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*
Division of Biology, California Institute of Technology, Pasadena, CA 91125; and
Stowers Institute for Medical Research, Kansas City, MO 64110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the regulation of IL-2 expression in response to stimulation has been studied in great detail in transient transfection assays (reviewed in Refs. 6, 7), we know very little about the mechanisms regulating IL-2 expression in a developmental context. Available cell lines are wholly inadequate to address this question by in vitro transfection studies because they do not represent normal developmental states. However, the well-characterized promoter/enhancer of the IL-2 gene is notoriously poor at allowing expression of reporter genes in transgenic mice. Use of the known promoter/enhancer (600 bp) to drive transgene expression resulted in only 1 of 17 and 2 of 26 founder mice expressing the transgene properly (8, 9). Use of a more extended 5' regulatory region (up to 2700 bp), including all sequences tested for function by transfection (10), resulted in expression in only two of five founders (11). In several of the transgenic founders that express the transgenes, there is evidence for insertional appropriation of host regulatory sequences: i.e., the transgene gives ectopic expression, causes lethality when homozygous, or both (8, 9, 11) (our unpublished results). In this context of variable expression, it has been impossible until now to attempt mapping sequence elements that restrict IL-2 expression to particular cell types.
Such a pattern of poor expression is characteristic of transgene
constructs that are known to contain incomplete positive regulatory
regions (12, 13, 14). The regulatory regions of these
transgenes are missing sequence elements that can cooperate with
proximal promoters/enhancers by opening the local chromatin and
maintaining a transcriptionally competent domain. Some such elements
are designated locus control regions
(LCRs),4 genetic
regulatory elements that confer tissue-specific and physiological
levels of transcription on linked genes irrespective of integration
site (15). It is only recently that the potential roles of
elements affecting chromatin structure have been addressed for
regulation of any cytokine genes (reviewed in Ref. 16).
Distal regulatory elements have been identified by DNase
hypersensitivity for the IL-3/GM-CSF locus (17, 18), and differential chromatin remodeling has been described
for IFN-
, IL-4, and IL-13 in Th1 vs
Th2 cells (19). In addition, histone acetylation has been
shown to be important for IL-4 inducibility in mature T cells
(20). The addition of a heterologous (CD2) LCR to the
2.7-kb IL-2 promoter/enhancer region was recently shown to yield a high
incidence of transgene expression, in 6 of 7 founders
(21), which suggests that an LCR-like element is indeed
missing from the IL-2 promoter regions tested to date. Constructs with
exogenous LCRs, however, cannot be assumed to recapitulate all aspects
of normal developmental regulation that may be mediated by different
protein-DNA interactions. Therefore, in this study, we sought molecular
and functional evidence to locate any natural LCR-like elements that
control the normal developmental regulation of the IL-2
gene.
Regions of DNA-protein interactions are often hypersensitive to DNase I digestion, and this characteristic has proven to be useful in detecting new and distant transcriptional regulatory sites. An early study using this approach found evidence for hypersensitive (HS) sites beyond the minimal enhancer of the human IL-2 gene, although they were not precisely mapped (22). We therefore used this method to find a series of new HS sites several kilobases upstream of the known IL-2 promoter/enhancer. The inclusion of these newly identified distal HS sites in green fluorescent protein (GFP) reporter gene constructs, along with the known IL-2 promoter/enhancer region, resulted in dramatically improved and consistent expression of GFP in independent transgenic founders over a wide range of transgene copy numbers. Expression was inducible and cell type specific in all expressing lines. This is the first study to identify a distal region of the IL-2 locus containing cis-acting elements that are sufficient for developmentally regulated expression of a transgene independent of the site of chromosomal insertion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Splenocytes and purified T cells were cultured in DMEM
supplemented with 10% FBS, 2 mM L-glutamine, 5 x
10-5 M 2-ME, 100 U/ml penicillin, and 100
µg/ml streptomycin (Life Technologies, Gaithersburg, MD) at 37°C in
6% CO2. For stimulations via the TCR, 96-well
flat-bottom plates (Corning, Corning, NY) were coated with 10 µg/ml
purified 
CD3 and CD28 (PharMingen, San Diego, CA) in PBS for
2 h at 37°C and washed with medium before the addition of
106 cells. For chemical stimulations,
106 cells were incubated with 175 nM A23187 and
10 ng/ml PMA, both from Sigma (St. Louis, MO), and prepared as stock
solutions in DMSO.
DNase I-hypersensitive assays
For purified T cells, C57BL/6 (B6) splenocytes were stained with biotin-conjugated anti-B220 Ab (PharMingen), incubated with streptavidin-conjugated MACs Microbeads, and passed over a VS+ MACS magnetic separation column (Miltenyi Biotec, Auburn, CA) to remove B cells. Purified T cells were then subjected to DNase I treatment either immediately or after 6 h of stimulation with PMA and A23187. Syngeneic melanoma MCA102 cells were used for a non-T cell line (kindly provided by Michael Nishimura, University of Chicago, Chicago, IL).
DNase I HS assays were conducted, as described previously
(23), using aliquots of 100 x
106 cells permeabilized with lysolecithin for
DNase digestion and DNA extraction. Twenty micrograms of each DNA
sample were digested with XbaI, electrophoresed in agarose
gels, blotted, and hybridized, as described previously. The three
probes used were as follows (shown in Fig. 2
): the distal upstream
region, the 842-bp EcoRI to XbaI fragment, the
promoter region, the 784-bp AccI to XbaI fragment
including part of the second intron and third exon, and for the
downstream region, the 727-bp XbaI to EcoRI
fragment in the third intron.
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DNA from the IL-2 upstream sequence was cloned from a
C57BL/6 
genomic library (Stratagene, La Jolla, CA) using a probe
specific for the proximal promoter region of the IL-2 gene.
Upstream sequences, from the XhoI site at -8.4 kb to the
PstI site at +45 bp, and from the HindIII site at
-2kb to the PstI site, were subcloned into an enhanced GFP
vector plasmid (EGFP-1) (Clontech Laboratories, Palo Alto, CA). In
addition, the SV40 poly(A) site in the vector was deleted from
NotI to AflII, and replaced with the 3'
splice and poly(A) site from human 
-globin, as the BamHI
to EcoRI fragment from plasmid pES4 (kindly provided by
Stephen Hedrick, University of California, San Diego, CA).
The 8.4-kb upstream IL-2 region used in this study was sequenced by subcloning progressive unidirectional deletions using the Erase-A-Base system (Promega, Madison, WI). Dideoxy sequencing on the subclones was then conducted using Applied Biosystems Prism Dye Terminator Cycle Sequencing Ready Reaction and an Applied Biosystems Prism 9600 Sequence Detector System kit (PE Applied Biosystems, Foster City, CA). These sequence data have been submitted to the GenBank database under accession number AF290391.
Generation and characterization of transgenic mice
Transgenic mice were generated in the Caltech Transgenic Animal Facility (Pasadena, CA) by pronuclear injection of (B6 x DBA/2)F2 zygotes using standard methods, and transgene positive mice were identified by PCR analysis of tail DNA. PCR primers in the IL-2 proximal promoter (IL2-1F: 5'-CATCCTTAGATGCAACCCTTCC-3') and the GFP coding sequence (GFP-1R: 5'-GCTGAACTTGTGGCCGTTTAC-3') were used, amplifying a 830-bp product in transgene-positive mice. PCR conditions were as follows: 94°C, 5 min, then 35 cycles of 93°C, 30 s; 62°C, 15 s; 72°C, 45 s, followed by a final 5 min at 72°C, using an MJ PTC-200 DNA Engine Thermal Cycler (MJ Research, Watertown, MA). Founder mice were propagated by serial backcrosses to C57BL/6 mice. All conventional transgenic mice used for this study were generated and maintained in the Caltech Transgenic and Knockout Core Facility under specific pathogen-free conditions. Transgene copy number was determined by Southern blot hybridization of an IL-2 promoter region probe to genomic DNA cleaved with XbaI and EcoRI. The 600-bp probe was generated by amplifying a PCR product using the tail-typing primers, which was then digested with PstI to remove the GFP sequence by gel purification. The intensities of the 3.7-kb transgenic and the 2.4-kb endogenous (two-copy) IL-2 bands were determined by use of a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software.
To obtain transgenic SCID mice, transgene-positive male mice from two founder lines (lines 12 and 4) were crossed with C57BL/6-scid/scid (B6-SCID) mice from the Rothenberg laboratory breeding colony at Caltech (24). To prevent infection of the immunodeficient animals, breeders and their pups were kept on prophylactic antibiotic (Baytril) treatment and transferred to sterile microisolator cages. Heterozygous offspring were typed for the transgene by PCR analysis and then backcrossed to B6-SCID mice. Homozygous scid/scid pups were selected by flow cytometry of peripheral blood samples to determine the absence of peripheral lymphocytes, and transgene-positive animals were identified by PCR analysis of tail DNA. The transgene-positive SCID lines were then maintained on sterile food, water, and bedding, in sterile microisolator cages in ventilated racks.
Flow cytometric staining and cell sorting
Single-cell suspensions of 106
RBC-depleted blood, splenocytes, or thymocytes were stained on ice in
96-well U-bottom plates. Abs were diluted in Coffman’s balanced salt
solution (CBSS) buffer (1.25x HBSS without phenol red) supplemented
with 0.25% BSA (CBSS/BSA) (Pentax Fraction V; Miles, Kankakee, IL)
with or without 0.1% sodium azide, as previously described
(24). Cells were preincubated in Fc block (PharMingen),
followed by staining with specific Abs and washing with CBSS/BSA. The
following staining reagents were used: CD3-APC, TCR-CyChrome,
CD44-PE, CD62L-PE, CD69-PE, NK1.1-PE, Thy-1-PE, CD8-APC, HSA-biotin,
streptavidin-APC (PharMingen), CD4-PE (Becton Dickinson, Mountain View,
CA), and Sca-1-TC (Caltag Laboratories, South San Francisco, CA). FACS
analysis was performed using a FACSCalibur (Becton Dickinson). Cells
were sorted using a Coulter Elite (Coulter, Hialeah, FL).
RNA purification and real-time quantitative RT-PCR
Total RNA was extracted using RNAzol (Leedo Medical, Houston,
TX) in accordance with the manufacturer’s instructions. Stimulated
cells were incubated for 6 h with plate-bound CD3/CD28. RNA
from 2 x 106 cells was treated with
RNase-free DNase to remove residual genomic DNA. First-strand cDNA
synthesis reactions were then conducted using Superscript reverse
transcriptase II (Life Technologies) and 250 ng random hexamers
(Pharmacia, Uppsala, Sweden) following standard protocols.
Real-time fluorescent PCR analysis (25) was conducted
using the Applied Biosystems Prism 7700 Sequence Detection System (PE
Applied Biosystems). Taqman probes and primers for murine GAPDH (VIC
labeled) and IL-2 (FAM labeled) were purchased as sets from PE Applied
Biosystems, and a GFP-specific oligo probe,
6FAM-GCTTTACTTGTACAGCTCGTCCATGCCGA-TAMRA, was custom synthesized by
the same company. Primers used to specifically amplify the transgenic
message crossed the intron between GFP
(5'-CACATGGTCCTGCTGGAGTTC-3') and the 3' -globin sequences
(5'-CAGCACACAGACCAGCACGTT-3'). Thirty-microliter reactions were
conducted in Taqman Universal PCR buffer (1x Taqman buffer A; 5 mM
MgCl2; 200 µM each of dATP, dTTP, and dGTP; 400
µM dUTP; 8% glycerol; 200 µM primers; 100 µM fluorescent-labeled
probe; 0.01 U/µl AmpErase UNG; 0.05 U/µl AmpliTaq Gold) using the
following thermocycling conditions: 50°C, 2 min; 95°C, 10 min; then
40 cycles of 95°C, 15 s and 60°C, 1 min. The cycle thresholds
(CT) were determined by measurement of the amount
of fluorescent dye released after PCR amplification at each cycle using
cDNA samples and specific primers and probes for GFP, IL-2, and GAPDH
in separate duplicate tubes and selecting a threshold in the linear
range of the amplification. Control GAPDH CTs
were subtracted from GFP and IL-2 CTs for each
sample (=
CT). Each value was then adjusted by
subtracting the CT for a reference sample
within each experiment (the transgene-negative control for IL-2 and
transgenic line 4 for GFP) (=CT). Relative
amounts of mRNA were calculated as
2-CT.
To convert GFP and IL-2 expression to an absolute scale, standard
curves of control cDNA plasmids were analyzed in parallel in some
experiments. The IL-2 cDNA standard was pGmIL2 (26). For
the transgene, a PCR product was amplified from cDNA of stimulated
8kbIL2p-GFP-175 splenocytes, using a GFP-specific primer incorporating
an XbaI site for cloning
(5'-AGACTCTAGACCACATGAAGCAGCACGACT-3'), and a reverse primer specific
for exon 3 of the -globin 3' end (5'-TGATAGGCAGCCTGCACTGGT-3'). The
product was then digested with XbaI and EcoRI to
generate a 562-bp product that was cloned into pBlueScript
(Stratagene).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because protein-DNA contacts in the known 2700 bp of
IL-2 5'-flanking DNA are insufficient to mediate full
LCR-like activity, we sought evidence for other regions of the gene
that might confer position-independent expression on transgenes. To
identify distal sites of potential protein-DNA interactions in the
IL-2 locus, we performed DNase I-hypersensitivity assays on
unstimulated and stimulated purified mouse splenic T cells.
Unstimulated T cells displayed a prominent HS site at 
4.5 kb
upstream of the IL-2 start site, with a series of five weaker HS sites
seen between -3 and -4.5 kb (Fig. 1A, left panel).
The same HS sites were also found in unstimulated EL4 thymoma cells
that express IL-2 only after stimulation (data not shown). These sites
appeared to be cell type specific; the region from -3 to -4.5 kb was
not hypersensitive in a syngeneic melanoma cell line (Fig. 1A, right panel). These six T cell-specific HS
sites are of particular interest as they indicate that the region of
the IL-2 gene upstream of -2 kb has an accessible chromatin structure
in resting T cells and is bound by putative transcription factors that
may play a role in maintaining the locus in an open but
transcriptionally silent state.
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A, middle panel). Four new HS sites appeared
between -2.5 and -3 kb. At the same time, the six HS sites found in
the unstimulated cells were still present, although the -4.5-kb site
became less prominent. Three new, more distal sites also appeared, one
strong band at approximately -8 kb, and two others at -7 and -10 kb.
Again, a similar pattern of HS sites was also found in stimulated EL4
cells (data not shown).
In sharp contrast, no HS sites were observed in the region from the
third intron to 5 kb 3' of the IL-2 gene in unstimulated or
stimulated purified T cells (Fig. 1B) or in EL4 cells (data
not shown). HS sites in the promoter-proximal region that have been
reported in human and mouse T cell lines (4, 22, 27) were
also observed in purified T cells but not in non-T cells, and no new
sites were revealed (Fig. 1C). A summary of IL-2
locus HS sites found in resting and activated T cells is shown in Fig. 2. Overall, these results show that the
distal upstream region of the IL-2 gene is maintained in an open
chromatin structure in resting peripheral T cells, but not in non-T
cells, and that this region undergoes rapid and extensive remodeling
upon activation. The presence of at least 13 new distal HS sites
upstream of the IL-2 gene suggests that this region has many
previously uncharacterized DNA-protein interactions that may play in
vivo roles in mediating IL-2 locus accessibility or
providing tissue-specific enhancer and/or silencer functions.
Construction and in vitro testing of the IL2p-GFP transgenes utilizing 8.4 kb vs 2 kb of upstream IL-2 sequence
To characterize and test this upstream region for function in
vitro and in vivo, 5'-flanking sequence from the IL-2 gene
was cloned from a C57BL/6 genomic DNA library and sequenced. Two
fragments, 2 and 8.4 kb, were subcloned into a GFP-expressing vector,
to give constructs designated 2kbIL2p-GFP and 8kbIL2p-GFP,
respectively. Both constructs include the proximal 2 kb of 5'-flanking
sequence (to the PstI site at +45) that was found to drive
optimal inducible expression in transiently transfected cells
(10). The 8kbIL2p-GFP construct also includes an
additional 6.4 kb of flanking DNA containing all of the novel HS sites
found in resting T cells, as well as most, but not all, of the
activation-induced HS sites (Fig. 2). The SV40 poly(A) in the original
plasmid was replaced with the human -globin 3'-splice site and
poly(A) tail, which reproducibly improved the inducible expression of
GFP by 2–2.5x after transient transfections in EL4 cells (data not
shown). The 2- and 8.4-kb GFP constructs gave similar GFP expression
levels (within 20%) in these assays (data not shown), indicating that
the distal sites included in 8kbIL2p-GFP do not mediate conventional
enhancer activity in a nondevelopmental, nonchromatin context.
The 8kbIL2p-GFP-transgenic mouse lines express the transgene more consistently than 2kbIL2p-GFP lines
The two constructs, 2kbIL2p-GFP and 8kbIL2p-GFP, were injected into mouse embryo pronuclei, and transgenic founders were produced. Eleven independent 8kbIL2p-GFP and twelve independent 2kbIL2p-GFP founders were obtained. Founders were bred to C57BL/6 mice and more detailed analyses performed on cells from transgene-positive founders and/or their progeny. Transgene copy numbers ranged from 1 to >40 for both constructs, as determined by Southern blot analysis.
Transgene-positive mice were screened for activation-induced GFP
expression among CD3+ cells. GFP expression was
readily detected in CD3-positive cells from both kinds of founders, as
shown in Fig. 3A. Although
spontaneous GFP expression was observed in some
CD3+ cells (see below), the percentage and mean
fluorescence of the GFP+ cells increased
dramatically with PMA/A23187 stimulation. Some
GFP+ CD3- cells were
observed in stimulated cells from all lines of both 2- and 8-kb
transgenics which are capable of expressing GFP, and there is no
consistent difference between the two constructs in the percentages of
these populations. These cells do not appear to be B cells or
macrophages (data not shown) and may represent T cells that have
down-regulated the TCR-CD3 complex. Ectopic transgene expression was
not observed with either construct, as major nonlymphoid organs of the
transgenic mice do not express GFP.
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B). The 8kbIL2p-GFP lines expressed GFP more consistently
than any previously reported IL-2 promoter transgenics, with
10 of 11 (91%) lines expressing GFP in stimulated
CD3+ cells (>3% positive under the conditions
used in Fig. 3A). The only 8kbIL2p-GFP line that failed to
express GFP carries only one copy of the transgene. By contrast, only 4
of 12 (33%) of the 2kbIL2p-GFP lines expressed GFP at that level; the
remainder expressed the transgene very poorly or failed to express it
at all over a wide range of copy numbers (Fig. 3B,
left panels). The characteristics of the expression patterns
seen in different 8kbIL2p-GFP lines were qualitatively very consistent
between lines and among the progeny of most individual lines (see
below, and M. A. Yui and E. V. Rothenberg, in preparation).
The expression in these lines seemed unlikely to be a result of
insertional mutagenesis, since there was no evidence of morbidity in
transgene-positive animals. These results show that the additional 6.4
kb of IL-2 gene upstream sequence in the 8kbIL2p-GFP transgene sharply
increases the probability that an individual cell will open and express
the locus, independent of integration site.
GFP induction in purified GFP- T cells
Transgenic TCR+ splenocytes that are
initially GFP negative synthesize GFP de novo in response both to TCR
ligation and to PMA/A23187 stimulation, as shown for two independent
8kbIL2p-GFP transgenic lines in Fig. 4. A
subset of TCR+ GFP-
cells was seen to express GFP by 16 h after stimulation, and the
percentage of GFP+ cells increased further by
44 h. GFP was readily induced using either plate-bound
anti-CD3/anti-CD28 or PMA/A23187 stimulation, although a higher
percentage of GFP+ cells was always observed in
PMA/A2387-stimulated cells, with the mean fluorescence reaching peak
levels at earlier time points. These results show that, as with
endogenous IL-2, a subset of T cells that are initially GFP negative
respond rapidly to specific activating signals by producing high levels
of GFP.
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To assess whether the 8kbIL2p-GFP transgene includes regulatory
elements allowing transgene expression at a level comparable with that
of the endogenous IL-2 genes, GFP and IL-2 mRNA were
quantitated using real time fluorescent RT-PCR (25) in
samples from TCR/CD28-stimulated cells. RNA was prepared from
splenocytes from nontransgenic and transgenic mice, with or without 6-h
stimulation with anti-CD3/anti-CD28, and cDNA from these samples was
analyzed in parallel with cDNA plasmid standards. Fig. 5A shows that with this assay,
the threshold cycle number (CT) (the PCR cycle at
which the product of each sample crossed a detection threshold) was
linear with the log of sample dilution over five orders of magnitude,
for three independent sets of plasmid standard dilutions. These
standards were used to calculate absolute levels of IL-2 and GFP mRNA
in the cDNA from transgenic and control mice. As shown in Fig. 5B, the lower copy number transgenic lines (three to four
copies) could express GFP mRNA at similar levels to the two-copy
endogenous IL-2 genes, with ratios of about 1. Higher
GFP:IL-2 ratios were seen at higher transgene copy numbers. This
finding suggests that the 8.4 kb of upstream sequence in the
8kbIL2p-GFP transgene encompasses most, if not all, of the positive
regulatory elements required for inducible expression, in addition to
conferring positional independence.
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C). Thus, the elements mapping
between -2 and -8.4 kb affect the likelihood that transgenes will be
expressed more than they affect the level of expression once
induced. Effect of copy number on expression of 8kbIL2p-GFP transgenes in response to stimulation
With the exception of line 175 (23 copies), the ratio of GFP:IL-2
expression showed only a modest increase over a 10-fold increase in
copy number (Fig. 5B). Such a nonlinear response to copy
number could be a result of competition for a limiting pool of
trans-acting positive regulatory factors. However, GFP
expression did not occur at the expense of endogenous IL-2 expression,
even at high transgene copy numbers (Fig. 5C, left
panel). In agreement with this result, we have found that at both
low and high copy numbers, IL-2 and GFP protein can both be detected in
individual stimulated T cells (data not shown).
GFP mRNA expression in the 8kbIL2p-GFP lines (Fig. 5C,
right panel) generally paralleled protein expression (Fig. 3B), reflecting a composite of the percentage of T cells
that becomes GFP+ and the mean fluorescence
intensities of the GFP+ cells. The two lines with
the lowest transgene copy numbers also displayed the lowest mean
fluorescence intensities among GFP+ T cells (Fig. 3B, right panel). However, the percentage of
cells expressing GFP did not show a consistent increase as a function
of copy number (Fig. 3B, left panel). This
suggests that the number of transgene copies (per integrated array) has
only limited effect on the likelihood that these genes will be
activated transcriptionally in response to a particular episode of
stimulation.
Spontaneous GFP+ cells have an effector/memory cell phenotype
Although expression of the GFP transgenes is highly responsive to
activation in the population as a whole, there is a reproducible
background of transgene expression, at both the RNA and protein levels,
before stimulation (Figs. 3B and 5C). This
background expression is seen in all the expressing transgenic lines,
regardless of whether they are derived with the 2-kb or the 8.4-kb
promoter construct. Fig. 5C shows that background GFP RNA
expression can be seen even in populations of unstimulated cells that
express no detectable IL-2 RNA (see data for lines 4, 8, and 214, one
experiment each, and for lines 186, 177, and 17). The spontaneous
transgene expression, however, is associated with the same cell types
that express IL-2 in response to induction. As shown in Fig. 3A (and data not shown), spontaneously
GFP+ cells are overwhelmingly concentrated in the
CD3+ population, in both
CD4+ and CD8+ subsets (see
below).
To determine whether this spontaneous expression of GFP is dependent on
prior immunological activation, freshly isolated transgenic splenocytes
were analyzed for surface phenotype markers that distinguish naive from
previously activated cells. All of the GFP+ cells
in the CD4+ population were found in the minority
subset that is CD44high (Fig. 6), and predominantly
CD62Llow (data not shown), in every 8kb- and
2kbIL2p-GFP line tested. This phenotype is a hallmark of previously
activated effector/memory cells. The majority of the
GFP+ cells were found to be small, resting cells,
and most were also CD69low, indicating that only
a minority of GFP+ cells were recently activated.
The cells expressing GFP spontaneously in vivo can still respond to TCR
stimulation in vitro by turning on even higher levels of GFP expression
(data not shown), which is consistent with a memory cell phenotype.
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and NKT cells, both of which are known to be
activatible by endogenous Ags (data not shown and M.A.Yui and
E.V.Rothenberg, in preparation). Expression of the 8kbIL2p-GFP transgene in a developmental context: expression in primitive thymocyte subsets in SCID-transgenic mice
IL-2 is spontaneously induced both in
TCRhigh thymocytes undergoing positive selection
to CD4 and CD8 lineages (2, 3, 4, 5) and in much more primitive
cells before TCR gene rearrangement (4). All of our
IL2-GFP-transgenic lines that express in activated peripheral T cells
also show spontaneous expression in TCRhigh
thymocytes (data not shown; and see below). It was of particular
interest to determine whether the 8kbIL2p-GFP transgene is also
expressed in the more primitive thymocytes, in which expression cannot
be driven by conventional TCR-dependent signals. However, the immature
populations in which IL-2 mRNA is found are rare, altogether
representing <0.1% of normal thymocytes (4). Therefore,
to enrich these subsets 100-fold, we crossed the 8kbIL2-GFP
transgenes from two separate founder lines onto the B6-SCID homozygous
background. GFP-expressing thymocytes from these mice could then be
identified by four-color flow cytometry, using Sca-1 (Ly6-A/E) and HSA
(CD24) expression for primary subdivision of cell types (reviewed in
Ref. 28), as shown in Fig. 7.
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. In each of these subsets,
GFP+ cells were typical of the predominant cell
type: the precursor-like Sca-1+ subset was
Thy-1+ and NK1.1- (Fig. 7A), while the NK-like Sca-1- subset
was Thy-1 heterogeneous and NK1.1+ (Fig. 7B). Most, but not all, of the GFP+
cells were also CD69+, again in agreement with
IL-2 mRNA expression data (4). In sharp contrast, the
population including the majority of transgenic SCID thymocytes, the
HSA+ subset, did not express the transgene (Fig. 7C). The HSA+ subset consists of cells
undergoing specification and commitment to the T cell lineage,
corresponding to the CD25+
CD44+/- pro-T cell populations in the
conventional fractionation scheme (24, 28). Despite the
more advanced developmental state of these cells, they do not express
IL-2 mRNA in vivo (4). Results in SCID progeny of the
other 8kbIL2p-GFP transgenic line, line 12, were similar except that
the percentage of cells expressing GFP in the
Sca-1+ population was about 3-fold lower (data
not shown). The expression of the transgene thus recapitulates
faithfully the developmental expression pattern of the endogenous gene,
even in a stage of lymphocyte development in which the inducing signal
must be TCR independent. Qualitative distortions in expression pattern of the 2kbIL2p-GFP transgene: preferential expression in CD8+ T cells
In those lines in which it is expressed, the 2kbIL2p-GFP transgene
correctly directs expression to T cells and thymocytes, similarly to
the 8kbIL2p-GFP transgene, but the detailed pattern of transgene
expression in T cell subsets differs. The most conspicuous difference
is that the shorter transgene frequently drives high level expression
in CD8+, but not CD4+ T
cells. Whereas transgenic lines utilizing the 8.4-kb promoter construct
showed similar low levels of background expression in
CD4+ and CD8+ T cells, a
distinctly biased pattern of endogenous expression of GFP was observed
in the 2-kb transgenic lines, favoring the CD8+
cells (Fig. 8). This contrasts with the
pattern of expression of endogenous IL-2, which is expressed at least
as efficiently in CD4+ cells
(29, 30, 31). Of the four 2kbIL2p-GFP-transgenic lines that
express GFP, three express the transgene in percentages of splenic
CD8+ cells 5–28 times higher than in
CD4+ cells (Fig. 8C). This effect is
most pronounced in 2kbIL2p-GFP line 227. By contrast,
8kbIL2p-GFP-transgenic lines express GFP in CD4+
and CD8+ cells at similar frequencies. Line 227
expresses GFP in 10 times more CD8+ cells than an
8kbIL2p-GFP line matched for spontaneous expression in
CD4+ cells (Fig. 8A).
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, B and D). In agreement with
the transient expression of endogenous IL-2 at this stage
(2, 3, 4, 5), small percentages of CD4+
and CD8+ single-positive thymocytes express GFP,
in all of the expression-positive transgenic lines with either the 8-
or 2-kb construct (Fig. 8, B and D; and data not
shown). However, the frequency of GFP expression in
CD8+ cells relative to CD4+
cells is elevated in three of four 2-kb lines (Fig. 8D), and
the percentage of GFP+ cells among
CD8+ thymocytes is greatly expanded in line 227
(Fig. 8B). These results suggest that IL-2 may be regulated in part through distinct mechanisms in CD4 vs CD8 cells. The additional 6.4 kb of upstream sequence appears to be needed to exert a negative regulatory effect to limit expression in CD8+, but not CD4+, cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inclusion of the novel upstream HS sites in a transgene confers strong, position-independent activity
The 8.4-kb flanking sequence transgene was designed to include a set of candidate regulatory sites that we have located by DNase-hypersensitivity analysis. The presence of at least 13 tissue-specific HS sites upstream of any previously identified regulatory sequences provided the first evidence that multiple, uncharacterized regulatory elements in this region may contribute to normal IL-2 gene regulation in vivo, although they do not appear to be important in transient transfection in vitro. Our transgenic studies confirm this prediction. A construct utilizing 2 kb of IL-2 upstream sequence results in transgene expression in only 4 of 12 independently derived lines, consistent with previous reports (11). In sharp contrast, when the new 8.4-kb transgene construct is used, GFP is expressed in 10 of 11 independent lines. Thus, the addition of 6.4 kb of upstream sequence, including all of the 5'-flanking HS sites found in resting T cells and most of the sites observed in stimulated T cells, greatly increases the likelihood of transgene expression, suggesting a relative independence from local integration site positional effects. By contrast, expression from the 2-kb promoter construct appears to depend acutely upon factors related to the site of integration. Furthermore, the 8.4-kb promoter transgene was found to be expressed over a broad range of copy numbers 3–45(3–45) in our studies, while the shorter 2-kb promoter transgene was not. Position-independent expression and resistance of transgenes to heterochromatization of high copy number arrays are both properties correlated with the presence of an LCR (13, 14, 32), or of an enhancer mediating LCR-like functions (33, 34).
Normally, sites of regulatory importance are phylogenetically
conserved, e.g., the murine and human versions of the proximal 600-bp
IL-2 promoter/enhancer, which are 85% identical. This
comparison cannot be made for most of the new upstream region, however,
because the human sequence is interrupted at about -3300 by a >6-kb
LINE element (35), and 5' sequence beyond this is
currently unavailable. Only one significantly conserved region is seen
between -2000 and this breakpoint, corresponding to -2206 to -2262
in the murine gene (27, 35). Transfection studies in a
transformed cell line have not revealed any net enhancer activity in
the new 6.4-kb upstream region (data not shown), but chromatin-opening
functions need not be detectable in transient expression assays. In
view of the activities reported for certain nuclear matrix attachment
regions (12), it is of interest that the 8.4-kb upstream
region of the murine IL-2 gene includes a potential matrix attachment
region (see Fig. 2). Alternatively, the new sequences could exert their
main effects through enhancer activity specific for particular, early
developmental stages in the thymus when IL-2 is first expressed (cf
Ref. 13).
GFP transgene expression in response to stimulation in vivo
Expression of the 8kbIL2p-GFP transgenes (and 2kbIL2p-GFP
transgenes, in lines that allow expression at all) is cell type
specific and inducible by TCR/CD28 ligation. All 8kbIL2p-GFP and
2kbILp-GFP transgenic lines that are capable of inducibly expressing
the transgene also express GFP spontaneously in specific subsets of
thymic and peripheral cells. In the thymus, these are the same cell
populations that are known to express IL-2 in response to developmental
signals (2, 3, 4, 5), including two subsets of TCR-negative
thymocytes, as well as mature TCR and TCR T cells (M.
A. Yui and E. V. Rothenberg, manuscript in preparation). Thus,
both transgenic constructs appear to be expressed in the compartments
in which IL-2 is normally expressed.
IL-2 regulation is complex, and it will require further work to determine all the respects in which the transgenes may or may not reproduce the regulation of the endogenous genes. However, based on quantitative PCR analysis of mRNA from 8-kb transgenic splenocytes after stimulation, GFP mRNA appears to be expressed from three to four transgene copies at a comparable level to IL-2 mRNA from the two endogenous gene copies. This suggests that the gross efficiency of 8-kb transgene expression can approach that of endogenous IL-2. Still under investigation are the responses of the transgenes to different stimulation conditions, and the role of transgene copy number in the magnitude of the response. Under certain conditions, the endogenous IL-2 gene can be expressed monoallelically (36), through a mechanism that is not understood. More recently, IL-2 expression has been found to be mono- or biallelic, depending on the conditions of stimulation and the timing of the assay (37, 38), suggesting independent and stochastic regulation of the two alleles. We do not know whether the allelic unit is each individual gene (or transgene) or a larger chromatin domain. Transgenes generally integrate at a single site in a tandem array that may act as a single locus, e.g., due to local chromatin effects, or each transgene copy may act independently. In addition, the orientation of the transgenes relative to one another may affect the efficiency of transcription. Such mechanisms could complicate the relationship between GFP and IL-2 expression and may help to explain why GFP does not show a strictly linear relationship with transgene copy number.
In one respect, expression of the transgenes appears to deviate from expression of endogenous IL-2. There is background GFP mRNA expression well above the detection threshold in freshly isolated splenocytes of every transgenic mouse tested, although in many of these samples there is no detectable IL-2 mRNA expression. A variable percentage of fresh splenic T cells also expresses low levels of GFP protein, albeit at much lower levels than seen in experimentally stimulated cells, in all transgene-expressing 2- and 8-kb lines. This analysis shows no evidence for spontaneous activation of the transgene in naive T cells in any of the transgenic lines. Instead, in the spleen, spontaneously GFP-expressing CD4+ cells were found to be confined to the minority subset that expresses effector/memory markers (39), apparently elicited as a result of specific immune responses to environmental Ags, or possibly including cells transiently activated in the course of homeostatic proliferation.
In contrast to our results, when a single copy of the GFP gene was knocked in to the IL-2 locus, background GFP was not observed (37). This could indicate that there are additional regulatory sequences mapping outside the 8.4 kb included in our transgene that are required for repression in memory cells or partially activated cells. The consistency of phenotype in our 8kbIL2p-GFP transgenics should be a substantial aid toward functional identification of such distal silencing sequences, if they exist. Other factors are also likely to contribute to the difference in background. The fluorescence intensity of GFP in our trangenics is much higher than that of the single knock-in allele, and this increased sensitivity could make the cells spontaneously expressing GFP easier to detect. GFP mRNA is also likely to be more stable than IL-2 mRNA, as it is designed to lack the 3' untranslated region destabilization motif found in the IL-2 gene (40, 41). This would exempt transgene expression from any negative regulatory mechanism in memory cells that acts at the level of IL-2 mRNA stability. In any case, the 8kbIL2p-GFP construct reveals a possible regulatory difference between naive and memory T cells, and offers a tool to dissect its mechanism.
Cell specificity in the 8kbIL2p-GFP and 2kbIL2p-GFP transgenic lines: differential regulation in CD4+ and CD8+ cells
In the few 2kbIL2p-GFP transgenic lines that do express the transgene, the inducibility of GFP and fidelity of transgene expression are largely similar to those of the 8kbIL2p-GFP transgenics. However, this work presents evidence of one regulatory defect shown by the 2kbIL2p-GFP lines even when they permit expression. Most 2kbIL2p-GFP lines have relatively high background levels of GFP in CD8+ cells in thymus as well as spleen, while expression in CD4+ cells in both tissues remains indistinguishable from that in the 8.4-kb lines. Although both CD4+ and CD8+ cells can express IL-2 normally (29, 30, 31), there is no evidence for any natural situation in which CD8+ cells express more IL-2 than CD4+ cells. This suggests that there is a regulatory element or elements with distinct activities in CD8+ and CD4+ cells, which map(s) between 2 and 8.4 kb upstream of the IL-2 promoter. Alternatively, the CD8+ cell bias could be an artifact of the integration sites permissive for 2kbIL2p-GFP expression. In either case, these results offer the first evidence that a component of IL-2 regulation in CD8+ cells can be mediated by cis elements that are physically separable from those that regulate expression in CD4+ cells.
A tool for analysis of IL-2 developmental regulation
IL-2 is expressed in various developmental contexts that may ultimately make it most interesting as a marker for early hemopoietic lineage decisions (4, 5, 42). The signals driving the earliest intrathymic IL-2 expression are unknown, but the transcription factors mediating this programmed induction could be identified, once the critical cis-regulatory sequences are characterized. The 8kbIL2p-GFP transgene clearly includes these sequences and can be used as a starting point for this search. In addition, the ability to isolate primitive IL-2-expressing cells, without perturbation, based on strong, unambiguous GFP fluorescence, will make it possible to determine precisely their range of developmental potentials.
In conclusion, the results shown in this study locate element(s) conferring efficient, reproducible, position-independent expression in vivo that is separate from most of the elements in the IL-2 gene that mediate inducibility and T cell specificity. The identification of novel regulatory sequences between -8.4 and -2 kb makes it possible to begin to dissect the molecular basis of in vivo developmental regulation of IL-2 for the first time. In addition, these new transgenic lines, which provide highly sensitive detection of individual live GFP/IL-2-expressing cells, should be a valuable tool to define the developmental significance of IL-2 induction and inducibility in lymphocyte subsets and lymphoid precursors.
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Acknowledgments |
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-globin 3' splicing and poly(A) addition sites
and Michael Nishimura for the melanoma cell line MCA102. We gratefully
acknowledge help from Robert Chen for screening the genomic library for
the IL-2 promoter clone and to Xiao Sun for sequencing assistance.
Shelley Diamond and Pat Koen of the Flow Cytometry Core Facility
provided invaluable help with flow cytometry and cell sorting. We also
thank Shirley Pease, Xin Yu, Bruce Kennedy, and Alba Granados of the
Caltech Transgenic and Knockout Core Facility for the generation and
maintenance of the transgenic mice. All primers were made at the
Caltech Biopolymer Synthesis Facility, and sequencing was performed at
the Caltech DNA Sequencing Core Facility. |
Footnotes |
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2 M.A.Y. and G.H.-H. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Ellen V. Rothenberg, Division of Biology, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125.
4 Abbreviations used in this paper: LCR, locus control region; CBSS, Coffman’s balanced salt solution; GFP, green flluorescent protein; HS, hypersensitive site; HSA, heat-stable Ag (CD24).
Received for publication August 22, 2000. Accepted for publication November 13, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). To
construct the transgenes used in this study, the 2-kb
HindIII-PstI and the 8.4-kb
XhoI-PstI fragments were subcloned into a
GFP reporter vector with a 1.7-kb
BamHI-PstI human 
5), while the
8kbIL2p-GFP lines generally express the transgene equally well in both
populations (ratios