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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 419-430
Transcriptional Regulation of vav, a Gene Expressed
Throughout the Hematopoietic Compartment
By
Sarah Ogilvy,
Andrew G. Elefanty,
Jane Visvader,
Mary L. Bath,
Alan W. Harris, and
Jerry M. Adams
From The Walter and Eliza Hall Institute of Medical Research,
Melbourne, Victoria, Australia.
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ABSTRACT |
The vav gene is expressed in all hematopoietic but few other
cell types. To explore its unusual compartment-wide regulation, we
cloned the murine gene, sequenced its promoter region, identified DNase
I hypersensitive (HS) sites in the chromatin, and tested their promoter
activity with a  -galactosidase (-gal) reporter gene in
cell lines and transgenic mice. Whereas fibroblasts had no HS sites, a
myeloid and an erythroid cell line contained five, located 0.2 kb
(HS1), 1.9 kb (HS2), and 3.6 kb (HS3) upstream from the transcription
start and 0.6 kb (HS4) and 10 kb (HS5) downstream. A vav DNA
fragment including HS1 promoted -gal expression in a myeloid but not
a fibroblast line. Expression in leukocytes of transgenic mice also
required HS2 and HS5. Only hematopoietic organs contained -gal, but
virtually all -gal+ cells were B or T
lymphocytes. Expression was always variegated (mosaic), and the
proportion of -gal+ cells declined with lymphoid
maturation and animal age. Thus, these vav regulatory elements
promoted hematopoietic-specific expression in vivo, at least in
lymphocytes, but the transgene was sporadically silenced. Maintaining
pan-hematopoietic expression may require additional vav
elements or an alternative reporter.
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INTRODUCTION |
THE vav GENE, DISCOVERED through
its inadvertent oncogenic activation during transfection experiments,
proved to be widely expressed in hematopoietic cells.1 Its
polypeptide sequence implicated Vav in signal transduction as a GTP/GDP
exchange factor regulating a G-protein of the Rho/Rac/CDC42
family.2 As reviewed by Bonnefoy-Bérard et
al,3 engagement of diverse receptors on hematopoietic cells
leads to rapid tyrosine phosphorylation of the 95-kD Vav polypeptide,
and the phosphorylated Vav has been shown recently to activate the
Rac/Jun kinase pathway.4 Gene disruption has established
that Vav is essential for signaling through the antigen receptors of
lymphocytes but not for the development of hematopoietic
cells.5-8
The unique feature of the vav gene is its expression pattern.
Virtually all hematopoietic cell lines, irrespective of lineage or
stage of development, contain vav mRNA and protein, as do all normal adult and fetal hematopoietic cell types.1,2,9,10 In
contrast, vav expression outside the hematopoietic system is confined to embryonic stem cells,11 the developing
tooth,10 testicular germ cells,12 and the
extra-embryonic trophoblast, in which vav expression is
essential for implantation.8
The cis-acting DNA elements that allow lineage-specific
expression of a gene are becoming better understood. As well as the proximal promoter, where the transcription initiation complex forms,
various enhancer elements within or near the gene augment transcription
in specific cell types. These elements can frequently be detected in
chromatin from the relevant cells by their hypersensitivity to
digestion with DNase I.13 Many enhancers can stimulate
transcription of a linked reporter when introduced into cell lines, but
the more rigorous context of the transgenic animal often shows the need
for additional elements. For example, a region of the -globin locus
marked by a cluster of DNase I hypersensitive (HS) sites in
erythroblasts is thought to establish a large domain of transcriptional competence.14 The presence of this locus control region
(LCR) within a transgene allows efficient expression in the relevant cells, regardless of its point of insertion into the
genome.15 Whereas both the enhancer and the LCR
traditionally have been thought to increase the frequency at which RNA
polymerase molecules initiate transcription, recent findings suggest
that they may instead increase the probability for a given cell that an
initiation complex will form.16,17 If the positive
regulatory elements carried by a transgene are not sufficiently potent,
repressive effects from surrounding chromatin can extinguish its
expression in clones of cells, producing a variegated or mosaic pattern
of expression (reviewed by Martin and Whitelaw18).
In contrast to the considerable progress on the regulation of genes
expressed in specific lineages, almost nothing is known about
compartment-wide regulation. For example, does control of vav
gene expression require a combination of promoters and enhancers specific for the individual hematopoietic lineages or does the vav locus contain some element common to all genes expressed in this compartment? Studying vav gene regulation should clarify such issues and eventually enable construction of a transgenic promoter
that can target the expression of any gene to the entire compartment.
To explore the regulation of vav expression, we cloned the
mouse gene, characterized its promoter, and searched for DNase I HS
sites in the chromatin surrounding the gene. Having identified five HS
sites in its 5 region, we then sought to establish whether fragments spanning various of these sites could drive
hematopoietic-specific expression, first in cell lines and then in
transgenic mice. For these tests, the vav locus fragments were
coupled to the Escherichia coli lacZ
(-galactosidase [-gal]) gene; the widely used
-gal reporter gene allows sensitive enzymatic tests for
promoter activity, including a flow cytometric assay well suited to
hematopoietic cells.19,20 Hematopoietic-specific activity
of the vav-gal reporter was indeed demonstrable both in cell
lines and mice, but the expression in vivo was unexpectedly
restricted to lymphocytes and appeared to be sporadically silenced.
(B) Nucleotide sequence of the murine
vav promoter. Arrows denote major sites of transcription
initiation, as determined by RNase protection in (A). Consensus binding
site sequences for transcription factors are indicated, as are the
regions corresponding to HS1a ( 150 bp to +1 bp) and HS1b (620
bp to 450 bp).
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MATERIALS AND METHODS |
Cloning the murine vav gene and analysis of HS sites.
A 2.5-kb mouse vav cDNA probe2 was used to screen
106 plaques from a mouse genomic library (Stratagene, La
Jolla, CA). Four clones spanning greater than 50 kb were
isolated. The approximate positions of vav exons were mapped by
Southern analysis of the genomic clones using fragments of the
vav cDNA as probes.
HS sites were detected by the protocol of Dr P. Cockerill (Hansen
Centre for Cancer Research, Institute for Medical and Veterinary Research, Adelaide, Australia). Cells (4 × 107) were
washed in cold phosphate-buffered saline (PBS), resuspended in 30 mL of
lysis buffer (60 mmol/L KCl, 15 mmol/L NaCl, 5 mmol/L MgCl2, 10 mmol/L Tris-HCl, pH 7.4, 300 mmol/L sucrose, 0.1 mmol/L EGTA, 0.1% NP40, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 5 µg/mL leupeptin, and 10 µg/mL aprotinin) and centrifuged at
400g for 5 minutes. The pelleted nuclei were adjusted with
nuclei buffer (lysis buffer containing 1 mmol/L CaCl2
without NP40 or protease inhibitors) to a DNA concentration of 0.4 mg/mL, and 500-mL aliquots were digested with 0 to 15 µg/mL DNase I
(Worthington Biochemical Corp, Freehold, NJ) at 22°C for 3 minutes.
The reaction was stopped by adding 3.5 mL stop buffer (0.3 mol/L sodium
acetate, 0.5% sodium dodecyl sulfate, 5 mmol/L EDTA, 200 µg/mL
proteinase K) and incubated at 55°C for 2 hours. The DNA was then
extracted and subjected to Southern analysis.
Sequencing and RNase protection.
The 987-bp Hpa I-Sac I fragment of vav genomic
DNA encompassing the proximal promoter and 5 untranslated region
was cloned into HincII-Sac I-digested Bluescript
SK (Stratagene) and sequenced on both strands.
Putative transcription factor binding sites were identified using a
database maintained by Dr D. Ghosh (National Institutes of Health,
Bethesda, MD). The plasmid was linearized with Acc
I and  32P-UTP-labeled antisense RNA probe of 356 bp
synthesized. The probe was hybridized to 2 µg poly(A)+
RNA from cell lines or 10 µg tRNA, and RNase-resistant products were
resolved on a 6% denaturing polyacrylamide gel.
Transgene construction.
The -galactosidase (-gal) gene, modified to include a
mammalian consensus sequence for translation initiation, was a kind gift from Dr E. Stanley (The Walter and Eliza Hall Institute of Medical
Research, Melbourne, Australia). The promoterless vector, -gal, which includes a 1.1-kb rabbit -globin
polyadenylation signal21 downstream of the reporter, was
the substrate for construction of the transgenes (see Figs 4 and 5).
HS1 vav-gal was constructed by introducing a 965-bp
Hpa I-Nae I fragment upstream of the reporter gene.
HS21 vav-gal included a 2.3-kb EcoRI-Nae I
vav promoter fragment, whereas HS321 vav-gal
contained 4 kb of vav sequence 5 of the Nae I
site. To produce HS1/5 vav-gal, a 2-kb BamHI HS5
fragment was cloned into HS1 vav-gal downstream of the
polyadenylation sequence. Similarly, a 3.7-kb Kpn I HS5
fragment was cloned into HS21 vav-gal and HS321
vav-gal to give HS21/5 vav-gal and HS321/5 vav-gal. To introduce HS4, a 0.7-kb Hpa
I-BamHI fragment was cloned between the -gal
polyadenylation sequence and HS5 to give HS21/45 vav-gal and
HS321/45 vav-gal. Thus, both HS4 and HS5 lie 3 to the
-gal transcription unit. The positive control vector, SR-gal, was constructed by cloning the SR
promoter22 into -gal. Luciferase reporter
constructs were made by cloning the appropriate vav promoter
fragments into the Sma I site of the pGL2-Basic vector
(Promega, Madison, WI). SR-lux and
pA3RSV-lux plasmids were gifts from Drs A. Trout and T. Kay
(The Walter and Eliza Hall Institute of Medical Research) and the
pGL2-Promoter vector was obtained from Promega.
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| Fig 4.
Activity of -gal reporter constructs in a stably
transfected hematopoietic cell line (FDC-P1) and NIH3T3 fibroblasts.
vav DNA fragments encompassing the indicated HS sites
(arrowheads) were linked to the -gal gene (lacZ) and
a -globin intron and polyadenylation signal (see the Materials and
Methods). The unfilled box denotes vav 5 untranslated
sequences. Where included, the HS5 intronic site was placed 3 of
the reporter polyadenylation site. The -gal activity in extracts
from three to five independent pools of FDC-P1 transfectants and two to
three pools of NIH3T3 transfectants (3 independent determinations per
pool) was related to that of a pool transfected with a construct
containing the SR promoter (assigned 100% activity). Means (±SEM)
of the results are shown.
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| Fig 5.
Expression of vav-gal transgenes in primary
transgenic animals. For the indicated reporter constructs, the
proportion of primary animals that showed any -gal+
cells in the blood is given. The constructs analyzed were those used in
the stable transfection experiments (see Fig 4), plus others containing
an additional intronic hypersensitive site (HS4), also placed 3
of the polyadenylation site in the reporter gene.
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Cells and transfections.
FDC-P1 myeloid cells, EL-4 T cells and NIH3T3 fibroblasts were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum. Stable transfectants were produced by
coelectroporation (FDC-P1 cells) or lipofection (NIH3T3 cells) of 10 µg linearized vav-gal plasmid and 0.5 µg pSV2neo. Pools of transfectants selected in 800 µg/mL G418 (Geneticin; GIBCO, Grand
Island, NY) for FDC-P1 cells or 400 µg/mL G418 for
NIH3T3 cells were analyzed for -gal activity after 2 weeks. For
transient transfections, cells were electroporated with 10 µg of a
construct and harvested after 18 (EL-4), 30 (FDC-P1), or 40 hours
(NIH3T3) for luciferase assay. A -gal plasmid was included
to control for transfection efficiency.
4-Methylumbelliferyl--D-galactoside (MUG) and luciferase assays.
The MUG assay was performed as described.23 An aliquot of
extract (1% to 10% of the lysate from 105 cells) was
assayed in a TKO mini-fluorimeter (Hoefer Pharmacia Biotech, San
Francisco, CA). For each experiment, a standard curve was produced with
known concentrations of the fluorescent product 4-methylumbelliferone
(Sigma, St Louis, MO). Luciferase assays were performed
as described.24 Cells were lysed by repeated freeze-thaw
cycles, and 5 µg of extract protein diluted in glycylglycine buffer
containing 60 µmol/L luciferin was assayed using a Lumat LB 9501 luminometer (Berthold, Wildbad, Germany).
Transgenic mice.
Each transgene fragment was separated from the plasmid vector by
electrophoresis of HindIII-digested plasmid DNA, slot-elution, and ethanol precipitation. The purified DNA was microinjected into the
male pronuclei of fertilized (C57BL/6J × SJL/J) F2 eggs, which
were then transferred to pseudo-pregnant females.25 To identify transgenic pups, DNA extracted from tail biopsies was dotted
in duplicate onto nitrocellulose, along with nontransgenic DNA spiked
with varying copy-number equivalents of the transgene and the blot
hybridized with a lacZ cDNA probe.
Fluorescence-activated cell sorting (FACS)-gal analysis of -gal
activity.
For analysis of leukocytes, blood samples were subjected to red blood
cell lysis in 156 mmol/L NH4Cl, 0.1 mmol/L EDTA, 12 mmol/L
NaHCO3; washed; and resuspended in cold PBS/2% calf serum (CS). -gal activity was determined as described.19
Briefly, a working solution of 2 mmol/L fluorescein di-galactoside
(FDG) in ethanol/dimethylsulfoxide/water (1:1:98) was warmed to
37°C for 5 minutes in the dark. Cells were incubated at 37°C
for 75 seconds with an equal volume of the prewarmed FDG, and substrate loading was stopped with 10 vol ice-cold PBS/2% CS. Propidium iodide
(PI) was added (final concentration, 1 µg/mL), and the cells were
incubated for 2 hours on ice. From 2,000 to 10,000 live leukocytes were
then analyzed on a FACScan (Becton Dickinson, San Jose, CA) with Lysis
II software. A forward scatter gate excluded debris and residual red
blood cells, whereas dead cells were excluded by PI uptake. Femoral
bone marrow cells and single-cell suspensions prepared from thymus,
spleen, and mesenteric lymph nodes by passage through wire mesh were
loaded with FDG and analyzed similarly. Samples from nontransgenic (C57 × SJL) F1 mice and Rosa26 -geo transgenic
mice26 provided routine controls.
Simultaneous FACS analysis for -gal and surface markers.
Cells loaded with FDG as described above were incubated in saturating
quantities of biotin-labeled anti-cell surface marker-specific antibodies,27 together with an anti-Fc receptor antibody
(2.4G2) to reduce nonspecific binding28 and
streptavidin-Texas red (Amersham, Little Chalfont, Bucks, UK) as the
secondary reagent. Monoclonal antibodies used included anti-B220
(RA3-6B2), anti-CD4 (H129.19.6.8), anti-CD8 (YTS-169), anti-Thy1
(T24.31.2) anti-IgM (anti-Cµ.5.1), anti-IgD (11-26C), anti-Mac1
(M1/70), anti-Gr1 (RB6-8C5), and anti-Ter119. After staining, cells
were washed and resuspended in a buffered salt solution29
containing 2% CS, 10 mmol/L NaN3, and 1 µg/mL PI. At
least 104 viable nucleated cells were analyzed, using a
FACStar+ or modified FACS-II flow cytometer.
Immunoblotting.
Tissue lysates were made by homogenizing 0.1 g of tissue in 1 mL RIPA
buffer (52 mmol/L Tris-HCl, 157 mmol/L NaCl, 1 mmol/L EDTA, 0.1%
Triton X-100) containing 1 tablet of Boehringer Mannheim Complete
Protease inhibitors (Boehringer Mannheim, Mannheim,
Germany) per 50 mL RIPA using a Dounce homogenizer.
Lysates were mixed at 4°C for 15 minutes and centrifuged at
10,000g for 10 minutes to remove nuclei. Protein concentrations
were determined using the Bio-Rad Detergent Compatible Protein assay
reagents (Bio-Rad, Hercules, CA) and bovine serum albumin
standards.
Cell lysates (50 µg protein) were electrophoresed in 7.5%
polyacrylamide gels and transferred to Hybond C Extra (Amersham). Membranes were blocked overnight at 4°C in 1% Blocking Reagent (Boehringer Mannheim) in PBS and then incubated for 1 hour in a 1:2,000
dilution of antihuman Vav (Upstate Biotechnology, Lake Placid, NY) or
anti--gal (Boehringer Mannheim) monoclonal antibodies. After
incubation with 1:1,000 sheep antimouse Ig monoclonal
antibody-horseradish peroxidase (Silenus, Melbourne,
Australia), the secondary antibody was detected using the Amersham ECL
system.
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RESULTS |
Hematopoietic-specific HS sites in the vav locus.
Our search for cis-acting elements regulating transcription of
the vav gene was initiated by cloning and characterizing the mouse vav genomic locus. The four phage clones obtained span
over 50 kb of genomic sequence (Fig 1).
Only the most 3 clone did not overlap with the others, and the
uncloned region was estimated by genomic Southern blot analysis to be
approximately 5 kb. Southern analysis of the clones, using vav
cDNA fragments as probes, indicated that the gene contains at least 14 exons (Fig 1).
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| Fig 1.
The murine vav locus and DNase I hypersensitive
sites. Cloned genomic sequences are shown as a solid line, with the
relevant phage clones above. An uncloned gap of approximately 5 kb lies between  7.2 and 20.1. Approximate positions of exons (shown as
boxes) were assigned by Southern blotting of enzyme-restricted phage
clones using cDNA fragments as probes; only the first and second of at
least 14 exons are numbered. The major DNase I hypersensitive sites are
indicated by large triangles and minor, inconsistent sites are
indicated by small triangles. The expanded map shows the positions of
the five major HS sites in more detail, with the 5 UTR of exon 1 depicted as an open box and the coding region filled. The bar below
indicates the sequenced proximal promoter region (see text).
Abbreviations: A, Ava I; B, BamHI; Bg, Bgl II;
Hp, Hpa I; H, HindIII; K, Kpn I; N, Nco
I; Ne, Nae I; R, EcoRI; S, Sac I; X,
Xba I. Not all sites for these enzymes are shown.
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We searched for potential regulatory regions within the gene by
sensitivity of the chromatin to DNase I. Because we expected HS sites
associated with relevant regulatory elements to be hematopoietic specific, cells of two hematopoietic lineages, FDC-P1 myeloid cells and
C88 erythroleukemia cells, were compared with NIH3T3 fibroblasts as a
representative nonhematopoietic cell type. Appropriate selection of
restriction enzyme digests and vav probes allowed us to screen
a 60-kb region of chromatin spanning the gene.
No HS sites were found in the fibroblasts, but both hematopoietic cell
lines contained several prominent sites (Figs 1 and 2), and a preliminary analysis of bone
marrow and thymus showed the same pattern. The shared HS sites argue
that vav transcription probably is regulated by mechanisms
common to diverse hematopoietic lineages. The HS sites were positioned
by mapping with several restriction enzymes and, wherever possible, by
probing with DNA fragments located both 5 and 3 of each
site. The sites denoted HS1, HS2, and HS3 mapped approximately 0.2, 1.9, and 3.6 kb upstream from the transcription start site
(Fig 3), whereas two others (HS4 and HS5) were
located within the first intron at +600 bp and +10 kb. HS1 and HS4 were
analyzed in most detail in the erythroleukemia cells (Fig 2D and E).
Higher resolution mapping of HS1 (Fig 2E) resolved it into HS1a, a
strong site centered around 0.1 kb (best seen in the Sac
I digest in Fig 2E), and HS1b, a region of weaker hypersensitivity
located near 0.5 kb (seen in the Hpa I digest in Fig
2E).
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| Fig 2.
DNAse I hypersensitive site mapping of the vav
genomic locus in hematopoietic cells and fibroblasts. (A) Diagrams of
the probes, digests, and observed fragment sizes (in kilobases) that
delineated the five major HS sites in (B) through (D). Exons are shown
as solid boxes. Nuclei from FDC-P1 cells and NIH3T3 fibroblasts (B and
C) or C88 murine erythroleukemia (MEL) cells (D) were digested with
increasing amounts of DNase I followed by Southern analysis of
extracted DNA, using the indicated restriction enzyme/probe combinations. The sizes of the germline fragments (G) and the DNase I
cleavage products at each HS site are indicated in kilobases. The
additional germline band of approximately 4.5 kb in NIH3T3 cells (B)
reflects a polymorphic Bgl II site approximately 1 kb 5
of the first vav exon. In (C), the 4.7-kb band (G 4.7)
reflects detection of the 4.7-kb germline Xba fragment (see
[A]) by exon I sequences in cDNA probe b. On prolonged exposure,
bands corresponding to HS2,1 and 4 were also seen. In (E), fine mapping
of the proximal vav promoter demonstrates that HS1 comprises
two sites approximately 400 bp apart. The positions of the stronger
HS1a and weaker HS1b sites and probes c and d are indicated on the
diagram. Abbreviations: X, Xba I; S, Sac I; Hp,
Hpa I; Bg, Bgl II.
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| Fig 3.
Characterization of the vav promoter. (A) RNase
protection analysis showing major and minor clusters of transcriptional
initiation sites for the vav gene in lymphoid (EL-4, W408, and
ABLS-8), erythroid (F4N), monocytic (RAW309 and J774), and early
myeloid (416B and FDC-P1) cells. No transcripts were detected in NIH3T3
fibroblasts. The length of each protected fragment, numbered from the
ATG translation start, was determined from a sequencing reaction run in
parallel (not shown). The undigested probe was 356 bp in length.
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Sequence of the vav promoter.
To delineate the initiation site for vav transcription, RNase
protection experiments were performed on mRNA from cell lines representative of diverse hematopoietic lineages. All these RNA samples
yielded a pattern of fragments corresponding to a cluster of major and
minor start sites 95 to 133 bp upstream of the translation initiation
codon (Fig 3A), near the multiple start sites mapped for the human
vav mRNA.30 Thus, a single vav promoter
appears to be operative throughout the hematopoietic compartment. In
agreement with this conclusion, tests for amplification of 5
ends of vav mRNA by RACE,31 using a primer located
in the second exon, generated a product of equivalent size from four
different hematopoietic cell types but none from NIH3T3 cells (data not
shown).
To facilitate further dissection of the promoter, we sequenced a 981-bp
Hpa I-Sac I fragment extending 5 of the
vav translation initiation codon (Fig 3B). The vav
promoter is GC rich (55% overall for the region from 870 to +1
bp) and lacks identifiable core promoter elements such as a TATA box or
an initiator. Consistent with other promoters of this type (reviewed by
Ernst and Smale32), it contains an Sp1 binding site 50 to
60 bp upstream of the cluster of major transcriptional start sites (Fig
3B). In the region 150 bp to +1 bp there is 61% identity with
the published human vav promoter sequences.30 This
region of high homology, which corresponds to the dominant HS1a region
of hypersensitivity (Fig 2E), includes the Sp1 consensus binding site.
The murine vav promoter sequence also contains potential
binding sites for a number of additional ubiquitous transcription
factors (eg, Ap-1, Ap-2, and E-box factors) as well as for
tissue-restricted ones such as GATA, Myb, Oct, and Ets proteins (Fig
3A). No direct evidence that they participate in vav gene
regulation is yet available.
Hematopoietic-specific expression in stably transfected cell lines.
To assess promoter activity, potential vav regulatory regions
bearing HS sites were coupled to a -gal reporter. FDC-P1 and NIH3T3 cells were stably transfected with each construct, as well as a
promoterless negative control and gal driven by the potent SR promoter.22 Figure 4 shows the -gal
activity in extracts of pools of the transfected cells, as determined
by a sensitive fluorescence (MUG) assay. In both cell types, the
positive control SR-gal gave 40-fold higher activity than
the promoterless reporter or the level in untransfected cells. All four
vav regions tested, which included one to three of the upstream
HS sites (HS1, HS21, or HS321) or HS1 plus intron site HS5 (HS1/5),
were active in FDC-P1 cells but inert in NIH3T3 cells. Thus, even the
proximal promoter fragment containing only HS1 exhibited marked
hematopoietic specificity. Indeed, the HS1 vav-gal construct
was nearly as active in FDC-P1 cells as SR-gal, and the
addition of the HS2 or HS5 region augmented activity to levels similar
to those observed with this powerful promoter. In contrast, the
inclusion of HS3 consistently decreased activity several fold. In
confirmation of these results, histochemical (X-gal) and flow
cytometric (FACS-gal) assays also showed that the vav promoter
fragments functioned only in the FDC-P1 transfectants, and the pools
bearing the HS1, HS21, and HS1/5 vav-gal constructs
exhibited much higher activity than those harboring the HS321 region
(data not shown). These data suggested that HS2 and HS5 might contain
enhancers, whereas a negative regulatory element might reside within
the HS3 segment.
Low vav promoter activity in transiently transfected cells.
To determine whether the vav promoter could also function in
transiently transfected cells, we first tested the HS1 and HS21 vav-gal constructs in FDC-P1 cells and the T-cell line EL-4. No activity was evident, but the efficiency of electroporation with
these lines was not high enough to have shown low activity. Hence, we
turned to the more sensitive luciferase (lux) reporter and
compared these promoter fragments with the SR and Rous Sarcoma virus
(RSV) promoter in FDC-P1, EL-4, and NIH3T3 cells (Table 1). Hematopoietic specificity was evident in that neither the HS1
vav-lux nor the HS21 vav-lux construct was active in
the fibroblasts, whereas they exhibited twofold to fivefold higher
activity than the basal SV40 promoter in the hematopoietic lines.
Nevertheless, their activity in those lines was much lower than that of
the SR or RSV promoter and, indeed, reached only a few percent of the SR value. To assess whether the vav regulatory elements
could act as transcriptional enhancers on a heterologous promoter, the HS1, HS21, and HS321 vav fragments were cloned upstream of the SV40 basal promoter. In the hematopoietic cell lines, the HS1 and HS21
vav SV40-lux constructs displayed weak activity,
comparable to that of the corresponding constructs without the SV40
promoter, and no activity was evident in the fibroblasts. Inclusion of
the HS3 region decreased activity, consistent with the negative
regulatory effect observed with it in stably transfected cells (Fig 4).
Thus, the vav promoter elements appear to be far more active
when stably introduced into cells (see Discussion).
Hematopoietic-specific expression in vav-gal mice.
To extend the analysis to in vivo regulation, we generated transgenic
mice bearing seven -gal reporter constructs of the type
tested in vitro (Fig 5). Analysis of peripheral blood
from primary transgenic animals showed some in which the transgene was
expressed (see below). Dot blot analyses showed comparable transgene
copy numbers in the expressing and nonexpressing animals. To allow more
extensive analysis, two to four animals for each construct were bred to
generate independent progeny lines. Southern blot analysis on DNA from
expressing lines confirmed that all had a low copy number, from 1 to 4 per haploid genome, as judged by phosphorimager analysis (data not
shown).
When transgenic lines exhibiting -gal activity in the blood had been
established, hematopoietic specificity was assessed by immunoblotting
extracts of organs of mice carrying HS21/5 vav-gal. These
and all subsequent experiments included nontransgenic negative controls
and positive controls from Rosa26 mice, in which a -gal fusion gene (-geo) is expressed
ubiquitously.26 The Western blots indicated that expression
of the vav-gal transgene probably was, as expected,
restricted to hematopoietic tissues
(Fig
6). In the thymus, where a high proportion of cells express the
transgene (see below), -gal was abundant, as was Vav. In spleen and
lymph nodes, -gal was discernible, but the band was weaker than that of Vav; results presented below suggest that this reflects a lower proportion of -gal-expressing cells. No -gal appeared in adult liver, muscle, brain, or lung (not shown), and the trace in the kidney
might reflect contaminating blood cells, because a trace of Vav was
also detected (Fig 6). Unexpectedly, no expression was observed in
fetal liver (data not shown), a rich source of erythroid cells.
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| Fig 6.
Immunoblots showing -gal and Vav proteins in tissue
extracts. The tissue extracts derived from HS21/5 vav-gal
mice, nontransgenic (negative control) mice, or Rosa26 (positive
control) animals. The blots were probed with monoclonal antibodies to
p95Vav or -gal and, as a loading control, antibody to
Hsp70, and bands were shown with a sheep antimouse Ig reagent. The
lacZ-neo gene fusion in Rosa26 animals consistently yields a
146-kD polypeptide (-geo) and a smaller product (125 kD), perhaps
related to the two transcripts observed in these mice.26
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These results were confirmed by staining organs of a HS21/5
vav-gal mouse with X-gal to show -gal activity (data not
shown). The thymus stained strongly and the spleen and lymph nodes
moderately, whereas the heart, liver, lung, brain, kidney, and muscle
failed to stain. Thus, expression of the vav transgene indeed
appeared to be restricted to hematopoietic tissues.
Delineation of the vav regions required for expression in
vivo.
-gal expression in the peripheral blood of primary transgenic mice
and their progeny was assessed by the sensitive FACS-gal assay.19,20 Upstream vav regulatory regions alone
proved insufficient. Whereas HS21 or HS321 vav-gal were
active in FDC-P1 cells (Fig 4), blood from a total of 15 primary
transgenic animals bearing these two constructs exhibited no
-gal+ cells (Fig 5). On the other hand, three of the
four constructs that also contained intron site HS5 did yield a number
of animals showing expression (Fig 5). Thus, this site appears vital
for in vivo function. Other sites clearly influenced the proportion of
mice that showed expression. The combination of HS5 plus the HS1-bearing promoter region appeared inadequate, although unfortunately only two HS1/5 vav-gal lines were obtained. In contrast,
HS21/5 vav-gal, bearing the two proximal upstream HS sites,
yielded -gal+ blood cells in three of eight animals, and
the further addition of intron site HS4 increased the frequency of
expression for HS21/45 vav-gal to 11 of 15 primary animals
(73%).
Thus, the minimal region required for in vivo activity encompassed HS1,
HS2, and HS5. Consistent with the cell line data, distal upstream site
HS3 had a negative influence, because none of 21 independent founders
bearing HS321/5 vav-gal had detectable -gal+
blood cells (Fig 5). Nevertheless, HS321/45 vav-gal, which
also includes the proximal intron site HS4, yielded activity in 4 of 15 founders. Hence, HS4 appears to partially counter HS3 and augment the
proportion of integration sites that allow expression (by  2 test, P < .02).
Only a proportion of blood cells exhibited vav-gal
expression.
All transgenic progeny of each primary mouse that showed expression
proved to express the transgene in peripheral blood. However, to our
surprise, only approximately 15% to 40% of the leukocytes from either
the founders or their progeny were -gal+. The Rosa26
results clearly showed that all white blood cells could express -gal
activity under our conditions (Fig 7A), but the
vav-gal leukocytes invariably fell into distinct expressing and nonexpressing populations (Fig 7B). This unexpected result might
indicate that transgene expression was (1) restricted to certain
hematopoietic lineages, (2) confined to certain developmental stages
within a lineage, or (3) mosaic (variegated) for a given cell type.
Remarkably, all three types of restriction have proven to be operative.
Because we also unexpectedly found that the proportion of
-gal+ blood cells decreased with age of the mice (see
below), routine analyses were confined to animals less than 10 weeks
old.
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| Fig 7.
Detection of -gal+ cells in the blood of
transgenic mice. FACS-gal analysis of viable peripheral blood
leukocytes from (A) a (C57BL/6 × SJL) F1 negative control mouse
(broken line) and a Rosa26 positive control (solid line) and (B) a
HS21/5 vav-gal transgenic mouse. Cells were analyzed for
-gal activity (represented on the X-axis by mean fluorescence
units). The proportion of expressing cells was determined after setting
the window to yield less than 5% -gal+ for a negative
control mouse run in parallel.
|
|
vav-gal activity was highest in the thymus.
To determine which hematopoietic organs expressed the transgenes, we
performed FACS-gal analysis on cell suspensions from tissues of mice.
With each of the three active vav-gal constructs, the
expression pattern and intensity of fluorescence were similar, irrespective of transgene copy number. The thymus displayed by far the
highest proportion of expressing cells (Table 2). The majority of thymocytes, and in occasional mice nearly the entire population, was -gal+. Thus, all three transgenes were
very active in immature T lymphocytes. In contrast only approximately
one third of all leukocytes in lymph node and roughly 20% of those in
bone marrow and spleen were positive, as in the peripheral blood.
Although the construct containing a single intron site (HS21/5) yielded
a significantly lower proportion of -gal+ cells in
thymus and lymph node than those having two intron sites (P < .05), no significant differences between the three constructs appeared
in the bone marrow or spleen.
View this table:
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|
Table 2.
Cells Expressing -gal Activity in Hematopoietic
Tissues of vav-gal Transgenic Mice Bearing
Three Different Constructs
|
|
Expression in bone marrow was most prominent in early B-lineage
cells.
The proportion of -gal+ cells within particular
hematopoietic lineages was determined by concomitant flow cytometric
analysis for lineage-specific surface markers and -gal expression
(Table 2), as shown for bone marrow in Fig 8. In this
site of B lymphopoiesis, a substantial portion of the B-lymphoid
(B220+) cells displayed enzymic activity. Hence, the
transgenes were active in the B as well as the T lineage. In sharp
contrast, few if any cells bearing Mac-1, Gr-1, or TER-119 were
-gal+, indicating that the transgenes were silent in the
monocytic, granulocytic, and erythroid lineages. That conclusion was
reinforced by staining sorted Mac-1+, Gr-1+, or
TER-119+ cells for -gal activity: none contained a
-gal+ subpopulation.
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| Fig 8.
Flow cytometric analysis of -gal expression in
transgenic bone marrow populations. The lineage of
-gal+ cells was determined by simultaneous analysis
for the indicated lineage-specific surface markers (see the Materials
and Methods): (A) Mac1, a myeloid cell marker; (B) the erythroid marker
Ter119; (C) B220, a B-lymphoid marker; and (D) IgM and (E) IgD, markers of mature B lymphocytes. Less than 1% of lineage marker-positive cells
in the negative control bone marrow were -gal+.
|
|
Unexpectedly, we found that the frequency of transgene-expressing cells
declined with B-cell maturation. In B-cell ontogeny, expression of B220
precedes surface IgM, whereas IgD is acquired only later in the spleen;
hence, the marrow cells bearing IgD represent older recirculating B
cells. Whereas approximately one half to two thirds of all
B220+ cells in the bone marrow expressed the transgene, the
proportion among those also bearing IgM was lower (approximately
one-third, albeit higher in HS21/5 vav-gal mice), and few if
any IgD+ bone marrow were -gal+ (Fig 8 and
Table 2). Thus, the activity of the transgene was mosaic (variegated)
within this lineage, and the frequency of positive cells fell with
maturation to background levels in the recirculating B-cell population.
Expression in peripheral hematopoietic tissues was restricted to T
cells.
In accordance with the bone marrow results, very few, if any
-gal+ B lymphocytes could be found in the spleen or
lymph nodes (Table 2), indicating that few expressing B cells leave the
bone marrow. However, activity in the T-cell lineage was retained in
the periphery (Table 2). Interestingly, in spleen and lymph node, a
significant proportion of both the CD4+ helper T-cell
population and the CD8+ cytotoxic T cells showed
expression, although the proportion of -gal+ cells in
each subset was somewhat lower than that in total thymocytes. Hence,
expression also declines during T-cell maturation, albeit much less
rapidly than in the B lineage. The variegated expression pattern in the
mature T cells clearly was not related to differentiation into the two
functional subsets but seemed instead to reflect random silencing in
both the CD4+ and CD8+ populations.
Only a minor population of fetal liver cells express
vav-gal.
The endogenous vav gene has been shown by in situ hybridization
to be expressed strongly between E12.5 and E17.5 in fetal liver, the
major site of fetal erythropoiesis.2,10 To determine whether vav-gal was also expressed at this stage, livers
from HS21/5 fetuses at E14.5 were subjected to FACS-gal analysis (data not shown). The vast majority of the cells were
-gal, confirming that the transgene was silent in
the erythroid lineage. However, approximately 1% of total fetal liver
cells were positive. Because these cells did not bear the Ter119, B220,
Thy1, Mac1, or Gr1 markers, they may represent very early B-lymphoid
precursors.
Expression in peripheral blood leukocytes decreased as animals aged.
While investigating transgene expression in peripheral blood, we
noticed a decrease in the proportion of -gal+ cells in
older animals. Figure 9 shows for one transgenic line the percentages of -gal+ cells for mice of three
age groups. Despite the considerable scatter in the individual values,
the average values clearly decreased and t-tests indicated that
the differences between all three groups were statistically
significant, with that between the youngest and oldest groups being
highly significant (P < .000005). Another line showed a
similar decrease, and a third line showed a less marked decline. Thus,
the transgenes in three independent lines appeared to be silenced in a
stochastic fashion as the mice aged.
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| Fig 9.
Decline with mouse age in the average percentage of
-gal+ cells in peripheral blood leukocytes. Values for
individual mice of a single HS21/5 transgenic line in three age groups
are shown, as well as the mean and 2 SEM above and below the mean.
|
|
| |
DISCUSSION |
The findings reported here lay the groundwork for clarifying how the
unique compartment-wide expression of the vav gene is achieved.
A screen of 60 kb of chromatin encompassing the murine gene, which was
found to comprise at least 14 exons, showed five major DNase I HS
sites, all lying in its 5 moiety (Fig 1). HS1 seems to
correspond to the proximal promoter, whereas HS2 and HS3 lie further
upstream and HS4 and HS5 reside in the first intron. These sites were
found in hematopoietic cells of three distinct lineages (myeloid,
erythroid, and lymphoid), but none was present in fibroblasts. Thus,
these five sites, which are spread over 14 kb of the vav locus,
are likely to represent the major regulatory sites for control of
vav gene expression.
Activity of the vav promoter requires integration into
chromatin.
In good agreement with similar studies in human cell
lines,30 RNase protection experiments showed a cluster of
apparent vav transcription start sites, located approximately
100 bp 5 of the initiating AUG (Fig 3A). Because both the mouse
and human promoter lack a TATA box, heterogeneous starts are expected.
The presence of the same pattern of presumptive start sites in mRNA from murine cell lines representative of several hematopoietic cell
types makes it very likely that a single promoter is operative in the
various lineages.
High resolution mapping of HS1 resolved it into HS1a, a strong site
within 0.15 kb of the transcription start site and the weaker HS1b,
around 0.5 kb upstream. Potential binding sites for transcription
factors expressed in hematopoietic cells were evident within the 1-kb
sequenced promoter region, but as the sequence of few of these sites is
conserved in the known corresponding human (0.4 kb)
sequence,30 their relevance is problematic. Thus, novel
transcription factors may be required to drive vav expression, and the localization of the HS1a and HS1b sites should aid in their
identification.
The
activity of vav reporter constructs differed markedly between
stably and transiently transfected cell lines. In stably transfected
FDC-P1 cells (Fig 4), vav promoter fragments spanning HS1 plus
HS2, or even HS1 alone, were nearly as active as SR, among the
strongest of promoters for hematopoietic cells.22 In
contrast, transient transfection of these constructs yielded no -gal
activity, and even with the more sensitive luciferase reporter the
promoter activity in hematopoietic cells was minimal (Table 1). A major
difference is that stable transfection entails integration into
chromatin, whereas transiently transfected DNA remains episomal. Thus,
the vav HS sites may function only within the context of
chromatin. Although few studies have made this direct comparison, a
clear precedent is provided by the CD34 promoter, which was active only
when stably introduced into cell lines.33 It may be
relevant that CD34 is expressed in primitive hematopoietic cells of
multiple lineages. Hence, the vav and CD34 promoters may be
representative of a novel type of promoter that functions only when
integrated into chromatin.
Elements required for expression in vivo.
As has frequently been observed, the requirements for expression in
vivo proved much more stringent than in transfected cell lines. Whereas
the promoter region alone (HS1) sufficed for high activity in the
FDC-P1 myeloid cell line (Fig 4), expression in the blood leukocytes of
transgenic mice also required HS2 (ie, 2.3 kb of upstream DNA) as well
as the 3.7-kb intron fragment spanning HS5. When intron site HS4 was
included as well, more than 70% of primary transgenic animals had
-gal+ cells in their blood (Fig 5). Thus, establishing a
domain of transcriptional competence in vivo appears to require HS1,
HS2, and HS5, whereas HS4 seems to have a facultative role. Curiously, the distal upstream site HS3 appeared to have a predominantly negative
influence, because its presence in transgenic constructs reduced
expression in FDC-P1 cells (Fig 4) as well as the frequency of
transgenic lines that yielded expression (Fig 5).
No evidence was found associating particular HS sites with expression
in one or other hematopoietic lineage. The same pattern of sites was
evident in different lineages and the addition of HS4, or HS4 plus HS3,
to the minimal active transgenic construct, HS21/5 vav-gal,
did not produce expression in the nonlymphoid lineages (Table 2). Thus,
the five HS sites may act together as a unit to establish or maintain
transcriptional competence rather than as separate enhancers for the
different hematopoietic lineages.
It remains a puzzle that vav-gal constructs stably
introduced into a myeloid cell line were highly expressed (Fig 4) but inert within myeloid cells in vivo (Table 2). It seems relevant that
the transfected cell lines were isolated by coexpression of a
selectable marker (a neomycin gene driven by an SV40 promoter). Because constructs cointroduced into a cell line commonly integrate into the same locus, the selection for G418 resistance may have imposed
selection for active chromatin domains. Hence, the vav-gal constructs may be less effective at opening chromatin in myeloid cells,
even though they are very active in those cells within the appropriate
chromatin environment.
Variegated expression of vav-gal transgenes.
Only a proportion of lymphocytes of a given type were
-gal+. The variegated expression pattern held for
multiple independent vav-gal transgenic strains bearing up
to all five HS sites (Table 2). Selection against -gal activity
cannot account for this phenomenon, because this enzymic activity is
ubiquitous even in older Rosa26 mice. In both lymphoid lineages the
frequency of -gal+ cells declined with maturation. The
B-lineage decline was dramatic, with very few -gal+ B
cells persisting outside the bone marrow, their tissue of origin. Because the decline in the T lineage was comparable for the
CD4+ and CD8+ subsets, it was unrelated to
mature T-cell function.
The proportion of -gal+ leukocytes also decreased with
age of the mice (Fig 9). Because all peripheral expressing cells seemed to be T cells, the decrease in the blood probably represented an
extension of the decrease in frequency observed between thymic and
peripheral T cells (Table 2). Because the activity in the residual
expressing cells did not decrease notably, the decline appeared to
reflect stochastic shut off in a proportion of the cells. All of the
vav-gal transgenes seemed subject to this random silencing.
For a given transgenic line, the founder and progeny of different
generations yielded similar results, suggesting that the silencing was
recapitulated in each generation.
In many past transgenic studies, low transgene expression has been
assumed to reflect a low level in each cell rather than absence of
expression in the vast majority, but as reviewed
recently18,34 several studies with reporters allowing
cell-by-cell analysis have shown variegated expression. Examples
include variegation in erythrocytes with well-studied globin regulatory
elements35-38 and in lymphocytes with the CD2
LCR.39 These observations and our findings favor the view
that the cluster of positive regulatory elements associated with a gene
acts in an all-or-none fashion to determine whether a given cell
activates transcription of the gene.17 The maturation- and
age-related decrease in expression observed here and in certain other
recent studies38,40 also emphasizes that the long-term
maintenance of transcriptional competence is likely to require elements
in addition to those needed to establish a competent state.
In principle, variegated expression of a transgene might be related to
the position of insertion, the copy number, an incomplete set of
regulatory elements, or the presence of sequences not from the
endogenous locus, such as the reporter.
Position of insertion undoubtedly strongly affects transgene
expression, and variegated transgene expression is often likened to
position effect variegation in Drosophila,18 where
an active gene shifted near heterochromatin is stochastically and
clonally inactivated. Insertion into heterochromatin appeared to
promote variegation with CD2 and -globin
transgenes.36,39 However, that mechanism is unlikely to
account for the variegation we observed, because it occurred in all 18 independent expressing transgenic founders and lines.
Variegation with Drosophila transgenes has been ascribed to
heterochromatinization promoted by the transgene array itself, with
higher copy numbers increasing the rate of silencing.41 Whereas some high-copy mouse transgenes show reduced
expression,18,41 extensive arrays are unlikely to account
for our observations, because all the expressing founder mice we
examined had low copy numbers (<4 per haploid genome) and all showed
a comparable extent of variegation. The copy number was also not a
major factor in the variegation observed with an -globin
promoter.38
Compromising effects of the -gal reporter.
The widespread use of -gal (lacZ) transgenes to
monitor promoter action in vivo, particularly in the embryo, has
included successful studies with several hematopoietic lineages,
including megakaryocytes,42 erythrocytes,43 and
myeloid cells.44,45 Nevertheless, it is of note that many
reports of poor or variegated expression involve -gal
transgenes.17,20,37,40 Indeed, -gal has failed
as a reporter with a number of regulatory elements that work well with
other reporters.20,46 Even with the potent -globin LCR
micro-locus and the entire -globin gene, a
-gal reporter compromised position-independent expression
and provoked variegation.37
In view of these concerns, we have recently engineered vav
promoter constructs with a eukaryotic reporter. Preliminary results indicate that these transgenes are expressed at readily detectable levels in most founder mice, often in most of the blood cells, including those of nonlymphoid lineages. Hence, in accord with the
results in the FDC-P1 cells (Fig 4), the vav regions defined here are sufficient to drive expression in the myeloid as well as
lymphoid lineages and may comprise all of its critical control elements. Detailed analysis of such mice should allow us to determine whether they are sufficient to establish and maintain pan-hematopoietic expression.
| |
FOOTNOTES |
Submitted June 24, 1997;
accepted September 8, 1997.
Supported by the National Health and Medical Research Council
(Canberra) and the US National Cancer Institute (CA12421).
Gen Bank accession number AF031651 Bank It 148623.
Address reprint requests to Jerry M. Adams, PhD, The Walter and Eliza
Hall Institute of Medical Research, Post Office, Royal Melbourne
Hospital, Victoria 3050, Australia.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are grateful to Drs Frank Battye and Andreas Strasser for
advice on flow cytometry and to Dr Strasser for antibodies, to Dr
William Kerr for Rosa26 mice, to Drs Peter Cockerill and Selina Raguz
for help with HS analysis, to Jodie De Winter and Kim Patane for animal
husbandry, to Margaret Santamaria for technical assistance, and to
Jeanette Tyers for preparation of the manuscript.
| |
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Abstract
Introduction
Abstract