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Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, Australia
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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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B, and AP1 families (5, 6) and constitutive factors
such as OCT1 (5, 6). Despite such extensive knowledge about the structure and function of the IL-2 gene and signals leading to its activation in T cells, relatively little information is available about regulation of the IL-2 gene at the chromatin level. Previous investigators have shown that the human IL-2 minimal enhancer undergoes specific increases in DNase I accessibility consistent with changes in chromatin structure upon activation (7). In contrast, a region between -313 and -570 is constitutively hypersensitive to DNase I in T cells and has been proposed to play a role in cell specificity (8). In addition, Ward et al. (8) have used DraI restriction enzyme accessibility studies to reveal that the murine IL-2 proximal promoter remains in a closed conformation until stimulation (8). There is no information available on the chromatin structure of the IL-2 gene in primary T cells nor how that structure may be remodeled in response to T cell activation in vivo.
The genomic DNA in the nucleus is organized into a highly complex structure known as chromatin, where the basic building block is the nucleosome (9, 10, 11). The higher order structure of chromatin represents an obstacle to the binding of transcription factors, the formation of preinitiation complexes at the TATA box and transcription start site and finally the elongation of RNA polymerase II. Recent studies have identified several ATP-dependent multiprotein complexes whose primary function is to alter chromatin structure so that its DNA sequence becomes transparent to the transcriptional apparatus (9, 10, 11). The modification of histone proteins by acetylation or phosphorylation is also thought to alter the packaging of nucleosomes (12, 13, 14).
It is now well documented that chromatin structure is perturbed in the neighborhood of expressed genes, and this is particularly significant in controlling their expression. Chromatin remodeling in response to cell activation has been well studied for the Saccharomyces cerevisiae Pho5 gene, HIV, and the murine mammary tumor virus (MMTV)2 promoters. Induction of the Pho5 promoter with low phosphate results in the selective remodeling of four nucleosomes in the vicinity of the promoter (15, 16). In a similar manner, induction of the MMTV promoter by the glucocorticoid receptor results in the selective remodeling of the nucleosome spanning the glucocorticoid receptor binding sites (17, 18, 19). By comparison, the HIV promoter is nucleosome free, but nucleosomes positioned downstream of the transcription start site undergo remodeling upon T cell activation (20, 21). These studies clearly establish that alterations in chromatin structure play an important role in the regulation of inducible transcription in eukaryotes.
Several recent studies have indicated that chromatin remodeling is
important for transcription of cytokine genes in mammalian cells,
including the genes that encode IL-4, IFN-
(22, 23, 24),
and IL-12 (25). Upon macrophage induction, a single
nucleosome, positioned upstream of the start site of the IL-12p40
promoter, is rapidly and selectively remodeled in a protein
synthesis-dependent manner (25). Agrawal and Rao
(23) have shown that chromatin remodeling of cytokine gene
loci is functionally associated with acquisition of the Th cell
phenotype. The differential expression of IL-4 and IFN- in Th cell
clones has been shown to correlate completely with differential
chromatin accessibility in specific regions of the IL-4 and IFN-
genetic loci (23).
In this report, we describe a novel assay, based on real-time PCR
analysis, to determine the chromatin remodeling events of the murine
IL-2 gene in both EL-4 T cells and primary T cells. This assay has been
designated as chromatin accessibility by real-time PCR (CHART-PCR) and
provides a comparatively rapid, sensitive, and quantitative means of
characterizing chromatin remodeling events for inducible genes in both
cell lines and primary cells. We show, using the CHART-PCR assay, that
the murine IL-2 gene undergoes substantial changes in chromatin
accessibility upon T cell activation in both T cell lines and, more
importantly, primary T cells. This remodeling is limited to a region of
300 bp across the proximal promoter region. The remodeling can be
inhibited by several pharmacological agents that are known to inhibit
specific transcription factors involved in IL-2 gene transcription.
Furthermore, we found that the kinetics of remodeling appear to
slightly precede IL-2 mRNA accumulation on both primary stimulation and
restimulation following withdrawal of the stimulus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EL-4.E1.F4 (EL-4) thymoma cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 50 µM 2-ME, and antibiotics. Cells in suspension were stimulated for an optimal period of 4 h at 1 x 106 cells/ml with PMA; Ca2+ ionophore, A23187 (I); an activating CD28 Ab (BD PharMingen, San Diego, CA); and plate-bound CD3e (BD PharMingen) at final concentrations of 10 ng/ml, 1 µM, 5 µg/ml, and 10 µg/ml, respectively. When inductions were performed in the presence of the inhibitors cyclosporin A (CsA; 0.5 µg/ml), cAMP (1 mM), and H89 (10 µM), cells were pretreated with the respective inhibitor for 30 min at 37°C before the addition of the stimulus. When using the deacteylase inhibitor, tricostatin A (TSA), cells were pretreated with TSA for 5 h before the addition of stimulus. A time course was initially conducted to determine this optimal period of 4 h for IL-2 mRNA production by semiquantitative PCR analysis.
Primary T cell preparation
All mice were maintained in a pathogen-free environment in barrier facilities. Spleens were isolated from C57BL/6 mice (5–6 wk old). The CD4+ cells were purified using MACS CD4+ (LT34) beads according to the manufacturer’s guidelines (Miltenyi Biotec, Auburn, CA). The cells were subsequently stained and analyzed by flow cytometry with T cell populations shown to be 90–95% pure using Abs against CD4+ T cells, CD8+ T cells, B cells, and macrophages.
Primer design for real-time PCR analysis (sequence 7700 detector)
Primers were designed for SYBR Green PCR analysis with the
sequence detector 7700 to perform 1) IL-2 mRNA quantification and 2)
CHART-PCR assays using the computer program Primer Express
(PerkinElmer/PE Biosytems, Foster City, CA). The sequences of all
primer sets used are detailed in Table I
. For amplification of IL-2
cDNA, primers were designed on an intron-exon junction to prevent
coamplification of genomic DNA. The melting temperature was 57–60°C.
The amplicon length was kept between 100 and 250 bp. For each primer
set, primer concentrations (50–900 nM) were tested to optimize the PCR
amplification. However for all primer sets identical thermocycler
conditions were used: stage 1, 50°C for 2 min for one cycle; stage 2,
95°C for 10 min for one cycle; and stage 3, 95°C for 15 s and
60°C for 1 min for 40 cycles. Since PCR amplification was being
monitored by SYBR Green, it was essential to confirm the absence of
nonspecific amplification by analyzing the PCR amplification products
on a 4% Nuseive (3/1) agarose gel (Perkin Elmer/PE Biosytems).
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RNA extraction and cDNA synthesis. Total RNA was extracted from stimulated and unstimulated T cells using the modified RNAzol B methodology described previously (26). The sample sizes were kept constant, with 1 x 107 cells in all experiments. RNA was quantified on a Gene Quant Pro RNA/DNA calculator (Amersham Pharmacia Biotec, Arlington Heights, IL), and the RNA (1–5 µg) was subsequently treated with DNase I (1 U/1 µg RNA). The DNase I-treated total RNA was reverse transcribed using 100 U Superscript II reverse transcriptase (Life Technologies, Grand Island, NY) as detailed in the manufacturer’s guidelines
SYBR Green PCR amplification.
PCR were performed on the Applied Biosystems PRISM 7700 sequence
detector (PerkinElmer/PE Biosytems). SYBR Green PCR were performed in a
total volume of 50 µl containing 10 ng cDNA as detailed in the
manufacturer’s guidelines (Perkin Elmer/PE Biosytems, protocol
04304965). Each PCR was performed in duplicate using the thermocycler
conditions detailed above. Controls were included for all PCRs to
exclude PCR amplification of contaminating genomic DNA and to ensure
that amplification was not due to contamination of other components
within the PCR mix. An aliquot of each sample was analyzed by
quantitative PCR for 
-actin to normalize for inefficiencies in cDNA
synthesis and RNA input amounts. To correlate the threshold (Ct) values
from the IL-2 cDNA amplification plots to copy number, a standard curve
was generated using a plasmid incorporating murine IL-2 cDNA.
CHART-PCR assay
Accessibility assays. Stimulated and unstimulated T cells were pelleted by centrifugation at 1500 rpm at 4°C. Cells (5 x 106 cells/sample) were washed once in ice-cold PBS. The cell pellet was resuspended in 1 ml ice-cold Nonidet P-40 lysis buffer (10 mM Tris (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, 0.15 mM spermine, and 0.5 mM spermidine) and incubated on ice for 5 min The suspension was centrifuged at 3000 rpm for 5 min to pellet the nuclei. The nuclei were subsequently washed in the respective digestion buffer (without CaCl2). DNase I, micrococcal nuclease (MNase), and restriction enzyme accessibility assays were performed essentially as previously described (25). A control without the accessibility agent was included from both the stimulated and unstimulated cells to monitor endonuclease activity. The genomic DNA was isolated using a QIAamp blood kit (Qiagen, Valencia, CA).
CHART-PCR assay. The DNA samples from the accessibility assays described above were quantified using the Gene Quant Pro RNA/DNA calculator (Amersham Pharmacia Biotec). One hundred nanograms of genomic DNA was used to perform SYBR Green real-time PCR. The PCR were performed in 50-µl reaction volumes as detailed in the manufacturer’s guidelines (PerkinElmer/PE Biosytems, protocol 04304965). Each PCR was performed in duplicate. To correlate the Ct values (threshold values) from the IL-2 CHART-PCR amplification plots to percent accessibility, a standard curve was generated using serial dilutions of genomic DNA and the appropriate primer sets using the procedures previously described (27). For restriction enzyme accessibility assays, the data are calculated and plotted as a percentage of the undigested sample for each cell treatment. For MNase accessibility, the data are plotted as a percentage of the accessibility observed in the unstimulated digested DNA sample.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies were conducted to establish the time point for optimal
IL-2 transcription in the EL-4 thymoma cell line, which has previously
been shown to express IL-2 (5, 28), using traditional
semiquantitative PCR and SYBR Green real-time PCR (Fig. 1). As shown in Fig. 1, low levels of
IL-2 mRNA were first detected after 1 h of stimulation of EL-4 T
cells with PMA/I. Real-time PCR analysis (Fig. 1B) indicated
that an 5-fold increase in IL-2 mRNA was observed compared with
unstimulated cells. At 1.5 h poststimulation increased quantities
of IL-2 mRNA were detected (70-fold; Fig. 1B), reaching
maximal levels (1050-fold; Fig. 1B) at 4 h (Fig. 1)
and declining to 40-fold at 16 h poststimulation. The highest
level of IL-2 mRNA was observed at 4 h for all the stimulants used
with real-time PCR (data not shown and see Fig. 3E). These
data are in agreement with data obtained by previous investigators
using Northern blotting and semiquantitative PCR analysis (28, 29).
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A). The procedure is
standardized by the use of appropriate calibration controls described
in Materials and Methods. This new assay procedure has been
designated CHART-PCR. The primer sets A–F (Fig. 2B and
Table I
) were used to perform the
CHART-PCR assay across the IL-2 gene. Using 100 ng genomic DNA from
unstimulated cells, the Ct values for the six primer sets all fall
within one Ct value of each other, ranging from 23 to 24 (Table I).
This indicates that these regions of the IL-2 gene are amplified with
similar efficiencies.
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B) located
within the promoter of the murine IL-2 gene becomes accessible upon
stimulation of T cells (8). Thus, to measure the
effectiveness of the CHART-PCR assay, accessibility studies were
initially performed with the DraI enzyme. In vitro
experiments were initially conducted with purified genomic DNA to
determine the parameters of digestion and to generate a standard curve
(Fig. 3A), which was linear
from 600 ng down to 1.5 ng input DNA. This represents a range in cell
numbers from 2.4 x 105 to 600 cells. All
experiments were conducted well within this linear range using 50–100
ng genomic DNA, which equates to 20,000–40,000 cells. Genomic DNA
isolated from EL-4 cells was incubated with DraI for 0–60
min in vitro and the amount of DraI digestion monitored by
real-time PCR using primer set C, and the results are plotted as a
percentage of the uncut sample (Fig. 3B). A substantial
decrease in the amount of PCR product was observed at 15, 30, and 60
min of DraI digestion, indicating that 30% of the DNA
was digested at 15 min, and almost complete digestion occurred at 60
min (Fig. 3B).
DraI accessibility studies were then conducted in cells by
incubating nuclei, prepared from stimulated and unstimulated EL-4
cells, with DraI enzyme for 0, 30, and 60 min, respectively
(Fig. 3C). We examined whether input DNA levels affect the
percent accessibility and found that across a range of input DNA from
25 to 200 ng the percent change observed from unstimulated to
stimulated samples remained the same. Since real-time PCR analysis was
being monitored by SYBR Green, the absence of nonspecific amplification
was confirmed by agarose gel electrophoresis where only one specific
band was seen for unstimulated, digested samples following 40 cycles of
PCR (Fig. 3D). One hundred nanograms of genomic DNA from the
stimulated and unstimulated EL-4 T cells was subjected to real-time PCR
analysis. The results were plotted as a percentage of the undigested
sample for each treatment. In unstimulated cells, no DraI
digestion was observed, indicating that this region of the promoter is
inaccessible (Fig. 3C). In EL-4 cells stimulated for 4
h with PMA/I there was an increase in DraI digestion, with
28 and 69% cutting at 30 and 60 min of digestion, respectively
(Fig. 3C). As an additional control, a region of IL-2
lacking a DraI site, encompassed by primer set F (Fig. 2B), was also amplified in unstimulated and stimulated
cells, and as predicted there was no change in accessibility in all
samples (data not shown).
As another measure of the effectiveness of the CHART-PCR assay,
accessibility measurements were conducted with the HinfI
enzyme, a site for which is located close to the DraI site
(-152–148; Fig. 2B). Fig. 3E shows that the
HinfI site becomes accessible to cleavage in activated EL-4
cells, and the extent of accessibility is dependent upon the nature of
the stimulus. The order of HinfI accessibility was I (25%
cutting) < PMA (50% cutting) < PMA/I (53%
cutting) < PMA/I/CD28 (60% cutting). These changes in
accessibility reflect the levels of IL-2 mRNA production that were
measured in these cells (Fig. 3E). These results show that
the CHART-PCR assay can be used to measure changes in restriction
enzyme accessibility in chromatin, and the data agree with previous
observations on DraI accessibility using primer extension
assays (8).
Chromatin remodeling is restricted to a 300-bp region across the IL-2 promoter region
Changes in restriction enzyme accessibility have been interpreted
as reflecting chromatin remodeling across a particular region of the
DNA (8, 17, 25). Thus, the data above indicate that
chromatin remodeling may be occurring across the proximal promoter
region of the IL-2 gene upon stimulation of EL-4 cells. To generate a
more comprehensive picture of changes in chromatin structure across the
control regions of the IL-2 gene, primer sets were designed across the
promoter (-14 to -330, sets B–D, Fig. 2B), downstream of
the start of transcription (+40 to +110, set A, Fig. 2B), at
a region shown to have T cell-specific constitutive DNase I footprints
(-460 to -550; set E, Fig. 2B), and in a far upstream
region (-1890 to -1980, set F, Fig. 2B).
MNase has a preference for digestion in regions of DNA that are
nucleosome-free and can thus be used to measure nucleosome remodeling
(25). The primer sets described above were used to measure
MNase accessibility across the IL-2 control regions using the CHART-PCR
assay. EL-4 cells were stimulated for 4 h with PMA/I. The nuclei
were isolated and incubated with MNase for 5 min at room temperature.
In unstimulated cells all the regions measured were inaccessible to
MNase as shown by the fact that the real-time PCR curves parallel the
curves for the samples without MNase digestion (data not shown). Since
real-time PCR analysis was being monitored by SYBR Green the absence of
nonspecific amplification was confirmed by agarose gel electrophoresis
of samples from unstimulated, MNase-digested cells amplified for 40
cycles (Fig. 4B). When cells
were stimulated for 4 h with PMA/I, significant changes in MNase
digestion were observed with the primer sets that spanned the promoter
(Fig. 4A, sets B–D), with increases of between 60 and 90%
relative to unstimulated samples. Analysis of the regions between -460
and -550 and between -1890 and -1980 showed only a small change
in digestion compared with the proximal promoter region
(Fig. 4A, sets E and F). Interestingly, the region
immediately downstream from the transcription start site also showed
relatively little change in MNase sensitivity following PMA/I
activation (Fig. 4A, set A). Thus, the major changes in
MNase accessibility upon stimulation of cells are limited to a region
of 300 bp upstream from the transcription start site.
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B) DNase I
accessibility across the IL-2 gene has been previously mapped by
conventional Southern blot analysis and LM-PCR (5). To
compare DNase I accessibility patterns across the IL-2 gene with the
new CHART-PCR assay, EL-4 cells were stimulated with PMA/I for 4
h, and nuclei were isolated and incubated with 30 U DNase I for 3 min
at 37°C. Digestion was measured using the CHART-PCR assays with each
of the primer sets shown in Fig. 2B. Since real-time PCR
analysis was being monitored by SYBR Green the absence of nonspecific
amplification was confirmed by agarose gel electrophoresis of the
amplified products (40 cycles) from unstimulated cells, (Fig. 4D). Unlike MNase, DNase I digested all regions of the gene
in unstimulated cells, with levels of digestion ranging from 30 to
60% compared with undigested samples (data not shown). This presumably
reflects the fact that nucleosome-assembled DNA is still accessible to
DNase I, but not MNase digestion. When cells were stimulated with PMA/I
for 4 h, the region spanning the proximal promoter became more
sensitive to DNase I digestion (Fig. 4C, primer sets B–D)
with increases in digestion, compared with unstimulated samples, of
between 45 and 75%. In comparison, much smaller changes were seen
across the +40 to +110 region (Fig. 4C, set A), the -460 to
-550 region (Fig. 4C, set E) or the -1890 to -1980 region
(Fig. 4C, set F). Thus, changes in DNase I sensitivity upon
stimulation of cells are limited to the proximal promoter and correlate
closely with the MNase accessibility analysis. Kinetics of chromatin remodeling of the IL-2 gene
To further characterize the remodeling events, the kinetics was
monitored by CHART-PCR assay using MNase (Fig. 5A) and restriction enzyme
(Fig. 5B) accessibility. EL-4 cells were stimulated with
PMA/I for various times ranging from 15 min to 16 h (Fig. 5, A and B). Nuclei were prepared from each time
point and incubated with the appropriate accessibility agent, and the
CHART-PCR assay was performed with primer sets B–D (Fig. 2B). As shown in Fig. 5A, significant MNase
accessibility across the proximal promoter was first detected 1.5
h after addition of PMA/I. This remodeled state appears to be
maintained for at least 6 h across the entire proximal promoter of
the IL-2 gene (Fig. 5A, sets B–D). Increases of 80–100%
MNase accessibility were observed during this time period across the
promoter (+1 to -300; Fig. 5A, primer sets B–D). By
16 h poststimulation a decline in accessibility was observed. The
region of the promoter detected by primer set C remained highly
accessible (75% accessibility) even at 16 h poststimulation (Fig. 5A). In comparison, accessibility was reduced to 40–50% in
the regions of the promoter detected by primer sets B and D (Fig. 5A). As a further measure of chromatin remodeling kinetics,
accessibility studies were conducted at the DraI site (-169
to -164, Fig. 2B) and the HinfI sites (-152 to
-148 and -102 to -98, Fig. 2B) respectively. As shown in
Fig. 5B, digestion was first observed 1 h
(HinfI) or 2 h (DraI), with maximum
digestion at 3 h poststimulation and declining at 16 h. Thus,
the remodeling kinetics observed with MNase and restriction enzymes
closely parallel each other.
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C). Chromatin remodeling occurs with altered kinetics in restimulated cells
It has been previously shown that upon stimulus removal and
restimulation, IL-2 gene transcription occurs with faster kinetics
(30, 31). It is possible that the faster kinetics of
transcription following restimulation is related to maintenance of
chromatin remodeling. We asked whether the chromatin structure of the
IL-2 proximal promoter returned to its inactive state following
stimulus withdrawal and also followed the kinetics of chromatin
remodeling upon restimulation of the cells. EL-4 cells were stimulated
with PMA/I for 4 h, when maximum levels of IL-2 mRNA were
observed (Fig. 6). Next the stimulus was
removed, and mRNA levels were measured by real-time PCR. Three hours
following stimulus removal, IL-2 mRNA levels had fallen by 5-fold,
and by 16 h IL-2 mRNA levels were lower than those in resting
cells (Fig. 6). When these cells were restimulated with PMA/I, IL-2
mRNA was detected as early as 15 min, and at 1 h mRNA levels
reached 3–4 times those seen at 4 h following the primary
stimulation (Fig. 6).
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, W1).
By 16 h following stimulus withdrawal, the set C and D regions
(-110 to -330) were completely inaccessible (Fig. 6, W2).
Interestingly, the set B region closest to the transcription start site
(-14 to -115) remained partially accessible (33%; Fig. 6, W2).
Within 15 min of restimulation each of the three regions began to
increase in accessibility to MNase, with levels similar to the 4-h
primary stimulation reached after 1 h of restimulation (Fig. 6).
The gradual nature of the appearance of accessibility following
restimulation is also in contrast with the primary stimulation where
accessibility to almost maximum levels occurred between 1 and 1.5
h (compare Figs. 5 and 6). Similar results were observed with
DraI accessibility and primer set C (data not shown).
These data show that the region of the IL-2 promoter closest to the transcription start site remains partially accessible following stimulus withdrawal and that chromatin remodeling occurs with distinct and faster kinetics following restimulation.
Cyclosporin A and cAMP inhibit chromatin remodeling across the IL-2 proximal promoter
Treatment of EL-4 cells with either CsA or cAMP has been shown to
inhibit PMA/I activation of IL-2 gene transcription (5, 32). This has been shown to occur through the inhibition of
specific transcription factors (5), which, in turn, may
function to recruit chromatin remodeling complexes. To determine
whether inhibition of specific transcription factors affected chromatin
remodeling across the IL-2 gene, EL-4 cells were stimulated with PMA/I
for 4 h with or without 30-min pretreatment with CsA or cAMP. The
nuclei were prepared from these samples and incubated with
DraI or HinfI restriction enzymes or MNase. As
shown above, PMA/I leads to an increase in DraI or
HinfI cutting of the genomic DNA in nuclei (60–70%
compared with unstimulated cells; Fig. 7A). When cells were treated
with cAMP together with PMA/I a significant decrease in enzyme
digestion was observed (down to 12–14% of unstimulated levels; Fig. 7A). Pretreatment with CsA before P/I activation for 4
h completely inhibited MNase accessibility for all three primer sets
spanning the promoter. The data for primer set B is shown (Fig. 7B), and sets C and D gave identical results (data not
shown). The inhibition of chromatin remodeling by CsA is paralleled by
an inhibition of IL-2 gene transcription, as expected from previous
studies (Fig. 7B). These results show that signals blocked
by cAMP and CsA are required for initiation of chromatin remodeling. To
determine whether signals blocked by CsA were also needed for
maintenance of the remodeled state, cells were treated with CsA 2
h following P/I activation, at a time when chromatin remodeling has
already occurred (see Fig. 5). In this case CsA reduced the amount of
remodeling measured at 6 h postactivation to 20% of the
control value (Fig. 7B). A similar pattern of inhibition was
seen across the entire promoter (data not shown). Since treatment of
preactivated cells with CsA has been shown to remove the NFAT proteins
from the nucleus within 30 min of treatment (33), we asked
whether chromatin remodeling was inhibited with the same
kinetics. When cells were treated with CsA 2 h after P/I
activation and chromatin remodeling was assayed 1, 3, and 6 h
later, it was seen that chromatin reverted gradually over this time
frame to a more inaccessible state (Fig. 7C).
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For some genes, chromatin remodeling has been shown to be dependent on
the modification of histone proteins by acetylation or phosphorylation
(34). It has not been shown whether these modifications
are involved in cytokine gene transcription in T cells. We next
examined the effect of TSA, a histone deacetylase inhibitor, on
chromatin remodeling and IL-2 gene transcription. When EL-4 cells were
treated for 1 h with P/I a very small increase (5-fold) in IL-2
mRNA levels was observed compared with that seen at 4 h of
activation (1000-fold) as described above (Fig. 7D).
However, if cells are pretreated for 5 h with TSA (500 ng/ml) a
much larger increase (127-fold) in mRNA levels was detected at 1 h
post P/I stimulation (Fig. 7D). This implies that
hyperacetylation of histone proteins, induced by inhibition of
deacetylases, leads to increased IL-2 transcription. Chromatin
remodeling is not normally observed at 1 h postactivation, so to
determine whether the hyperacetylation induced by TSA pretreatment led
to earlier chromatin remodeling, we measured MNase accessibility at
1 h in the presence or the absence of TSA. Surprisingly, chromatin
remodeling measured by either MNase or restriction enzyme accessibility
(Fig. 7D and data not shown) was not increased at 1 h
postactivation in the presence of TSA.
It has recently been shown the human MSK1 is a histone H3 kinase, and
phosphorylation of histone H3 by MSK1 leads to immediate-early gene
induction in response to a wide variety of stimuli (34).
By searching mouse EST databases, we found ESTs that matched the human
MSK1 sequence and designed primers to amplify the mouse MSK1 cDNA. We
then showed that EL-4 T cells and CD4+ T cells
expressed mRNA for MSK1 and that the level of mRNA was not increased by
P/I activation (data not shown). Pretreatment of EL-4 T cells with H89,
a specific inhibitor of MSK1 at the concentrations used
(34), inhibited IL-2 mRNA levels by 7-fold (Fig. 7E), but did not have any effect on chromatin remodeling
across the promoter (Fig. 7E).
These data show that while histone acetylation and phosphorylation may play a role in IL-2 gene transcription, they do not affect the chromatin accessibility of the IL-2 promoter as measured by the CHART-PCR assay.
Chromatin remodeling is restricted to the IL-2 minimal promoter in primary T cells
The CHART-PCR assay described above is of significant value in
assessing the chromatin structure of the IL-2 gene in primary T cells
where the requirement for large numbers of cells has previously
hampered detailed chromatin analysis. To test the efficacy of the
method in primary T cells, DraI accessibility studies were
performed using nuclei prepared from stimulated and unstimulated mouse
primary splenic T cells. Treatment of splenic T cells with PMA/I, P/I
and CD28 or CD3 and CD28 leads to increases in IL-2 mRNA levels (Fig. 8A). DraI
accessibility studies of cells from two different populations of mice
showed an increase in DraI digestion for each of the stimuli
used (Fig. 8A). The combination of CD3 and CD28 activation
was the most effective at remodeling chromatin at least across the set
C region (Fig. 8), although it lead to the lowest level of mRNA
accumulation. The level of accessibility in the
CD4+ cells was generally lower than that seen in
the EL-4 cell population, which may indicate that a smaller percentage
of the primary cell population is activated to express IL-2. The
results, however, indicate that it is possible to accurately measure
chromatin changes in primary cells with the CHART-PCR assay.
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B shows that the
proximal promoter of the IL-2 gene (-14 to -330, primer sets B–D) is
selectively remodeled upon stimulation in primary splenic T cells. As
seen in EL-4 cells, the regions encompassed by primer sets A and E
showed smaller changes in accessibility, and the region of primer set F
was not altered by stimulation (Fig. 8B). Thus, for the
first time we show that chromatin accessibility across the IL-2
proximal promoter changes following activation of primary T cells and
that this change in accessibility is restricted to the 300-bp proximal
promoter region. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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300 bp upstream of the transcription start
site in both EL-4 T cells and, more importantly, primary T cells.
Reproducing the DraI accessibility studies by Ward et al.
(8) of the murine IL-2 gene with the mouse
thymoma EL-4 cell line validated the CHART-PCR method. The
CHART-PCR assay provides a rapid and sensitive means of identifying
chromatin remodeling events for inducible genes, requiring only small
numbers of cells. Thus, this assay should enable us in the future to
perform chromatin remodeling studies on specific populations of primary
T cell where only small numbers of cells are available. A region of the promoter extending from -14 to -330 is specifically remodeled following T cell activation in either EL-4s or in CD4+ primary T cells. It is interesting that no remodeling was observed downstream of the start of transcription, implying that polymerase movement along the DNA does not bring about the same type of disruption as does the assembly of transcription factor complexes. It has been shown that bacterial polymerases in vitro can transcribe through a nucleosome and move it backward along the DNA without complete disruption (35, 36). In eukaryotic systems it has been shown that chromatin disruption complexes accompany the RNA polymerase as it moves along the DNA (37), suggesting a possible transient disruption that may not be detected in this assay. The region of the IL-2 gene that becomes accessible to MNase corresponds closely with the region of the gene that has been shown to have altered sensitivity to DNase I and dimethyl sulfoxide in in vivo footprinting assays (8). This 300-bp region has functional transcription factor binding sites across its entire length and corresponds to the minimal functional region in reporter assays (38).
Our results using MNase accessibility imply that nucleosomes may be
located across the entire IL-2 gene in unstimulated cells and that
those nucleosomes that are located across the proximal promoter region
are selectively remodeled or disrupted following activation. We have
recently shown that a highly positioned nucleosome can be assembled in
vitro across the region from -60 to -200 of the IL-2 promoter (R.
Reeves, J. Attema, and M. F. Shannon, unpublished data).
Such a positioned nucleosome(s) may exist in cells and be remodeled
following T cell activation as observed in the MNase and restriction
enzyme accessibility assays described here. Recently it has been shown
that nucleosomes positioned across the control regions of several
genes, including the IL-12p40 gene (25), the IL-2R 
promoter (39), the IFN- gene (40, 41, 42), the
proto-oncogenes c-fos and c-myc
(43), and the steroid-inducible MMTV promoter (17, 18), are all remodeled following cell activation. Thus, a common
feature of many of the inducible genes studied to date, including now
the IL-2 proximal promoter, is the selective remodeling of chromatin
structure across the control regions upon cellular activation. This
suggests that chromatin remodeling complexes are most likely recruited
to these promoters with precision.
Time course experiments were conducted to determine the kinetics of the accessibility changes within the IL-2 proximal promoter. Upon exposure of T cells to PMA/I, significant remodeling was first observed 1.5 h postinduction. The chromatin remodeling observed following 1.5-h stimulation with PMA/I remained at a similar level for at least 6 h and started to decline after 16 h of exposure to stimulus. The kinetics of chromatin remodeling occur slightly in advance of in vivo transcription factor binding and IL-2 mRNA transcription kinetics (5). In vivo footprinting analysis indicates that maximal levels of regulatory region occupancy are obtained after 2 h of stimulation and persist for at least an additional 9 h (5). These results imply that chromatin remodeling is required before the assembly of active transcription factor complexes that, in turn, give rise to the large increase in IL-2 mRNA seen between 1.5 and 3 h. An accumulation of recent data suggests that gene-specific transcription factors are required for recruitment of chromatin remodeling complexes to promoter/enhancer regions, followed by the binding of other transcription factors to generate active transcription complexes (9, 10). In the few genes studied in detail, however, the order of events appears to be gene specific (41, 44).
Either of two models could explain the 1.5-h lag phase observed before
the detection of IL-2 chromatin remodeling. In the first model
chromatin remodeling is dependent on the presence of specific
transcription factors that recruit remodeling complexes to the promoter
region (45, 46). The binding of factors to the IL-2
promoter is strictly coordinated and probably occurs by a cooperative
mechanism (8, 47, 48, 49). Thus, this implies that no factors
bind stably to the IL-2 promoter unless all factors are present and
competent to bind. Our finding that both cAMP and CsA block chromatin
remodeling of the IL-2 minimal promoter provides support for the model
above. cAMP has been shown to inhibit transcription by preventing the
binding of NFAT and NF-B factors to the IL-2 promoter
(32). CsA has been shown to inhibit NFAT translocation to
the nucleus by blocking calcineurin phosphatase activity (33, 50). Factors such as NF-B and NFAT, translocate to the
nucleus upon activation with very fast kinetics (6, 40).
Thus, the availability of these proteins is unlikely to be the limiting
event. In addition, brahma-related gene product-containing
SWI/SNF complexes have been shown to rapidly translocate to chromatin
compartments within 10 min of T cell activation (46). It
is unlikely that this recruitment occurs specifically at the proximal
promoter region within this time frame, since we do not observe
remodeling until 1.5 h poststimulation. The limiting factor in
this model may be the requirement of de novo synthesis of certain
transcription factors. Factors such as Fos family proteins
have been shown to be synthesized de novo upon T cell activation
(6) and thus may constitute the limiting event in this
model. CsA inhibits not only the initiation, but also the maintenance
of chromatin remodeling. Thus, if transcription complexes are
destabilized by the removal of a single transcription factor, in this
case NFAT, then the chromatin appears to revert slowly to its resting
state. These results agree with the results reported by Rothenberg and
colleagues (5, 8, 47) showing that the formation of
transcription factor complexes on the IL-2 promoter is highly
cooperative, and in the absence of one factor the complexes become
unstable.
In the second model recruitment of remodeling complexes is preceded by other events on the chromatin such as post-translational modifications, for example by acetylation or phosphorylation, of the histone tails of nucleosomes on the IL-2 promoter (41, 42). This would then facilitate the recruitment and subsequent binding of transcription factors on to the IL-2 proximal promoter, which, in turn, would lead to remodeling complex recruitment. Our results using inhibitors of histone deacetylation and phosphorylation do not support a role for these modifications in chromatin remodeling across the IL-2 proximal promoter, although there appears to be a role for these events in controlling the transcription rate from the IL-2 gene. We do not know, however, if this is a direct or an indirect effect, as the acetylation status of nucleosomes across the IL-2 gene has yet to be determined.
The remodeling of chromatin across the IL-2 promoter needs the continuous presence of signal. Once the stimulus was withdrawn, the chromatin reverted slowly to its resting state, with complete reversal seen by 16 h at least for the region of the gene from -100 to -300. The region from +1 to -100 did not revert to a resting state at least in the time frame assayed here. This agrees with the CsA results, which show that the continuous presence of transcription factors inhibited by CsA is required for maintenance of the remodeled state. A similar situation has been described for glucocorticoid induction of the tryosine aminotransferase gene (18). In this case removal of the stimulus led to a rapid reversal of DNase I-hypersensitive sites. The slow reversal seen on the IL-2 promoter implies that the factors involved in maintenance of remodeling have a longer half-life or that reversion may be related to cell division events.
It has been previously shown that transcription from the IL-2 gene occurs at an earlier time and at a higher level in restimulated T cells (31). Our data here support the idea that chromatin remodeling also occurs at an earlier time following restimulation. It is also interesting that remodeling occurs gradually from 15 min following restimulation. This implies that the limiting factors for remodeling gradually accumulate in the cell population following restimulation and allow remodeling to reach maximum levels by 1 h. When the stimulus was withdrawn after 4 h of activation, the region of the gene immediately upstream from the start of transcription remained partially accessible to MNase digestion. Transcription factors or components of the basal transcription machinery may remain bound to this region of the promoter, which contains the TATA box and the ARRE1 region (binds OCT, NFAT, and AP1 factors) (6). The partial accessibility remaining in this region may be important in the faster kinetics of chromatin remodeling and transcription from the IL-2 gene following restimulation.
In summary, in this study we show that significant chromatin remodeling occurs selectively across the IL-2 promoter upon T cell activation in both murine primary CD4+ T cells and EL-4 T cells. Overall, there appears to be an interdependence between chromatin remodeling and the formation and maintenance of stable transcription factor complexes on the promoter.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: MMTV, murine mammary tumor virus; CHART-PCR, chromatin accessibility by real-time PCR; CsA, cyclosporin A; Ct, threshold; I, Ca2+ ionophore, A23187; MNase, micrococcal nuclease; TSA, tricostatin A.
Received for publication May 15, 2001. Accepted for publication August 17, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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enhancer activation occurs via a conformational change of a pre-assembled nucleo-protein complex. EMBO J. 19:2034.[Medline]
enhanceosome is required for synergistic activation of transcription. Mol. Cell. 1:277.[Medline]
promoter. Cell 103:667.[Medline]
promoter. Mol. Cell. 3:125.[Medline]
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), and the amplified fragments are shown (arrows). The
positions of the DraI site (-169 to -164) and the two
HinfI sites (-152 to -148; -24 to -20) are also
shown.
