). In CD34– primitive c-Kit+, Sca-1+, Lin– (KSL) mouse bone marrow (BM) cells, Abcg2 mRNA was highly expressed; however, when these HSCs differentiated to a CD34+ phenotype, the expression levels of Abcg2 transcript decreased significantly (3). Expression of ABCG2 is sharply up-regulated again during erythroid differentiation, and significant levels of protein expression are detected on mature red blood cells (5). It is likely that the expression of ABCG2 in these hematopoietic cells also plays a protective role against both endogenous and exogenous toxins. Although all the substrates of ABCG2 transporter are not yet identified, erythrocytes from Abcg2 null mice were found to have increased levels of protoporphyrin IX (PPIX), and the mice were very sensitive to the dietary chlorophyll breakdown product pheophorbide a, having a phenotype similar to erythropoietic protoporphyria (6). The accumulation of PPIX in erythroid cells from Abcg2 null mice has been shown to be due to loss of active efflux of PPIX mediated by Abcg2 as PPIX is a direct substrate for this transporter (5).

To date, little is known about the regulation of ABCG2 expression. The human ABCG2 promoter lacks a TATA box and contains five putative Sp1 and several AP-1 binding sites downstream from a putative CpG island (7). Recently, a novel estrogen-response element and one hypoxia-inducible factor (HIF-1) binding site were found within this promoter, indicating that ABCG2 expression could be regulated by estrogen and hypoxia in vitro (8, 9). However, these findings do not explain the developmental regulation of ABCG2 expression profile during hematopoiesis. In this study, we analyzed the structure of mouse Abcg2 mRNA transcripts by expressed sequence tag (EST) data base searches, identified three novel 5'-noncoding exons, and examined the expression pattern of Abcg2 mRNA isoforms in mouse HSCs and erythroid cells. Our results show that alternative use of multiple promoters and leader exons plays a role in the developmental regulation of Abcg2 expression during hematopoiesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Data base Searches and Computer Analysis of 5'-Untranslated Region (5'-UTR) of Abcg2 Gene—Searches of the EST data base (dbEST) and Unigene data base for sequences containing the first coding exon of mouse Abcg2 and its human homolog were performed using the nucleotide-nucleotide BLAST program. Putative promoter regions were identified by analysis of the 5'-flanking genomic sequence of mouse Abcg2 with ProScan software (Version 1.7) (10). Putative transcription factor binding sites were found by TESS (11) and MatInspector programs (12).

Magnetic Activated and Fluorescence-activated Cell Sorting— Murine BM cells were harvested by standard methods from the tibias and femurs of 6–10-week-old female C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME). After staining cells with phycoerythrin-conjugated anti-mouse Sca-1 monoclonal antibody (Pharmingen) followed by incubation at 4 °C for 15 min with anti-phycoerythrin magnetic microbeads (Miltenyi Biotec, Auburn, CA), positive selection for Sca-1+ BM cells was performed on autoMACSTM separator (Miltenyi Biotec). Then the recovered cells were labeled with allophycocyanin (APC)-conjugated anti-mouse c-Kit and fluorescein isothiocyanate-conjugated lineage antibody mixture consisting of anti-mouse B220, CD4, CD8, Gr-1, Mac-1, Ter119 (Pharmingen). After washing, KSL cells were sorted by flow cytometry (BD Biosciences). For comparison, Ter119+ erythroid BM cells were isolated by flow cytometry after staining BM cells with phycoerythrin-conjugated anti-mouse Ter119 monoclonal antibody (Pharmingen). For isolation of CD34– or CD34+ KSL cells, Sca-1+ BM cells were first enriched by antoMACS as described above and were stained with fluorescein isothiocyanate-conjugated anti-mouse CD34 (eBioscience, San Diego, CA), APC-conjugated anti-mouse c-Kit, and biotin-conjugated mouse lineage markers (Pharmingen) including CD3e, B220, Gr-1, Mac-1, and Ter119 and then incubated with streptavidin-APC-Cy7 (Pharmingen).

RNA Isolation and Reverse Transcription (RT)-PCR—Total RNA was extracted from cells and mouse tissues using RNA STAT-60TM reagent (Tel-Test, Friendswood, TX) according to the manufacturer's instructions. The first-strand cDNAs were synthesized using oligo(dT)15 primer (Promega, Madison, WI) by superscriptTM II reverse transcriptase (Invitrogen). Equal aliquots of the cDNA products were subjected to 32–38 cycles of PCR with isoform-specific primer sets. PCRs were run with 2.5 units of TaqDNA polymerase (Qiagen, Valencia, CA), 10 pmol of each primer, and 0.2 mM each dNTP in a final volume of 50 µl. The PCR products were then electrophoresed on 1% agarose gel. The sequences of PCR products were determined by either sequencing directly with the primers described or subcloning using TOPO TA cloning kits (Invitrogen) and sequencing with universal primers.

The distinct forward primers used for mouse Abcg2 isoforms were as follows: E1A, 5'-TCTGTCTTCCTGGTCCTCTC-3'; E1B, 5'-AGCGTGTGCAGGTCTGAGTG-3'; E1C, 5'-GAAGAACCACACCAATAAGG-3'; and E2, 5'-ATGAACTCCAGAGCCGTTAG-3'. The common reverse primer, 5'-TTGAAATGGGCAGGTTGAGG-3', was located in exon 4 of mouse Abcg2. Another primer pair, 5'-TCTGTCTTCCTGGTCCTCTC-3' and 5'-CAGGGCCACATGATTCTTCC-3', derived respectively from exon 1A and exon 16 of mouse Abcg2, was used to amplify the full-length isoform A of mouse Abcg2 mRNA.

Quantitative Real-time RT-PCR—First-strand cDNAs were synthesized as described above. Quantitative real-time PCR was carried out on ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). Primers designed to discriminate three different mouse Abcg2 isoforms were as follows: E1A forward, 5'-CCTTTTGACACCTCATTACACATAGC-3'; E1B forward, 5'-CCAGGGACCGCGAGAAAG-3'; E1C forward, 5'-GGAAATTTTAACTGCACATTGAGAGA-3', and the common reverse primer, 5'-GTCCTAACGGCTCTGGAGTTCA-3', was derived from exon 2 of mouse Abcg2. Taqman probe 5'-VIC-CATAAATCCTAAAGATGTCTTCTAMRA-3' binds to the beginning of exon 2. Mouse glyceraldehydes-3-phosphate dehydrogenase (GAPDH) expression was determined using the specific primers and probe (Applied Biosystems), taken as the endogenous control.

Real-time PCR with serial diluted samples was first performed using SYBR Green I dye (Applied Biosystems) to demonstrate that single bands of double-stranded DNA were generated by each PCR reaction. Thereafter, the PCR mixtures containing cDNAs, primers (0.4 µM each), probe (0.25 µM), and Taqman universal PCR master mix (Applied Biosystems) were preheated at 50 °C for 2 min and then incubated at 95 °C for 10 min to activate AmpliTaq Gold DNA polymerase followed by 50 cycles alternating between 95 °C for 15 s and 60 °C for 1 min. Results were collected and analyzed using the ABI Prism 7700 sequence detection system software (Applied Biosystems). Dilution of a cDNA sample prepared from MEL cells total RNA was used to create standard curves for the GAPDH amplification. The expression of each mouse Abcg2 isoform was normalized with GAPDH endogenous control and calculated using standard curve method, in which standard curves of Abcg2 isoform amplification were generated with serial diluted plasmid DNA templates.

5'-Rapid Amplification of cDNA Ends (5'-RACE)—5'-RACE was performed using SMARTTM RACE cDNA amplification kit (Clontech) according to the manufacturer's instructions. Briefly, 0.1–1 µg of total RNA was reverse-transcribed into 5'-RACE-ready cDNA using PowerScript reverse transcriptase, SMART II A oligonucleotide, and 5'-CDS primer. The mouse Abcg2 gene-specific reverse primer 5'-CTCACTGTCAGGGTGCCCATCACAAC-3' was derived from exon 5 of mouse Abcg2. Touch-down PCR was performed using Advantage 2 DNA polymerase (Clontech). The reaction conditions were as follows: an initial denaturation at 94 °C for 1 min, then five cycles of amplification (94 °C for 30 s and 72 °C for 2 min), five cycles of amplification (94 °C for 30 s, 71 °C for 30 s, and 72 °C for 2 min) followed by 25 cycles of amplification (94 °C for 30 s, 69 °C for 30 s, and 72 °C for 2 min), and a final extension step at 72 °C for 10 min. The RACE products were ligated into pCR4-TOPO vector (Invitrogen) by TA cloning. Positive clones were screened by digestion with EcoRI, and the DNA inserts were sequenced using universal primers.


Figure 1
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  		FIGURE 1.
Organization of mouse Abcg2 gene. Mouse Abcg2 gene locus has three different leader exons, resulting in distinct Abcg2 transcript isoforms. All intron/exon boundaries display canonical GT/AG sequences. The translational start codon localizes in exon 2. Three EST clones are indicated.

 
Construction of Abcg2 E1A Distal Promoter-EGFP Lentiviral Vector—Genomic DNA was isolated from the tails of C57BL/6J mice using a Puregene DNA purification kit (Gentra Systems, Minneapolis, MN). To clone the Abcg2 E1A 5'-flanking region, primers containing BamHI and MluI or NcoI restriction enzyme sites at their 5'-ends, 5'-TCGGATCCACGCGTGTACAGAGGCAGTAGG-3' and 5'-CATGCCATGGTGTGGGTTCCAACTCAAGAC-3', were used for PCR amplification with Pfu DNA polymerase (Stratagene, La Jolla, CA). The PCR products were fractionated on 1% agarose gel, purified and digested with BamHI and NcoI, and then subcloned into a shuttle vector containing the EGFP cDNA. The cloned Abcg2 promoter region together with EGFP cDNA were excised with MluI and NotI and inserted into a self-inactivated lentiviral vector pCL20cw INS1R MpGFP (a modified version of pCL20c INS1R MpGFP plasmid (13) with the woodchuck hepatitis post-transcriptional regulatory element) by replacement of the internal murine stem cell virus retroviral vector long terminal repeat and EGFP cDNA fragments. For negative control, a promoter-less vector was constructed by removal of the internal murine stem cell virus retroviral vector long terminal repeat fragment from pCL20cw INS1R MpGFP vector by partial digestion with NcoI and MluI and recircularization with T4 DNA ligase.

Preparation of Lentiviral Stocks—Lentiviral stocks were prepared as described previously (13). In brief, 5 x 106 293T cells were transfected on a 10-cm dish with a mixture of plasmid DNA consisting of 6 µg of pCAGkGP1R (Gag/Pol), 2 µg of pCAG4-RTR2 (Rev/Tat), 2 µg of pCAG-VSVG (VSV-G envelope) plasmids, and 10 µg of Abcg2 distal promoter-EGFP lentiviral vector using the calcium phosphate precipitation technique. At 18 h after transfection, the 293T cells were washed with phosphate-buffered saline twice and then cultured for an additional 24 h in fresh medium. Then the supernatant containing vector particles was harvested, filtered through a 0.45-µm pore-sized hydrophilic polyvinylidene difluoride filter, and concentrated by ultracentrifugation and then quick-frozen in aliquots and stored at –80 °C.

Cell Culture, Lentiviral Transduction, and GFP Reporter Assay—Murine erythroleukemic MEL cells, 293T cells, and NIH3T3 cells were grown in Dulbecco's modified Eagle's medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Mouse BM cells were harvested 3 days after treatment with 150 mg/kg 5-fluorouracil and were prestimulated in Dulbecco's modified Eagle's medium with 15% fetal bovine serum, 20 ng/ml murine interleukin-3, 50 ng/ml human interleukin-6, and 50 ng/ml murine stem cell factor for 48 h. Lentiviral transduction of adherent cells were carried out as described previously (13). For suspension-cultured cells, 3 x 105 cells were seeded into each well of the 6-well plates coated with 100 µg/ml RetroNectin (Takara, Madison, WI) and were transduced 2–3 times at intervals of 24 h in a mixture containing the above concentrations of serum/cytokines, concentrated lentiviral particles, and 6 µg/ml Polybrene. Following the final transduction, the cells were grown in fresh culture medium for an additional 7–14 days. Thereafter, GFP expression in transduced cells was analyzed by flow cytometry after exclusion of dead cells by propidium iodide staining.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Three Isoforms of Mouse Abcg2 mRNA Differ in Their 5'-UTR—Screening the NCBI mouse EST data base using the sequence of the first coding exon of mouse Abcg2 resulted in identification of three different EST clones (BQ561807 [GenBank] , AA008579 [GenBank] , and AI226912 [GenBank] ), all of which contain the Abcg2 first coding exon but with distinct 5' upstream regions, indicating the presence of alternative leader exons in mouse Abcg2 gene. Comparison of the sequences of these EST clones with mouse genomic DNA sequences revealed that the distance of these leader exons (termed E1A, E1B, and E1C) from the first coding exon (designated as E2) in genomic DNA is 58.5 kb for E1A, 15.0 kb for E1B, and 5.1 kb for E1C, respectively (Fig. 1). On the basis of this DNA sequence information, we designed PCR forward primers specific for each leader exon, together with a common reverse primer, which was derived from exon 4 of mouse Abcg2, and used RT-PCR and sequencing to confirm the physical existence of three isoforms of mouse Abcg2 mRNA. The results of the RT-PCR assay showed that all three Abcg2 transcript isoforms were expressed in mouse brain, adult liver, and kidney, whereas the E1B-containing Abcg2 transcript was the only isoform detected in mouse small intestine (Fig. 2a). Sequencing analysis of RT-PCR products confirmed the structures of three isoforms shown in Fig. 1. These data demonstrated that mouse Abcg2 gene has three novel leader exons that are alternatively used during transcription, giving rise to distinct isoforms of Abcg2 mRNA that differ in their 5'-UTR.


Figure 2
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  		FIGURE 2.
The expression pattern of mouse Abcg2 mRNA isoforms. a, expression of Abcg2 mRNA isoforms in normal murine tissues. E1A represents the E1A-containing transcript; E1B represents the E1B-containing transcript; E1C represents the E1C-containing transcript; and E2 represents the total Abcg2 transcripts as a positive control. Primers designed for specific transcript isoforms and a positive control are shown. b, the differential expression pattern of Abcg2 mRNA isoforms between mouse erythroid cells and HSCs. c, RT-PCR yields a full-length mouse Abcg2 transcript containing E1A.

 
Differential Expression of Abcg2 Isoforms in Mouse HSCs and Erythroid Cells—To define the expression pattern of three Abcg2 mRNA isoforms in hematopoietic cells, we isolated primary Ter119+ erythroid cells from normal murine BM by fluorescence-activated cell sorting. For comparison, first-stand cDNA was synthesized from total RNA of KSL BM cells, a population devoid of erythroid cells but highly enriched for repopulating HSCs (14). RT-PCR analysis showed that the transcript containing E1B exon was the only isoform detected in a mouse MEL erythroleukemic cell line (Fig. 2b). Moreover, the E1B-containing transcript was expressed at significantly higher levels relative to other two isoforms in mouse fetal liver (Fig. 2b), which predominantly consists of erythroid cells (15). Consistently, in Ter119+ erythroid BM cells, the E1B-containing transcript was the predominant isoform of Abcg2 mRNA (Fig. 2b); the Abcg2 transcript containing E1A exon was also barely detected in Ter119+ erythroid cells by RT-PCR. In contrast, the E1A-containing transcript was relative highly expressed in KSL BM cells, although the E1B-containing transcript was also present (Fig. 2b). These data suggest that the E1B-containg Abcg2 isoform is relatively erythroid-specific and the E1A-containing transcript is HSC-specific.

To determine whether the E1A-containg Abcg2 isoform is a fulllength transcript, we used another primer pair derived from exon 1A and exon 16 of mouse Abcg2, respectively, to perform the RT-PCR. The PCR amplification yielded a single band at expected size (2.1 kb) on agarose gel electrophoresis (Fig. 2c). The sequencing analysis showed the amplified E1A-containg Abcg2 isoform to be a full-length transcript without internal alternative splicing, indicating that the translation of the E1A-containg Abcg2 transcript will result in a full-length transporter protein.

To obtain more information about the expression of Abcg2 mRNA isoforms in mouse HSCs and erythroid cells, we developed a quantitative real-time PCR assay using isoform-specific primers and Taqman probe. An amplicon generated by another primer pair derived from exon 14 and exon 15 of mouse Abcg2 (sequences available upon request), which represented all three mRNA isoforms, was used to compare the expression of total Abcg2 transcripts with the comparative Ct (cycle threshold) method. Consistent with the previous finding (3, 4), we found that the expression levels of total Abcg2 transcript decreased significantly when CD34– KSL BM cells differentiated and became CD34-positive; however, when cells displayed Ter119+ erythroid phenotype, the expression of total Abcg2 mRNA was sharply up-regulated (data not shown). Importantly, our results of real-time PCR showed that the E1A-containing transcript was expressed at the highest level in CD34– KSL cells, modestly down-regulated in CD34+ KSL cells, but hardly detected in Ter119+ erythroid BM cells and MEL cells (Fig. 3a). In contrast, the E1B-containing transcript and the E1C-containing transcript were highly expressed in Ter119+ BM cells (Fig. 3, b and c), which was consistent with our RT-PCR data (Fig. 2b).


Figure 3
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  		FIGURE 3.
Quantitative analysis of the expression of mouse Abcg2 mRNA isoforms in different subsets of bone marrow cells. a– c, quantitative real-time RT-PCR analysis of the E1A-containing Abcg2 transcript (a), the E1B-containing transcript (b), and the E1C-containing transcript (c) in CD34– or CD34+ KSL BM, Ter119+ erythroid cells and MEL cells. The copy numbers of each Abcg2 mRNA isoform were quantified by comparison with standard curves. Values were normalized to mouse GAPDH expression, which was used as the endogenous control. The results were generated from two independent sorting samples. The values with statistical significance (p < 0.05) are indicated. Error bars represent the standard deviation from five separate experiments.

 


Figure 4
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  		FIGURE 4.
Mouse Abcg2 transcripts are initiated from staggered transcription start sites from multiple leader exons. 5'-RACE and sequencing analysis of RACE products showed mouse fetal liver and KSL BM cells differentially transcribed Abcg2 mRNA from multiple leader exons with staggered transcription start sites. Each row represents a distinct 5'-RACE product. The numbers of randomly selected positive clones are indicated.

 
Staggered Transcription Start Sites From Multiple Leader Exons of Mouse Abcg2 Gene—5'-RACE of cDNA derived from mouse fetal liver gave rise to a single band of about 500 bp (Fig. 4). After subcloning of the RACE products into the pCR4-TOPO vector, sequencing of the inserts from nine positive clones showed that transcription of mouse Abcg2 mRNA in fetal liver was exclusively initiated from E1B, and the length of E1B fragments included in the inserts varied from 23–82 bp (Fig. 4), indicating the staggered transcription start sites of mouse Abcg2 gene. In contrast, the 5'-RACE products of mouse KSL BM cells showed two broad bands on agarose gel (Fig. 4). In addition to the 500-bp DNA fragments that were also observed in mouse fetal liver sample, there was a subset of DNA molecules with larger size (about 1.0 kb) in 5'-RACE products of mouse KSL cells. Plasmid cloning of the entire pool of 5'-RACE products from KSL cells and subsequent sequencing analysis demonstrated that 6 out of 11 randomly selected clones contained the inserts consisting of the variable fragments of E1A, 4 out of these 11 clones contained the variable fragments of E1B, and the other clone contained the sequence of 141 bp from E1C (Fig. 4). These results confirmed that mouse Abcg2 mRNA can be transcribed from three different leader exons, and there was a significant differential expression pattern of Abcg2 mRNA isoforms between mouse HSCs and erythroid cells.


Figure 5
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  		FIGURE 5.
Genomic nucleotide sequence of the 5'-flanking region of exon 1A of mouse Abcg2 gene. TSS represents the distal transcription start site (designated as base 1) from exon 1A identified by 5'-RACE of cDNA from KSL cells. The partial sequence of exon 1A is depicted in italic letters. Putative transcription factors binding sites identified by TESS or MatInspector programs are underlined. The beginning and end of the cloned promoter region for functional analysis are indicated.

 
Transcriptional Activity of the Distal Promoter of Mouse Abcg2 Gene—Because the E1A-containing Abcg2 mRNA was preferentially expressed in mouse HSCs, it is very likely that the 5'-flanking region upstream of E1A contains a promoter that controls the transcription of Abcg2 in mouse HSCs. Bioinformatic analysis of this 5'-flanking region using ProScan software (10) showed that a putative promoter region localized –245 – +5 bp relative to the distal transcription start site from exon 1A, which lacked for canonical TATA and CCAAT boxes. Several Sp1 putative binding sites and one AP-2 putative binding site were found within this 5'-flanking region, and multiple hematopoietic transcription factors such as AML1, GATA-1, GATA-2, and Pu.1 (16) putative binding sites were identified by TESS and MatInspector computer software (Fig. 5).

To determine whether this putative promoter fragment of mouse Abcg2 confer promoter activity, we cloned the 1.3-kb E1A upstream region containing the putative promoter with the flanking sequence (Fig. 5) and inserted this fragment into a self-inactivated lentiviral construct (Fig. 6a) containing 1.2-kb fragment hypersensitive site 4 (HS4) insulator from the chicken beta-globin locus control region (17), which is used to shield the EGFP reporter gene from adjacent cellular regulatory elements so that the GFP expression will be only controlled by the cloned Abcg2 promoter after integration of lentiviral DNA into host cells genome. Flow cytometry analysis of Abcg2 distal promoter-EGFP lentiviral transduced cells showed that significant GFP expression was observed in murine BM cells, MEL cells, and 293T cells as well as NIH3T3 cells (Fig. 6b and data not shown), indicating that the cloned distal promoter of mouse Abcg2 had constitutive transcriptional activity. To exclude the possibility of leaking expression of GFP, an internal promoterless vector was constructed (Fig. 6a). We found that there was no GFP expression in MEL cells, 293T cells, or mouse BM cells transduced by the promoter-less vector (Fig. 6b), whereas integration of lentiviral DNA was confirmed by Southern blot analysis (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The previously described mouse Abcg2 gene contains 15 coding exons on chromosome 6, which encodes an ABC transporter of 657 amino acids (18). In this study, we found three novel noncoding exons of mouse Abcg2 located in 5'-UTR by EST data base searches and RT-PCR. 5'-RACE analysis also confirmed the alternative use of these noncoding exons during Abcg2 transcription. Previous studies reported that the human ABCG2 gene spans over 66 kb on human chromosome 4q22 and consists of 16 exons and 15 introns. The translational start site was found in the second exon, and the majority of the 5'-UTR is in exon 1 (1, 7). The most recent study of Nakanishi et al. (19) showed that there was alternative splicing within the previously described first exon of human ABCG2, and another different leader exon was found to localize 291 bp upstream of this previously described first exon. Because of the 5'-end heterogeneity in mouse Abcg2 gene, we also analyzed the human EST and Unigene data base and found a EST clone (BQ889670 [GenBank] ) that contains exon 2 of human ABCG2 but has distinct 5' upstream sequences when compared with those two previously described noncoding exons (7, 19). The expression of these different human ABCG2 transcript isoforms was further confirmed by RT-PCR and sequencing (data not shown), suggesting that human ABCG2 gene also has multiple alternative leader exons with a similar organization as its murine counterpart. In addition, a recent report described that murine Abcg1 gene also had a similar organization with alternative first exons, resulting in three different isoforms of Abcg1 transcripts (20). Moreover, the 5'-end heterogeneity was also observed in human MDR1 (ABCB1) gene (21), which indicates that this structure feature may be a general character of certain members of mammalian ABC transporters.


Figure 6
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  		FIGURE 6.
Functional analysis of the distal promoter region of mouse Abcg2. a, the schematic maps of the self-inactivated (SIN) lentiviral reporter vectors containing hypersensitive site 4 (HS4) insulator with or without the cloned distal promoter of mouse Abcg2. LTR, long terminal repeat. RRE, rev-responsive element; WPRE, woodchuck hepatitis post-transcriptional regulatory element. b, flow cytometry analysis of GFP expression in Abcg2 distal promoter-EGFP lentiviral transduced cells (solid line) and promoter-less vector transduced cells (dotted line) relative to untransduced cells (shaded area).

 
The existence of multiple leader exons is usually associated with alternative usage of multiple promoters during transcription, which is often responsible for tissue-specific or developmental stage-restricted gene expression (22). For instance, it has been reported that human stem cell surface protein AC133 gene had at least nine different leader exons, whereas only exon 1A-containing AC133 transcript was specifically expressed in human CD34+ cord blood cells (23). The results of our studies showed that in mouse erythroid cells, the E1B-containing transcript was the predominant form of Abcg2 mRNA and the E1A transcript was hardly detected, whereas in mouse HSC-enriched subpopulation, the E1A-containing transcript was relatively highly expressed. This differential expression pattern of Abcg2 mRNA isoform between mouse HSCs and erythroid cells indicates that these two distinct subsets of hematopoietic cells use different promoters to control the initiation of Abcg2 transcription. This promoter switch explains the developmental expression profile of mouse Abcg2 during hematopoiesis, suggesting that the expression of Abcg2 on mouse hematopoietic cells is regulated at the level of transcription in a hematopoietic stage-specific manner.

Because we found that E1A-containing Abcg2 mRNA isoform was preferentially expressed in mouse HSCs and that the E1A isoform was a full-length transcript, it appears that the 5'-flanking region of E1A contains HSC-specific promoter. However, functional analysis of this region using an EGFP reporter lentiviral vector showed that the cloned distal promoter of mouse Abcg2 had ubiquitous transcriptional activity, even in transduced NIH3T3 fibroblast cells where endogenous Abcg2 expression should not be observed. There are several reports of tissue-specific expressed genes whose promoters had housekeeping-type constitutive activity in vitro. Such examples include the mouse CD24 (24), rat neuronal SCG10 (25), mouse Thy-1 (26), human {alpha}-globin (27), and endothelial nitric-oxide synthase (28). For most of these genes, tissue-specific suppressor or enhancer elements responsible for precise regulation of spatial and temporal expression were found in the flanking DNA or distal sequences (25–27). Given the paradox of HSC-specific expression of E1A-containing Abcg2 transcript in the context of the constitutive transcriptional activity of its distal promoter, it is possible that an HSC-specific regulatory element is present in the E1A-flanking region, which may preferentially suppress the Abcg2 distal promoter activity in mature hematopoietic cells. Meanwhile, it is important to note that the expression of E1A-containing Abcg2 transcript is not restricted to HSCs since we found that the endogenous expression of this Abcg2 mRNA isoform was also observed in several kidney and hepatic cell lines (data not shown).

Like the previously described human ABCG2, MDR/(ABCB1), and MRP1 (ABCC1) promoters (7, 29, 30), computer analysis of mouse E1A 5'-flanking region revealed the absence of the consensus TATA box in the distal promoter of mouse Abcg2. Genes with TATA-less promoters often have multiple transcription start sites. Consistently, our 5'-RACE data showed that transcription of mouse Abcg2 was initiated at staggered transcription start sites from multiple leader exons. The sequence analysis of the E1A 5'-flanking region found several Sp1 putative binding sites, which may be necessary for the basal promoter activity. Interestingly, multiple hematopoietic-specific transcription factor putative binding sites were found to be present within the 5'-flanking region of mouse Abcg2 E1A. Although all of them are potential binding sites and further experiments need to be done to define their functional significance, it is important to note that some of these transcription factors themselves, such as murine AML1, GATA-1, and GATA-2, also have alternative leader exons and multiple promoters, leading to cell type-specific expression in hematopoietic cells (31–33). Moreover, it is known that the alternative use of multiple promoters and leader exons may affect stability or translation efficiency of mRNA variants (22). For example, AML1 mRNA initiated from the proximal promoter bears a long 5'-UTR, which contains a functional internal ribosome entry site and mediates cap-independent translation (34). These findings implicate much diversity and complexity in the regulatory mechanisms for fine-tuning of Abcg2 expression during hematopoiesis.

In this study, we reported three novel leader exons of mouse Abcg2 gene, which yield distinct isoforms of Abcg2 mRNA, and the differential expression patterns of Abcg2 mRNA isoforms were observed between mouse HSCs and erythroid cells. These findings not only explain the developmental regulation of Abcg2 expression during hematopoiesis but may also provide an assay for HSC quantitation using the HSC-specific E1A-containing Abcg2 mRNA isoform.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant R01 HL67366 (to B. P. S.) and by a grant from the American Lebanese Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 A recipient of the Hal and Alma Reagan Fellowship from the University of Tennessee Health Science Center. To whom correspondence may be addressed: Division of Experimental Hematology, Dept. of Hematology-Oncology, St. Jude Children's Research Hospital, 332 N Lauderdale St., Memphis, TN 38105. Tel.: 901-495-2727; Fax: 901-495-2176; E-mail: E-mail: yang.zong{at}stjude.org. 2To whom correspondence may be addressed: Division of Experimental Hematology, Dept. of Hematology-Oncology, St. Jude Children's Research Hospital, 332 N Lauderdale St., Memphis, TN 38105. Tel.: 901-495-2727; Fax: 901-495-2176. E-mail: brian.sorrentino{at}stjude.org.

3 The abbreviations used are: ABC, ATP binding cassette; Abcg2, ABC transporter G2; HSC, hematopoietic stem cell; KSL, c-Kit+, Sca-1+, Lin– cells; PPIX, protoporphyrin IX; EST, expressed sequence tag; 5'-UTR, 5'-untranslated region; RT, reverse transcription; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; 5'-RACE, 5'-rapid amplification of cDNA ends; MEL, mouse erythroid cell line; BM, bone marrow; APC, allophycocyanin; GFP, green fluorescent protein; EGFP, enhanced GFP. Back


   ACKNOWLEDGMENTS
 
We thank the members of Sorrentino laboratory for technique help, the Flow Cytometry Core for sample analysis, Dr. Marguerite Evans-Galea for lentiviral vector pCL20cw INS1R MpGFP, and Dr. John Cunningham and Xiaohua Chen for scientific discussion.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
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