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Prepublished online as a Blood First Edition Paper on August 22, 2002; DOI 10.1182/blood-2002-03-0788.
HEMATOPOIESIS
From the Hematology/Oncology Division, Harvard
Institutes of Medicine; and the Department of Cancer Immunology and
AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston,
MA.
The elements regulating gene expression in hematopoietic stem cells
are still poorly understood. We previously reported that a 141-kilobase
(kb) human CD34 transgene confers properly regulated human CD34
expression in transgenic mice. A construct with only the human CD34
promoter and 3' enhancer region is not sufficient, suggesting that
critical distal elements are necessary for expression of the human CD34
gene. To further localize such elements, we analyzed deletion
constructs of the human CD34 gene and evaluated their function in
transgenic mice. Constructs harboring as little as 18 kb of 5' and 26 kb of 3' human CD34 flanking sequence conferred human expression in
tissues of transgenic mice with a pattern similar to that of the 141-kb
human transgene. In contrast, a construct harboring 10 kb of 5' and 17 kb of 3' human CD34 flanking sequence gave no expression. These data
demonstrate that regions between 10 to 18 kb upstream and/or 17 to 26 kb downstream of the human CD34 gene contain critical elements for
human CD34 expression in vivo. Further functional analysis
of these regions in transgenic mice will be crucial for understanding
CD34 gene expression in hematopoietic stem and progenitor cells.
(Blood. 2002;100:4420-4426) Human CD34 is a transmembrane protein
expressed on the surface of stem and early hematopoietic
cells1-5; expression occurs on a low percentage of bone
marrow cells and declines during hematopoietic differentiation.
Therefore, human CD34 represents a good model for understanding stem
cell gene regulation. Furthermore, identification of human CD34
regulatory elements may allow identification of transcription factors
necessary for stem cell expression, as well as facilitate
regulated expression of heterologous genes in hematopoietic stem cells.
Initial analysis of the murine CD34 gene demonstrated that the
relative conservation with human CD34 decreased from the carboxyl toward the amino terminus of the coding region.6-8 In
addition, in contrast to many other hematopoietic
genes,9-11 there is no conservation of sequence or
regulatory elements in the proximal promoter or 5' untranslated
region.12-18 These results suggested that distal elements
might play a more important role in CD34 regulation. Indeed, we
reported that a human CD34 minigene including a 4.5-kb CD34 5' promoter
fragment and a 3' enhancer active in transient
transfections12 failed to express human CD34 in transgenic mice.19
In order to delineate the important distal control elements of human
CD34, we used the technique of transgenic analysis using large genomic
clones, which has been successful in expression of a number of
hematopoietic genes.20-22 It has been demonstrated that transgenic mouse models made with yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-based artificial chromosome (PAC) clones bearing large genomic sequences are
powerful tools for reproducing proper expression of human genes as well as delineating critical distal elements necessary for proper gene regulation. Therefore, we isolated a human PAC clone including 110 kb
of 5' upstream flanking sequence and 26 kb of 3' downstream flanking
sequence.19 Transgenic mice harboring a 141-kb fragment from this PAC expressed human CD34 mRNA and protein in nonhematopoietic tissues in a manner similar to that of murine CD34.23 (See
the accompanying paper by Radomska et al24 that describes
the use of these CD34 regulatory elements to express heterologous genes in early hematopoietic cells, beginning on page 4410.) However, consistent with reports that murine CD34 may not be highly expressed on
stem cells functionally defined by transplantation to lethally irradiated recipients,25-27 transgenic mice harboring this
PAC express human and murine CD34 differently in the bone
marrow. Only human and not murine CD34 targets true
repopulating hematopoietic stem cells in these transgenic mice, while
common myeloid progenitors (CMP), granulocyte macrophage progenitors
(GMP), common lymphoid progenitors (CLP), and thymic T-cell progenitors
express both human and murine CD34.28,29 The elements
mediating the difference in expression between human and murine CD34
are still unknown. Therefore, in this report, we made a number of
transgenic lines with constructs containing various lengths of 5'
upstream and 3' downstream human CD34 flanking sequences in order to
locate critical cis distal elements.
Extraction and purification of PAC clones containing the entire
human CD34 genomic sequence
Transgenic mice
Genotyping human CD34 transgenic mice with Southern blot
hybridization and PCR
RNA extraction and Northern blot analysis RNA from tissues of transgenic mice were homogenized with 4 M guanidine isothiocyanate solution and put onto a cushion of cesium trifluoroacetate (Amersham Pharmacia Biotech, Piscataway, NJ) diluted with 1× TE to adjust the density to 1.60. After ultracentrifugation, RNAs were dissolved in autoclaved water. From each tissue, 10 µg of RNA was electrophoresed in 1% agarose 2.2 M formaldehyde gels. RNAs were transferred to nylon membranes (Biotrans Plus) with 20× SSC and immobilized with a UV cross-linker (Stratalinker). For human CD34 Northern blots, a 0.5 kb HindIII-BamHI fragment from the 3' untranslated region of the human CD34 cDNA was used as a probe.19 For detection of murine CD34 RNA, a 500-bp fragment derived from the 3' region of the murine CD34 cDNA, which was not conserved between human and murine CD34, was amplified by PCR using primers 5'-TGCAGGAAAGTGGCATCTCTTG-3' and 5'-CAAGCTACTTGGAAGCCTAAAGA-3'. A murine glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA fragment was used for normalization of RNA. Membranes were hybridized at 65°C in 7% SDS, 0.5 M NaPO4 pH 7.2, 1% bovine serum albumin (BSA) (Pentax fraction V),32 washed with 0.2× SSC/0.1% SDS at 65°C, and exposed to film using BioMax intensifying screens (Kodak).FACS analysis of bone marrow cells from transgenic mice Bone marrow cells were flushed from femurs and tibias of 2- to 3-month-old transgenic mice, suspended in phosphate buffered saline (PBS) with 2% fetal bovine serum, and filtered through 40 µm nylon mesh (Becton Dickinson, Franklin Lakes, NJ). One million cells of this single cell suspension were stained with phycoerythrin (PE)-conjugated anti-human CD34 antibody or fluorescein isothiocyanate (FITC)-conjugated anti-human CD34 antibody (HPCA-2, Becton Dickinson) and FITC-conjugated anti-mouse CD34, Gr-1, Sca-1 (BD Pharmingen, San Diego, CA), B220, Mac-1 (Caltag Laboratories, San Francisco, CA) antibodies, or PE-conjugated anti-c-kit, CD4, CD8, TER119 antibodies (BD Pharmingen). Cells were washed with PBS twice and resuspended in PBS with 2 µg/mL propidium iodide (PI; Sigma, St Louis, MO). Fluorescence-activated cell-sorter (FACS) analysis was done with a FACScan (Becton Dickinson, San Jose, CA).Quantitation of human and murine RNA expression by real-time PCR Multiplex PCR with amplification of 18S RNA in the same tube for quantitation of human and murine CD34 expression, TaqMan analysis, and subsequent calculations were performed with an ABI Prism 7700 sequence detection system (Perkin Elmer, Foster City, CA), which detects the signal from a fluorogenic internal probe. For each sample, 100 ng cDNA was subjected to PCR with primers 5'-AAACTACAACACCTAGTACCCTTGGAA-3' and 5'-GAATTTGACTGTCGTTTCTGTGATG-3' for human CD34 expression, according to protocols provided by the manufacturer of the Taqman system (ABI, Foster City, CA). The sequence of the double-labeled oligonucleotide used as probe was FAM-CCCTGTGTCTCAACATGGCAATGAGGCC-TAMRA. Amplification of 18S RNA was performed in the same reaction tubes as an internal standard with an alternatively labeled probe to distinguish its product from that derived from CD34 RNA (multiplex PCR). For murine CD34, the primers were 5'-TCTTCTGCTCCGAGTGCCA-3' and 5'-CCTGGGCCAACCTCACTTC-3', and the sequence of the double-labeled oligonucleotide used as probe was FAM-TAAGGGAGAAATCAAATGCTCTGGAATCCG-TAMRA. Experiments were performed in quadduplicates for each standard and bone marrow sample.Colony assay Bone marrow cells were isolated from femurs and tibias from 4-month-old mice, suspended in PBS with 2% fetal bovine serum, and stained with PE-conjugated anti-human CD34 antibody (HPCA-2) and FITC-conjugated anti-mouse CD34 antibody (Pharmingen). Cells were washed with PBS twice and resuspended in PBS with 2 µg/mL PI. PI-negative cells of human CD34+/mouse CD34 ,
human CD34+/mouse CD34+, human
CD34/mouse CD34+, and human
CD34/mouse CD34 populations were sorted
using a high-speed cell sorter (Moflo-MLS, Cytomation, Fort Collins,
CO). Each cell population was then inoculated in Methocult H4100 (Stem
Cell Technologies, Vancouver, BC) supplemented with 30% fetal
calf serum, 1% bovine serum albumin, 20 ng/mL stem cell
factor, 20 ng/mL interleukin-3 (IL-3), 10 ng/mL
IL-11, 10 ng/mL granulocyte-macrophage colony-stimulating
factor, 1 U/mL erythropoietin, 10 ng/mL thrombopoietin (R&D
Systems, Minneapolis, MN), 2 mM L-glutamine (Stem
Cell Technologies), 100 U/mL penicillin, and 100 µg/mL streptomycin
(GIBCO BRL, Carlsbad, CA). Colony numbers were counted after
5-6 days of culture.
Generation of transgenic mice with constructs harboring human CD34 genomic sequences of various lengths In order to characterize the regulatory elements of the human CD34 gene, we generated several constructs that included, in addition to all exons and introns, various lengths of 5' upstream and 3' downstream flanking sequences of the hCD34 gene (Figure 1). PAC7H11 and PAC54A19 included all exons and introns of human CD34 gene, 23 kb and 18 kb 5' upstream flanking sequence, and 95 kb and 43 kb 3' downstream sequence, respectively. Constructs with 48 kb and 31 kb of 5' upstream flanking sequences were derived from PAC88L2 DNA using different restriction enzyme digests. The construct with 10 kb of 5' upstream and 17 kb of 3' downstream flanking sequences was derived from PAC54A19 DNA following digestion with XhoI. We used FIGE to analyze these fragments, which can be greater than 100 kb in size, and to separate them from the PAC vector DNA. The yields of transgenic mice with DNA fragments greater than 100 kb were low. The number of transgenic mouse founders was inversely correlated with the size of the fragment (Figure 1).To confirm the structure of the human CD34 transgene in transgenic mice, we performed Southern blot analysis of the human CD34 transgene with a probe bearing all of exon 1 and intron 1, as well as PCR corresponding to 4 different regions of the human CD34 genomic sequence, as described in "Materials and methods." The 5-kb EcoRI fragment detected by Southern blot with the exon 1/intron 1 probe was intact in all transgenic mice founders (data not shown). The PCR corresponding to the 3' human CD34 genomic sequence was consistent with the transgenes including 5 kb of sequences downstream of the transcription termination site. Thus, the transgenic mice include all human CD34 exons and introns. In order to check the status of the 5' upstream flanking sequence, 2 different regions were amplified by PCR. All transgenic lines except for the line with only 10 kb of 5' upstream flanking sequence included the region corresponding to the sequence 11 kb upstream (data not shown). Only one line, containing a construct including 48 kb of 5' upstream flanking sequence, was positive for PCR using primers corresponding to 38 kb upstream of the transcription start site. These results are consistent with the transgenes harboring the structures shown in Figure 1. Transgenic mice with human CD34 constructs ranging from 18 to 48 kb of 5' upstream flanking sequences express human CD34 RNA In order to delineate the important cis regulatory elements in human CD34, we assessed human CD34 RNA expression of each founder by Northern blot analysis. All constructs including 18 to 48 kb of 5' upstream sequence confer human CD34 expression in various tissues. Figure 2 demonstrates the pattern of human and murine CD34 expression in a transgene with PAC54A19, which includes 18 kb of 5' upstream and 43 kb of 3' downstream flanking genomic sequence. High levels of RNA were detected in heart, brain, kidney, spleen, and lung, similar to that observed with a larger, 141-kb construct.23,24,29 The expression pattern of human CD34 was very similar to that of murine CD34, with the exception of lung. This expression pattern was observed in all transgenic lines shown in Figure 1, with the exception of the XhoI-digested PAC54A19 construct, containing 10 kb of upstream and 17 kb of downstream flanking sequences. Therefore, these results indicate that elements between 18 kb upstream and 26 kb downstream are sufficient for directing human CD34 expression in vivo.
We also asked whether the expression of human CD34 was dependent on
copy number. While the number of transgenic lines was relatively low,
the level of human CD34 RNA in heart tissue of the 4 transgenic
founders with PAC54A19, which includes 18 kb of 5' upstream and 26-43 kb of 3' downstream flanking genomic sequences, appeared to be gene
copy number dependent (Figure 2B, Transgenic mice with human CD34 constructs ranging from 18 to 48 kb of 5' upstream flanking sequences express human CD34 on the surface of immature bone marrow cells Because less than 5% of bone marrow cells express human CD34, human CD34 RNA levels in bone marrow were too low to compare among transgenic mouse lines with different constructs. Therefore, we measured human CD34 surface expression by fluorescence-activated cytometry. Transgenic mice made with all of the constructs except for the one with 10 kb of upstream sequence expressed human CD34 protein on the surface of 2% to 4% of bone marrow cells (Figure 3). Of the 2% to 4% of bone marrow cells that expressed human CD34, 42% to 79% also expressed early markers such as murine CD34, Sca-1, and c-Kit (Figure 3). Conversely, the erythroid differentiation marker TER119 was not coexpressed with the human CD34+ cell population. Gr-1, Mac-1, and CD8 antigens were expressed in a smaller fraction of the human CD34+ cells, indicating that these cells might represent progenitors. We did observe significant coexpression of human CD34 with B220, consistent with the findings of others and expression of human CD34 in pro-B cells.29,33,34 The expression patterns of a second founder line made with this same construct (MluI-digested PAC54A19) were almost identical (data not shown).
In order to compare the expression of different constructs in the bone
marrow, we measured expression of human CD34 antigen from transgenic
mice made with human CD34 genomic DNA constructs harboring different
amounts of 5' flanking sequence. As shown in Figure
4A, constructs with 48, 31, 23, or 18 kb
of 5' upstream and either 26 kb or 95 kb of 3' downstream flanking
sequence confer expression of human CD34 antigen in bone marrow cells
of transgenic mice. Therefore, sequences critical for in vivo
expression of the human CD34 transgene are located from
Upstream sequences extending from  18 to 10
and/or from +17 to +26 led to complete loss of RNA expression in
multiple transgenic lines (Figure 5). In
order to exclude the possibility that this construct was still
expressing human CD34 on bone marrow progenitors, we performed
fluorescence-activated cytometry. In contrast to what was
observed with PAC54A19 and larger constructs (Figures 3 and 4), 3 different transgenic mouse founders with 10 kb of 5' upstream and 17 kb
of 3' downstream flanking sequence failed to express human CD34 protein
in bone marrow cells (Figure 6). These
data indicate that the critical elements for human CD34 expression are
located between 18 and 10 kb and/or from +17 to +26 kb.
The human CD34 transgene is expressed in the most immature progenitor cells The data in Figures 3 and 4 demonstrate that human CD34 is expressed in cells with an immature phenotype. Therefore, we wanted to test whether human CD34 was selectively expressed on cells that function as immature progenitors. As shown in Figure 3, hematopoietic cells from MluI-digested PAC54A19, which includes 18 kb of upstream and 43 kb of downstream flanking sequences, can be subdivided into 4 populations based on expression of human and murine CD34: human CD34+/murine CD34; human
CD34+/murine CD34+; hCD34/murine
CD34+; and hCD34/murine CD34.
To assess the function of these 4 cell populations, we sorted the cells
and performed colony-forming assays with each population. As shown in
Table 1, all CD34+ cell
populations gave rise to colony-forming units in culture (CFU-Cs),
whereas no CFU activity was observed in human CD34/murine
CD34 cells. The majority of CFU-Cs, as well as
CFU-granulocytes, erythrocytes, monocyte/macrophages, and
megakaryocytes (CFU-GEMMs, the most immature multipotent progenitor
cells), were found in the human CD34+/murine
CD34+ cell population, whereas a relatively small number of
GEMMs were observed to be human CD34+/murine
CD34. In contrast, no GEMM colonies were observed in
cells that lacked expression of human CD34. In summary, all very
immature CFUs, the CFU-GEMMs, expressed human CD34, whereas some of the
more mature CFUs, the CFU-Cs, did not, consistent with expression of human CD34 in the earliest progenitors.
We and others have studied the regulation of the CD34 gene as a means of understanding gene regulation in hematopoietic stem cells, identifying the promoter region of human and murine CD34,12,16,17,30 a cell type-specific enhancer element at the 3' end of the human gene,12,19 and an enhancer at the 5' end of the murine gene.14 DNaseI hypersensitivity assays spanning a 43-kb region extending from 8.5 kb upstream to 9.5 kb downstream of the gene identified a number of CD34-specific hypersensitive sites, including several within 3 kb of the transcription start and termination sites, as well as in introns 1 and 4.13,19 In addition, a number of transcription factors, including c-myb,35,36 ets-2,36 MZF-1,15,37 Sp1 and Sp3,17 and NFY18 were demonstrated to bind to their cognate sites and modulate the activity of the human and/or murine promoters and/or 5' untranslated regions. However, in most vertebrate genes, several kbs of promoter region often fail to confer proper gene expression,22 as they lack distal regulatory elements located far upstream,38,39 within introns,40 or 3' of the coding region.41,42 This was the case for human CD34: a minigene construct including 4.5 kb of human CD34 promoter as well as the 3' enhancer, which included the previously identified DNaseI hypersensitive sites described above, failed to express human CD34 mRNA in transgenic mice.19 In recent years, transgenic mice models made with YAC, BAC, or PAC
clones bearing large genomic sequences have been shown to be powerful
tools for studying human gene regulatory elements in transgenic
mice.20-22 With this in mind, we generated transgenic mice
with a large PAC clone that included 141 kb encompassing the entire
human CD34 genomic region. This PAC expressed high levels of human CD34
mRNA and protein,23,24 demonstrating that other distal
elements were critical for human CD34 gene expression in transgenic
mice. To identify such critical distal elements, we analyzed a series
of deletion constructs of the human CD34 gene in transgenic mice. As
little as 18 kb upstream and 26 kb downstream sequences were sufficient
for expression of human CD34 RNA and protein in several tissues,
including bone marrow, at levels with a specificity similar to that of
transgenic mice made with the 141-kb human CD34 genomic
clone.29 The expression levels of the 18-kb construct
appear to be gene copy number dependent, suggesting that this construct
includes all the critical elements needed for human CD34 expression in
various tissues, including bone marrow cells. In contrast, further
deletion of both an upstream and downstream region eliminated human
CD34 expression in all tissues examined. These data strongly suggested
that distal critical cis elements are located between With both the larger 141-kb PAC88L2 transgene and the smaller PAC54A19
constructs, human CD34 protein was expressed on 2% to 4% of total
bone marrow cells, a slightly lower frequency than that of the murine
CD34 gene (5% to 7%). Analysis of human CD34 expression in
progenitors demonstrated that all CFU-GEMMs expressed human CD34. These
data strongly suggested that critical distal cis elements
necessary for proper expression of human CD34 in bone marrow cells are
contained within the PAC88L2 (141 kb) and PAC54A19 (86 kb) transgenes
and that important cis elements are located within the Further delineation of the regulatory regions will require smaller deletions and assessment of function in transgenic animals, as well as testing the ability of smaller regulatory elements to complement the ability of the human CD34 promoter to express human CD34 in vivo. The results of such studies will lead to further insights into the regulation of stem cell genes, including identification of the transcription factors mediating this specificity, as well as potentially provide the means to develop vectors that can target heterologous genes to stem cells.24
We thank Maris Fenyus for expert animal husbandry and genotyping, other labmates from the Tenen lab for their support, Joel Lawitts for production of transgenic mice, Bertie Göttgens and Tony Green for assistance with sequence comparisons, Linda Clayton for thoughtful discussions, and Mary Singleton and Alison Lugay for expert assistance with preparation of the manuscript.
Submitted March 13, 2002; accepted July 17, 2002.
Prepublished online as Blood First Edition Paper, August 22, 2002; DOI 10.1182/blood-2002-03-0788.
Supported by The Golden Family Foundation (H.S.R.), an international (JC 2000) fellowship from the Jose Carreras Leukemia Foundation (C.S.H.), and National Institutes of Health grant DK48660 (D.G.T.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Daniel G. Tenen, Harvard Institutes of Medicine, Rm 954, 77 Avenue Louis Pasteur, Boston, MA 02115; e-mail: dtenen{at}caregroup.harvard.edu.
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© 2002 by The American Society of Hematology.
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Top
Abstract
Introduction
-agarase
(New England Biolabs, Beverly, MA), and DNAs were purified and
resuspended in 0.1× TE. The DNA was injected into fertilized oocytes
of FVB/N mice and implanted in uteri of pseudopregnant FVB/N mice
according to standard procedures.
) was determined by ImageQuant
(Molecular Dynamics, Sunnyvale, CA) and standardized to GAPDH
expression. The gene copy number was determined by Southern blot
analysis. 
represents one transgenic founder containing 141 kb of
human CD34 genomic DNA (PAC88L2), as previously
reported.
regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells.
Blood.
1996;88:1234-1247