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HEMATOPOIESIS
From the Departments of Molecular Genetics and Cell
Biology and Medicine, and Howard Hughes Medical Institute, University
of Chicago, Chicago, IL; and the Department of Molecular and Structural
Biology, University of Aarhus, Aarhus, Denmark.
The c-fes proto-oncogene encodes a 92-kd protein
tyrosine kinase whose expression is restricted largely to myeloid and
endothelial cells in adult mammals. A 13.2-kilobase (kb) human
c-fes genomic fragment was previously shown to contain
cis-acting element(s) sufficient for a locus control
function in bone marrow macrophages. Locus control regions (LCRs)
confer transgene expression in mice that is integration site
independent, copy number dependent, and similar to endogenous murine
messenger RNA levels. To identify sequences required for this LCR,
c-fes transgenes were analyzed in mice.
Myeloid-cell-specific, deoxyribonuclease-I-hypersensitive sites localized to the 3' boundary of exon 1 and intron 3 are required to confer high-level transgene expression comparable to
endogenous c-fes, independent of integration site. We
define a minimal LCR element as DNA sequences (nucleotides +28 to +2523 relative to the transcription start site) located within intron 1 to
intron 3 of the human locus. When this 2.5-kb DNA fragment was linked
to a c-fes complementary DNA regulated by its own
446-base-pair promoter, integration-site-independent,
copy-number-dependent transcription was observed in myeloid cells in
transgenic mice. Furthermore, this 2.5-kb cassette directed expression
of a heterologous gene (enhanced green fluorescent protein) exclusively
in myeloid cells. The c-fes regulatory unit represents a
novel reagent for targeting gene expression to macrophages and
neutrophils in transgenic mice.
(Blood. 2000;96:3040-3048) Hematopoietic cells of the myeloid lineages
(monocytes/macrophages and neutrophils) are likely to be derived from a
common multipotent progenitor cell. c-fes, the cellular
homologue of an oncogene transduced in numerous feline and avian
retroviruses, is preferentially expressed in hematopoietic progenitor
cells and mature cells of the myeloid lineages.1-7
The mammalian c-fes proto-oncogene encodes a 92-kd
cytoplasmic protein tyrosine kinase (p92c-fes)
thought to regulate proliferation and differentiation during myelopoiesis. In adult animals, peritoneal macrophages and
bone-marrow-derived monocytes, macrophages, and granulocytes
demonstrate high levels of c-fes messenger RNA (mRNA) and
p92c-fes protein.1,4,5,8 There has
also been detection of c-fes mRNA in highly purified
CD34+ hematopoietic stem cells.8
Interestingly, c-fes expression remains constant during
myelomonocytic differentiation but decreases and is extinguished upon
erythroid maturation. Greer et al7 have also demonstrated
c-fes expression in adult human and murine vascular
endothelial cells. During early embryonic development, c-fes
mRNA has been detected in multiple fetal tissues derived from all 3 germ layers.8 However, prominent
p92c-fes expression becomes more limited at
later stages of development and is largely restricted to myeloid and
vascular endothelial cells in the adult.9
A critical role for p92c-fes in myeloid
development has been suggested by a variety of experiments. For
example, K562 leukemic cells (expressing undetectable levels of
p92c-fes) spontaneously undergo myeloid
differentiation upon stable transfection with a 13.2-kilobase (kb)
human c-fes genomic construct.10 Inhibition of
c-fes expression in HL60 cells with antisense
oligonucleotides results in apoptosis during granulocytic
differentiation or a block in macrophage production during treatment
with vitamin D3.11-13 The protein
p92c-fes is tyrosine phosphorylated and
catalytically activated in response to granulocyte-macrophage
colony-stimulating factor (GM-CSF),13,14 which (along with
interleukin [IL]-3) is a potent enhancer of neutrophilic and
monocytic development from hematopoietic progenitors. A direct
association between phosphorylated p92c-fes and
the common The transcription of most genes introduced into transgenic mice is
influenced by the surrounding chromatin at the site of integration.
Remarkably, a 13.2-kb human c-fes transgene is expressed in
mice in a tissue-specific manner irrespective of integration site and
proportional to transgene copy number.1 Therefore, the human c-fes transcription unit includes
cis-regulatory DNA elements sufficient for a locus control
region (LCR). LCRs were first described for the human Active genes are typically located within regions of general
deoxyribonuclease I (DNase I) sensitivity. Interestingly, the c-fes locus contains 3 myeloid-cell-specific
Dnase-I-hypersensitive sites (HSs).30 Here, we show that
all 3 sites are essential for full locus control activity. The
c-fes LCR is located within DNA sequences +28 to +2523.
Thus, like many other genes containing LCRs
( Generation of transgenic constructs and animals
An alternative approach was used to develop a second series of
transgenic constructs (Figure 5). All of these plasmids contain the
c-fes promoter (nucleotides All plasmids were purified twice over cesium chloride gradients,
digested with EcoRI to remove plasmid DNA, and isolated from agarose gels with GeneClean (Bio101, Vista, CA). Following
microinjection into male pronuclei,33 surviving fertilized
eggs were transferred into pseudopregnant females.34
Ribonuclease protection assays
RNA was isolated from 6-week-old transgenic founder animals with the
exception of the 13.2 and Generation of the c-fes EGFP construct and transgenic animals The 0.5-kb HSabc construct was digested completely with SpeI and partially with XbaI to remove the c-fes cDNA sequence and to insert a new polylinker. This polylinker re-established the SpeI site and disrupted the XbaI site and consists of recognition sites for the following unique restriction enzymes: SpeI-SalI-MluI-ClaI-NotI-XhoI. For simplicity, this construct is now called the c-fes cassette. An NheI-XhoI fragment from pEGFP-C1 (Clontech) containing the EGFP gene was cloned into the SpeI/XhoI sites of the c-fes cassette. The function of the c-fes EGFP construct was verified by transient transfection of the murine myeloid FDC-P1 cell line (from ATCC; Rockville, MD) by means of electroporation, essentially as described,39 followed by fluorescence microscopy after 24 hours. We identified 8 EGFP transgenic founders by dot blot analysis on purified tail DNA (Dneasy tissue kit, Qiagen, Valencia, CA) using an EGFP-specific probe. The transgenic status of the founders was subsequently verified by Southern analysis.Flow cytometry analysis of c-fes EGFP mice Cells from bone marrow, spleen, or thymus were hemolyzed (with the use of NH4Cl) to remove erythrocytes and washed twice in flow buffer (phosphate-buffered saline with 2% fetal calf serum and 2 mmol/L NaN3), and 1 million leukocytes were preincubated for 5 minutes on ice with Fc-Block (Becton Dickinson, San Diego, CA) to prevent nonspecific binding of antibody and were subsequently incubated for 30 to 45 minutes on ice with either the monoclonal antibodies Gr-1 (phycoerythrin [PE] conjugated), Mac-1 (PE or PE-Cy5), and B220 (PE-Cy5), or the matching isotype controls (Cedarlane Laboratories, Hornby, ON, Canada). The cells were washed twice in flow buffer and dissolved in flow buffer containing 1% formaldehyde to fix the cells prior to flow cytometry analysis. Initial screening for transgene-expressing lines was done by analyzing bone marrow cells for EGFP expression. The samples were analyzed on a Coulter XL flow cytometer (Beckman Coulter, Fullerton, CA) by means of the fluorescein isothiocyanate channel green fluorescence. We analyzed 50 000 counts from each sample. List-mode analysis was done with the use of Coulter software version 2.
Generation of transgenic mice carrying the human c-fes locus A human 13.2-kb EcoRI restriction fragment contains all 18 c-fes coding exons, the first noncoding exon, 446-bp 5' flanking sequences, and 1.5-kb 3' flanking nucleotides (Figure 1A). Greer et al1 determined that this relatively short 13.2-kb construct is likely to contain an LCR when introduced into transgenic animals. Their report analyzed transgene expression in a large number of murine tissues, including bone marrow, spleen, thymus, heart, lung, kidney, liver, brain, testes, and muscle. We generated 3 13.2-kb transgenic founder lines with 3, 23, and 88 copies each of the human c-fes genomic fragment and tested bone marrow RNA for human c-fes transcripts by RPAs. Transgene copy number for each founder animal was determined by DNA hybridization analysis of tail DNA samples and comparison with the endogenous murine gene. The human c-fes-specific riboprobe was prepared with the use of a 372-bp AflII-NaeI genomic fragment that includes most of exon 19; human transcripts protect a 273-nucleotide fragment derived from this probe (see "Materials and methods"). A 314-bp murine c-fes cDNA fragment yielding a 288-nucleotide protected band was used to detect endogenous mouse mRNA. As shown in Figure 1B, all 3 founders expressed human c-fes mRNA in the bone marrow. Consistent with previous observations,1 transgene expression correlated well with copy number. For example, compared with 2 copies of the murine c-fes locus, founder line 3 carrying 88 copies of the human transgene expressed 43 to 44 times as much human c-fes RNA. No human c-fes transcripts were detected in nontransgenic CD1 mice (Figure 3A).
Further analysis revealed that human c-fes mRNA was present in bone marrow, spleen, brain, lung, and thymus (Figure 1C). On the basis of numerous in situ hybridization and immunohistochemical analyses, these transcripts arise from infiltrating myeloid cells, such as alveolar macrophages in the lung and microglial cells in the central nervous system.1,7,9 RNase assays for murine c-fes transcripts allow careful quantitation of circulating myeloid cells within these tissues (Figure 1C). Densitometric scanning with a Phosphorimager revealed that human transcripts were proportional to murine c-fes mRNA (within a factor of 3) for all 3 founder lines. Therefore, the 13.2-kb construct appears to be expressed independently of integration site and dependent on copy number. These results are consistent with those of Greer et al1 and support their observation that an LCR active in myeloid cells resides within the human c-fes locus. Because c-fes is a proto-oncogene, careful histopathological assessment of all transgenic mice was performed; however, no tissue hyperplasia, neoplasia, or abnormal morphology was detected. Genomic sequences downstream of exon 19 and between nucleotides +3767 and +10738 are not required for c-fes LCR activity To determine if sequences 3' to exon 19 include cis-acting elements that contribute to locus control function, we generated transgenic mice with the 3' region deleted ( 3') (Figure 2). As shown in Figure
3A, this construct still contains an
intact LCR. We found that 3' transgenic animals, harboring 1, 18, 30, and 32 copies of transgenic DNA, expressed human c-fes
at levels comparable to the full-length 13.2-kb construct (Figures 2
and 3A). Furthermore, based on RPAs of additional tissues (brain, lung,
and thymus), the 3' construct was expressed only in cells where the
endogenous murine c-fes message was also detected (data not
shown). We concluded that DNA 3' to c-fes exon 19 is
dispensable for LCR activity.
To rapidly localize domains within the 13.2-kb DNA element encompassing
the c-fes LCR, we generated constructs with internal DNA
sequences deleted by restriction-enzyme digestion of the full-length plasmid. Importantly, all constructs in this series maintain the integrity of exon 19 and could be assayed for expression by means of
the probe descibed in Figure 1. Analyses of mice harboring 3 integration events of the
The promoter is required but not sufficient for LCR function We have previously shown that the 446-bp c-fes 5' flanking region contains a myeloid-cell-specific promoter element.28 To determine if the 446-bp promoter provided LCR activity, a construct was generated that includes c-fes 5' flanking sequences, a human c-fes cDNA, and 5 hGH exons to provide splicing and polyadenylation signals (0.5-kb, Figure 4C). 5 integration events were analyzed, and all transgenic animals demonstrated low levels of human c-fes mRNA and expression in ectopic locations (Figure 5). For example, the founder animal depicted in Figure 4C exhibited inappropriately high levels of human c-fes mRNA in the lung. Furthermore, regardless of copy numbers ranging from 3 to 195, each transgenic founder expressed equivalent amounts of human c-fes (data not shown). Therefore, although the 446-bp c-fes promoter region is active when introduced into chromatin, it does not provide copy-number-dependent transcription in the appropriate cell types. These data are consistent with all 3 myeloid-cell-specific DNase I HSs being essential for locus control function.
Production of a c-fes minilocus that retains LCR activity The production of a c-fes minilocus was accomplished by a combination of the 2 preceding transgenic strategies. To restore LCR activity to the c-fes 446-bp promoter/cDNA construct, we introduced sequences containing different HSs into plasmid 0.5 kb. Six transgenic founder animals containing the 446-bp
5' region, the cDNA, and +28 bp to +863 bp of human c-fes
starting from exon 1 (0.5 kb HSa) were tested. However, as summarized
in Figure 5, all 6 transgenics ranging in copy number from 2 to 25 exhibited ectopic expression in tissues such as the thymus and brain.
Furthermore, transgene expression was not proportional to copy number.
These results clearly indicate that the HSa site is not sufficient for
LCR characteristics in transgenic mice. Therefore, the HS sites located
within intron 3 appeared most likely to contribute to LCR function and
were further analyzed. The 0.5-kb HSbc plasmid contains
nucleotides +1094 to +2519 of the human locus, including small portions
of exons 3 and 4 and all of intron 3. All 4 of the integration events expressed human c-fes in transgenic animals (Figure 5).
However, transgene expression was neither copy number dependent nor
tissue specific (data not shown). We concluded from these results that the HS sites in either intron 1 or intron 3 were not capable of conferring locus control function on their own.
Finally, we tested the hypothesis that all 3 tissue-specific HS sites
are necessary for full c-fes LCR activity. The 0.5-kb HSabc
construct shown in Figure 5 harbors all 3 myeloid-specific HS sites.
Eight transgenic founders ranging in copy number from 1 to 26 exhibited
human c-fes expression that was copy number dependent,
restricted to myeloid cells, and at levels similar to the murine
c-fes locus (Figure
6A). As shown in Figure 6B, splenic RNA
samples from all 8 0.5-kb HSabc founders transcribed human
c-fes at levels consistent with their copy number
within a factor of 4. Analysis of additional tissues (bone marrow,
brain, lung, and thymus) confirmed that transgene expression was mostly in the appropriate cell types, on the basis of murine c-fes
mRNA levels (Table 1). Three transgenic
mice (numbers 1, 23, and 25) expressed only 10% of the expected levels
of human c-fes RNA in the bone marrow (Table 1). However,
all other tissues analyzed showed copy-number-dependent mRNA
production. Comparison with murine c-fes mRNA
levels demonstrated that the levels of human c-fes RNA are
well within a factor of 3 of transgene copy numbers for all 8 of these
founders (Table 1). In summary, the data from the transgenic strategy
described above clearly support the notion that DNA localized around HS
sites within introns 1 and 3, in conjunction with the promoter, contain
the human c-fes LCR.
Myeloid-specific expression of a heterologous gene (encoding the EGFP) in transgenic mice by means of the minimal c-fes expression cassette To assess the usefulness of the minimal c-fes expression cassette to drive expression of a heterologous gene in the myeloid compartment in transgenic mice, we removed the c-fes cDNA from the 0.5-kb HSabc construct, introduced a new polylinker, and inserted the gene encoding the EGFP. The function of this construct was evaluated by transient transfection of the bone-marrow-derived myeloid cell line FDC-P1 and subsequent fluorescence microscopy and flow cytometry. Transfected FDC-P1 cells expressed high levels of green fluorescent protein (data not shown). After microinjection of the EGFP construct, we obtained 8 transgenic founders. F1 animals from all 8 lines were analyzed for EGFP expression by flow cytometry. Surprisingly, EGFP expression was detected in only 3 of the transgenic lines. Apparently, the integration-site-independent expression observed with the 0.5-kb HSabc construct was lost when a heterologous gene was substituted for the c-fes cDNA, which may indicate that the LCR acts in conjunction with elements in the cDNA sequence. The apparent loss of integration-site independence is also observed when other genes are expressed in mice by means of this cassette (as evaluated by RNase protection; data not shown).To determine the cell-type specificity of EGFP transgene expression, we
analyzed cells from bone marrow, spleen, and thymus by flow cytometry.
The cells were incubated with myeloid (Gr-1 and Mac-1) or B-lymphoid
(B220) lineage markers prior to analysis, and expression of these
markers was evaluated in combination with EGFP expression.
Representative data from the transgenic line with highest EGFP
expression are depicted in Figure 7.
Expression of EGFP is highest in bone marrow (Figure 7A), lower but
significant in spleen (Figure 7B), and essentially absent in thymus
(Figure 7C). B220+ cells did not express EGFP, indicating
that the transgene is not expressed in the B-cell lineage. In
constrast, 50% of Gr-1+ (granulocytes) or
Mac-1+ (granulocytes, monocytes, macrophages) bone marrow
cells were EGFP+. Similarly, 30% to 50% of splenic
myeloid cells expressed the transgene. These percentages of
Gr-1+ and Mac-1+ bone marrow cells may
represent the total number of cells actually expressing endogenous
c-fes. Thioglycollate mobilization of peritoneal cells also
revealed EGFP expression in macrophages (not shown). The absence or
very low level of EGFP expression in the thymus demonstrates that the
c-fes expression cassette is not active in T cells. A
myeloid restricted-expression pattern was also observed in 2 additional
transgenic c-fes EGFP lines, demonstrating the consistency
of this minimal c-fes expression cassette (data not shown).
LCRs confer integration-site-independent, copy-number-dependent expression at high levels on transgenes. Previous experiments strongly suggested that the 13.2-kb human c-fes gene included such a dominant, myeloid-specific LCR element. Therefore, the relatively short c-fes genomic locus must include all necessary cis-acting DNA elements for high levels of myeloid-cell-specific expression. Surprisingly, this 13.2-kb genomic fragment contains only 446 bp of 5' and 1.4 kb of 3' flanking sequences, respectively. To locate where cis-acting sequences reside within the 13.2-kb DNA element, we have studied the expression of various c-fes constructs in a large number of transgenic mice. Two series of transgenic constructs convincingly demonstrate that the c-fes LCR lies within introns 1 and 3. These sequences direct integration-site-independent and copy-number-dependent expression in transgenic mice in conjunction with the myeloid-specific c-fes promoter. The deletion series diagrammed in Figure 2 established that sequences between +3767 and +10738 and downstream of +11377 are unnecessary for LCR activity. Furthermore, the 0.5-kb HSabc minilocus construct confirms that DNA sequences between +28 and +2523 in conjunction with the 446-bp promoter are sufficient for LCR regulation of the c-fes cDNA. All minilocus integration events express the transgene, demonstrating integration-site-independent expression. Furthermore, ribonuclease protection analyses of splenic RNA obtained from the 8 animals containing this minilocus support copy-number-dependent expression in a tissue-specific manner. RNase protection analyses revealed that expression of the 0.5-kb HSabc construct in the spleen is comparable to bone marrow expression. When used to express a heterologous gene (EGFP), this minimal construct directs myeloid-specific expression in hematopoietic tissues in transgenic mice. LCR characteristics are probably mediated by higher-order chromatin
structure.17,22,40-42 The colocalization of DNase I HSs to
LCR elements in a number of genes, such as Similar expression cassettes have previously been used for
analysis of the PML-RAR In contrast to the 0.5-kb HSabc construct where integration-site-independent expression was observed, only 3 out of 8 established lines expressed EGFP, as evaluated by flow cytometry. One likely explanation is that sequences in the c-fes cDNA are important for the integration-site-independent expression. Another explanation for the lack of expression for some EGFP lines is that F1 animals and not transgenic founders were analyzed. Perhaps some EGFP transgenes were methylated upon germ-line transmission. Finally, RPAs may be more sensitive than flow cytometry. The myeloid- restricted expression pattern of the cassette is, however, maintained after substitution of the c-fes cDNA, and we still observe a copy-number dependency for expression levels (data not shown). The usefulness of the c-fes cassette will be further enhanced by precisely defining the pattern of expression during embryonic development, together with an exact description of the expressing cell types in the adult mouse. We are currently investigating these issues. The identification of the c-fes minimal expression cassette
also permits myeloid-cell-specific knockouts using the cre/lox system
(reviewed in Marth53). An obvious use for such a strategy is to overcome embryonic lethality of particular mutants. Even if a
null allele is not lethal, it may be desirable to investigate the
cell-specific deletion of certain proteins in an otherwise normal
cellular environment. Cell-intrinsic vs cell-extrinsic questions can be
addressed in such a cell-type-specific chimeric animal. The
c-fes cassette can be used in such a deletion strategy to
study the role of many genes: for example, the C/EBP In summary, using a significant number54 of transgenic mice, we have successfully located the myeloid-specific LCR within the human c-fes locus and determined that it resides in intron 1 and intron 3. This DNA element, encompassing approximately 2.5 kb of c-fes genomic sequences, is necessary and sufficient for conferring integration-site-independent, copy-number-dependent expression on a cDNA construct in conjunction with the c-fes promoter. We have used this minimal expression cassette to drive transcription of the gene encoding EGFP in a myeloid-specific manner. Apparently, integration-site-independent expression is lost when the c-fes cDNA is substituted for a heterologous gene. However, the cassette is still capable of directing copy-number-dependent and myeloid-specific expression in transgenic mice. It thus provides a unique tool for expression of nonmyeloid genes or oncogenes in the monocytic and neutrophilic hematopoietic lineages. Furthermore, the human c-fes construct will be highly useful for conducting tissue-specific, myeloid-lineage gene targeting experiments.
Submitted December 23, 1998; accepted July 5, 2000.
Supported by National Institutes of Health grant R01 HL52094; Howard Hughes Medical Institute; The Danish Medical Research Council; and The Karen Elise Jensen Foundation.
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: M. Celeste Simon, Howard Hughes Medical Institute, Abramson Cancer Research Institute, University of Pennsylvania Cancer Center, Biomedical Research Bldg II/III, Rm 456, 421 Curie Blvd, Philadelphia, PA 19104; e-mail: celeste2{at}mail.med.upenn.edu.
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Top
Abstract
Introduction
chain shared by the IL-3 and GM-CSF receptors has
previously been reported. GM-CSF treatment induces the formation of a
multiprotein complex (consisting of the 
-globin,
locus.
), noncoding
regions (
), and myeloid-cell-specific DNase I HS sites (HSa, HSb,
and HSc). The positions of translational initiation and termination
codons are also shown. The 5' and 3' EcoRI
restriction sites (E) are located 0.446 kb upstream of exon 1 and 1.5 kb downstream of exon 19, respectively. Multiple transcription
initiation sites occur within the first exon; +1 corresponds to the
first and most prominent mRNA cap site. (B) Production of
c-fes mRNA in bone marrow obtained from multiple transgenic
mice generated with the 13.2-kb EcoRI human genomic fragment
depicted in panel A. Two progeny animals obtained from founder no. 3 (88 copies) in addition to founders harboring 23 and 3 copies each of
the 13.2-kb construct were analyzed. Ten micrograms of total RNA were
hybridized to the 372-bp human c-fes and the 314-bp murine
c-fes riboprobes. The 273-bp and 288-bp protected fragments
are indicated. Included as negative and positive controls,
respectively, were 25 µg yeast transfer-RNA and total RNA
harvested from a mouse macrophage cell line (J774.1) and a human
monoblastic cell line (THP-1). (C) Production of c-fes mRNA
in various tissues harvested from the same transgenic mouse analyzed in
lane 1 of panel B. To control for RNA loading, 5 µg RNA were
hybridized to a murine 
represents the
c-fes cDNA, and 
represents hGH exons. The data columns
are explained in Figure 
. (B) Spleen. Approximately 5% of the cells express
the transgene, and expression is observed only in Gr-1+ or
in Mac-1+ cells. (C) Thymus. Expression of EGFP is absent
or very low, indicating that the c-fes expression cassette
is inactive in T cells. In the double histograms, the numbers indicate
percentages of cells present in the given quadrant. Cell-density
scaling parameters are identical for all double histograms from the
same tissue, but differ slightly between bone marrow and spleen. There
were 50 000 counts from each sample analyzed.