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The Human IL-3/Granulocyte-Macrophage
Colony-Stimulating Factor Locus Is
Epigenetically Silent in Immature
Thymocytes and Is Progressively Activated
during T Cell Development
Fabio Mirabella, Euan W. Baxter, Marjorie Boissinot, Sally
R. James and Peter N. Cockerill
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Copyright © 2010 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2010; 184:3043-3054; Prepublished online 10
February 2010;
doi: 10.4049/jimmunol.0901364
http://www.jimmunol.org/content/184/6/3043
The Journal of Immunology
The Human IL-3/Granulocyte-Macrophage
Colony-Stimulating Factor Locus Is Epigenetically Silent
in Immature Thymocytes and Is Progressively Activated
during T Cell Development
Fabio Mirabella,1 Euan W. Baxter,2 Marjorie Boissinot, Sally R. James, and
Peter N. Cockerill
T
he closely related IL-3 and GM-CSF (or CSF2) genes have
arisen by a gene duplication event, and are activated via the
same signaling pathways (1). These genes are highly inducible, being activated by TCR signaling pathways in T cells and
equivalent pathways in mast cells (1–4). To identify locations of
potential regulatory elements, we have previously mapped DNase I
hypersensitive sites (DHSs) throughout the human IL-3/GM-CSF
locus (1) (see also Fig. 2A for a summary). These analyses revealed
two inducible DHSs associated with conserved upstream sequences
located 4.5 kb upstream of the IL-3 gene and 3 kb upstream of the
GM-CSF gene. These DHSs function as inducible enhancers (1, 3,
5) and encompass binding sites for NFAT and AP-1 family proteins,
which respond to Ca2+ and kinase signaling pathways linked to the
TCR receptor, plus sites for RUNX, Sp1, and GATA family proteins
Experimental Haematology, Leeds Institute of Molecular Medicine, University of
Leeds, St James’s University Hospital, Leeds, United Kingdom
1
Current address: Haemato-Oncology, Institute of Cancer Research, Sutton, United
Kingdom.
2
Current address: Experimental Oncology, Leeds Institute of Molecular Medicine,
University of Leeds, St James’s University Hospital, Leeds, United Kingdom.
Received for publication April 30, 2009. Accepted for publication January 8, 2010.
This work was supported by the Association for International Cancer Research,
Yorkshire Cancer Research, and Leukaemia Research.
Address correspondence and reprint requests to Peter N. Cockerill, Experimental
Haematology, Leeds Institute of Molecular Medicine, Wellcome Trust Brenner
Building, St James’s University Hospital, Leeds LS9 7TF, United Kingdom. E-mail
address: [email protected]
Abbreviations used in this paper: 2MeK4, histone H3 di-methylation of lysine 4;
3MeK27, trimethyl lysine 27 on histone H3; 3MeK9, trimethyl lysine 9 on histone
H3; A, anti-sense; AcK9, acetylation of lysine 9; BAC, bacterial artificial chromosome; ChIP, chromatin immunoprecipitation; DHS, DNase I hypersensitive site; DP,
double-positive; ncRNA, noncoding RNA; S, sense; SP, single positive.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901364
(1–8). The IL-3 gene has an additional nonconserved enhancer located 14 kb upstream (9), but it is doubtful that this element has
much significance in vivo. The function of this 214 kb element as an
enhancer, and the inducible formation of a DHS at this location,
have been detected in only a minority of leukemic T cell lines, and
not in normal T cells (3).
In addition to the inducible enhancer DHSs, the IL-3 gene is also
associated with two prominent constitutive tissue-specific DHSs
located 1.5 and 4.1 kb upstream of the promoter. Although they lack
classical enhancer activity, the presence of these sites is closely
correlated with the potential of cells to express IL-3 (3). The 24.1
kb DHS is present in T cells, myeloid precursors, and mast cells,
and the 21.5 kb DHS is present exclusively in T cells. The
function of these sites is unknown, but they might be part of the
process of priming the locus for subsequent activation by TCRinducible pathways. In addition, there are also other inducible
DHSs of unknown function located 5.5 kb upstream and 4.5 kb
downstream of the IL-3 gene transcription start site (3, 7).
Despite their close proximity, it is likely that the IL-3 and GM-CSF
genes are independently regulated by their own specific enhancers.
This may be necessary because these genes have overlapping but not
identical tissue-specific expression patterns (1), and there is evidence from the b-globin locus that a single enhancer acts on only
one gene at a time (10). It is already established that the human GMCSF gene is regulated independently of the IL-3 gene, because a 10kb genomic fragment of just the GM-CSF gene and enhancer is
sufficient to reproducibly direct correct regulation of GM-CSF expression in transgenic mice (7). Furthermore, we recently demonstrated that the IL-3 and GM-CSF genes are segregated by an
enhancer-blocking insulator that recruits the insulator factor CTCF
together with Cohesin (11). This insulator might provide a mechanism that allows these two genes to be controlled in an independent
fashion by their individual upstream enhancers. The insulator exists
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The closely linked IL-3 and GM-CSF genes are located within a cluster of cytokine genes co-expressed in activated T cells. Their
activation in response to TCR signaling pathways is controlled by specific, inducible upstream enhancers. To study the developmental regulation of this locus in T lineage cells, we created a transgenic mouse model encompassing the human IL-3 and GM-CSF
genes plus the known enhancers. We demonstrated that the IL-3/GM-CSF locus undergoes progressive stages of activation, with
stepwise increases in active modifications and the proportion of cytokine-expressing cells, throughout the course of T cell
differentiation. Looking first at immature cells, we found that the IL-3/GM-CSF locus was epigenetically silent in CD4/CD8
double positive thymocytes, thereby minimizing the potential for inappropriate activation during the course of TCR selection.
Furthermore, we demonstrated that the locus did not reach its maximal transcriptional potential until after T cells had undergone
blast cell transformation to become fully activated proliferating T cells. Inducible locus activation in mature T cells was accompanied by noncoding transcription initiating within the enhancer elements. Significantly, we also found that memory CD4 positive
T cells, but not naive T cells, maintain a remodeled chromatin structure resembling that seen in T blast cells. The Journal of
Immunology, 2010, 184: 3043–3054.
3044
Materials and Methods
Creation of transgenic mice
An intact 130 kb AgeI genomic DNA fragment of the human IL-3/GM-CSF
locus was purified from the bacterial artificial chromosome (BAC) clone
CTD2004C12 (Invitrogen, Carlsbad, CA). This clone represents a fully
sequenced clone used in the compilation and FISH analysis of the human
genome sequence (http://genome.cse.ucsc.edu). The purified DNA was in-
jected into CBA/C57 Black 6 hybrid mouse embryos, and all experiments
were performed using progeny of a transgenic founder named C42. Quantitative Southern blotting was used to estimate that line C42 contained six
copies of the locus. DNA from this line was assayed with probes throughout
the locus to confirm that all regions of the 130 kb fragment were represented,
intact, and inserted at a single locus. Two additional lines, B38 and D48,
were derived from the same BAC DNA at the same time and used just for the
original RNA analyses to confirm that similar levels of expression were
obtained at different chromosomal sites of insertion.
Cell culture and purification
To prepare mouse thymocyte and T cell cultures, C42 transgenic mouse
thymi and spleens were disrupted in 5 ml of media (IMDM plus GlutaMaxI
Life Technologies) and the isolated released cells were obtained by passage
through a 70-mm cell strainer. CD4 and CD8 positive T cells were purified
from freshly prepared C42 mouse splenocytes using MACS CD4 or CD8
T cell negative isolation kits (Miltenyi Biotec, Auburn, CA) according to
the manufacturer’s recommendations.
Mouse T lymphoblasts were generated from filtered splenic cells by
treatment with specific Ab-coupled beads as follows: cells were cultured at
a concentration of 106 cells/ml with magnetic Mouse CD3/CD28 T cell
expander Dynabeads (Dynal) (Invitrogen) at a bead-to-cell ratio of 1:1 in
medium supplemented with 10 U/ml recombinant mouse IL-2 (Roche,
Basel Switzerland). This procedure specifically activates and expands the
T lymphocyte population and induces transformation into proliferating
blast cells. After 2 d, the cell cultures were diluted with four volumes of
IL-2–supplemented media. On day 3, the magnetic beads were removed
with the use of a strong magnetic plate. The transformed blast cells were
then maintained in culture in media containing 10 U/ml IL-2 at a concentration of 0.5–2 3 106 cells/ml for an additional 2–4 d, when they were
harvested for the experiments. Human T blast cells were generated by PHA
stimulation of mononuclear cells purified from peripheral blood as previously described (4).
FACS purification of mouse thymocyte subpopulations
Freshly prepared thymocytes were resuspended in medium at a concentration of 1 3 108 cells/ml, and the MHCII-expressing myeloid cells and
B220-expressing B cells were removed with the aid of specific Abs plus
complement as follows. Cells were incubated with mAbs against MHCII
(M5/114.15.2) and B220 (RA3-3A1/6.1) for 20 min on ice and then incubated with an equal volume of Low-Tox-M Rabbit Complement (Cedarlane Laboratories, Hornby, Ontario, Canada) at 37˚C for 40 min. Dead
cells and debris were removed by density separation using Lympholyte-M
(Cedarlane Laboratories). Cells were washed and finally resuspended in 5
ml PBS plus 0.5% BSA. Cells were immunostained for 30 min on ice using
10 ml CD4 Ab conjugated with FITC (Miltenyi Biotec) and 10 ml CD8 Ab
conjugated with PE (BD Pharmingen, San Diego, CA) for every 1.5 to 2 3
107 cells, washed in PBS, and sorted by flow cytometry.
Intracellular cytokine FACS staining
Intracellular staining of permeabilized cells, which had been stimulated in
the presence of Brefeldin A, was performed using a BD Biosciences
Cytofix/Cytoperm Plus kit (no. 555028). Freshly isolated spleen T cells
were purified using a Pan T cell MACS purification kit (Miltenyi Pan T cell
kit no. 130-090-861). Cells were stimulated for 4 h in the presence or
absence of 20 ng/ml PMA and 2 mM ionophore A23187 (PMA/I), together
with 10 ml Golgi Plug reagent (Brefeldin A) to block the export of synthesized proteins from the Golgi apparatus. Cells were then incubated for
20 min at 4˚C in PBS with 1% FCS and 5% mouse serum (Sigma-Aldrich,
St. Louis, MO) to block Fc receptors. Cells were resuspended in 250 ml
Fix/Perm permeabilization solution (BD Biosciences Cytofix/CytopermTM Plus kit no. 555028) and incubated 20 min at 4˚C. Cells were
washed twice with 1 ml Perm/Wash solution and resuspended in 50 ml
before staining. The following Abs were used alone or in combination:
IgG2a FITC (1 ml/tube; BD Biosciences [San Jose, CA] #554688), GMCSF FITC (3 ml/tube; Santa-Cruz sc-52530), IgG1kappa PE (1 ml/tube;
BD Biosciences no. 554685) and IL-3 PE (1 ml/tube; BD Biosciences no.
554676). Cells were incubated for 30 min at 4˚C, washed twice in Perm/
Wash solution, and resuspended in 500 ml PBS 1% FCS. Samples were
analyzed on an LSRII cytometer (BD Biosciences) and with the BD Biosciences FACsDiva software.
Preparation of Th1 and Th2 cells
Freshly purified CD4-positive splenic T cells were resuspended at a concentration of ∼0.5–1 3 106 cells/ml and stimulated under polarizing
conditions without IL-2 for 3 d with CD3/CD28 magnetic Dynabeads and
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as a cluster of three ubiquitous DHSs located 2.9, 4.2, and 4.9 kb
downstream of the IL-3 gene transcription start site, each of which
encompass a single, conserved CTCF binding site. Curiously, the IL-3
+4.5 kb inducible DHS is located between the +4.2 and +4.9 kb CTCF
sites. However, the +4.9 kb element is a weak CTCF site that does not
consistently form a DHS (Fig. 7), so the +4.5 kb DHS might actually
be outside of the region that supports strong insulator activity.
Little is known about the developmental stages at which the GMCSF/IL-3 locus becomes active during T cell development, or the
chromatin modifications that accompany locus activation. Most of
the available information has been derived either from cell lines or
studies of fully activated T blast cells. In comparison, studies of
other T cell-derived cytokine genes have revealed that the IL-2 and
IL-4 genes are silent in the thymus at the stage when cells undergo
TCR selection, and they become active in mature thymocytes and
T cells (12, 13). It has also been shown that the IL-2 promoter
undergoes a specific DNA-demethylation event during the transition from naive to memory T cells (14). In addition, memory
Th2 cells maintain histone H3 lysine 4 methylation in the IL-4
gene (15). Hence, it is possible that the IL-3/GM-CSF locus might
follow a similar pattern of regulation.
The IL-3 and GM-CSF genes are themselves part of a larger
cytokine gene cluster that also contains the closely related IL-5,
IL-4, and IL-13 genes (1). Furthermore, it is well established that
the IL-4, IL-5, and IL-13 genes are preferentially activated in Th2
T cells relative to Th1 T cells, and this cluster of genes acquires
specific DHSs during Th2 cell differentiation (16–19). Th2 differentiation is driven in large part by GATA-3 (18, 19), raising the
possibility that GATA elements found in the IL-3 and GM-CSF
promoters, and enhancers may also be preferentially activated in
Th2 cells (1, 2). Interestingly, there is evidence that the closely
linked IL-4 and IL-13 genes are activated in a stochastic manner
within T cells whereby one, both, or neither of these genes can be
expressed on one or both alleles when assayed on an individual
cell basis within a population of T cells (20).
In this study, we created a transgenic mouse model of the intact
human IL-3/GM-CSF locus to investigate the activation of this
locus during the course of T cell development and differentiation.
We followed the acquisition of DHSs and other active chromatin
marks, and we determined the stages at which these genes become
competent to efficiently express IL-3 and GM-CSF upon stimulation. We have found that, like the IL-2 and IL-4 genes, the IL-3
and GM-CSF genes also exist in a silent state in the thymus at the
stage of TCR selection, suggesting that a global mechanism exists
to prevent inappropriate proinflammatory cytokine secretion in the
thymus. In contrast, we found that regulatory elements in mature
T cells progressively acquire active chromatin modifications that
are absent in the thymus and that chromatin activation was accompanied by the expression of inducible noncoding RNAs across
the locus. Finally, our parallel studies of human peripheral blood
T cells indicated that naive T cells maintain an inactive chromatin
structure across the locus, whereas memory T cells are able to
maintain DHSs representing an active chromatin structure upstream of the IL-3 gene. Hence, memory T cells are indeed able to
maintain modified chromatin structures within previously activated genes.
REGULATION OF THE IL-3/GM-CSF LOCUS IN T CELLS
The Journal of Immunology
then expanded for several more days, as described above; 10 U/ml IL-2
was included in the cultures after 3 d when the beads were removed. Cells
were also then diluted with an equal volume of media and maintained for
an additional 3–4 d at 0.5–2 3 106 cells/ml.
For Th1 polarization, the cell cultures also contained 10 ng/ml mouse IL12 (Roche) plus 10 mg/ml anti-mouse IL-4 Ab (eBioscience, San Diego,
CA) for the first 2 d. On subsequent days, the cultures were diluted with
media containing 10 ng/ml IL-12 and 2 mg/ml anti–IL-4 Ab.
For Th2 cell polarization, the cultures contained 2 ng/ml mouse IL-4
(R&D Systems, Minneapolis, MN), 5 mg/ml anti-mouse IL-12 Ab (R&D
Systems), and 10 mg/ml anti-mouse IFN-g Ab (eBioscience) for the first 2
d. After 2 d, the cells were diluted with media containing 2 ng/ml IL-4, 1
mg/ml IL-12 Ab, and 2 mg/ml IFN-g Ab. On subsequent days, the cells
were diluted with media containing 2 ng/ml IL-4 and 1 mg/ml IL-12 Ab.
Preparation of human naive and memory CD4 positive Th cells
DNase I hypersensitive site analysis
DHSs were mapped as previously described using either isolated nuclei (21)
or NP40-permeabilized cells (2) digested briefly for 3 min at 22˚C with
DNase I. DHSs upstream of the human IL-3 gene were mapped from either
a SpeI site 6.6 kb upstream of the gene or an internal EcoRI site, using
previously defined probes (3). DHSs downstream of the IL-3 gene were
mapped from a BamHI site ∼6.4 kb downstream of the transcription start
site, using a 1 kb BglI–BamHI fragment of the locus (7). The data presented below represent just the optimum DNase I digestion point from
a DNase I titration series.
mRNA expression analyses
mRNA was extracted from cells using Trizol (Invitrogen) according to the
manufacturer’s instructions. cDNA was generated from purified mRNA
using M-MLV Reverse Transcriptase (Invitrogen) according to the manufacturer’s recommendation. All of the gene expression analyses were
performed by quantitative real-time PCR using SYBR Green I reagents
(Applied Biosystems, Foster City, CA) and ABI Prism 7500 and 7900
sequence detection machines. The data from each set of primers were
analyzed against a standard curve made with a serial dilution of cDNA
from PMA/I stimulated T-blasts. Each single sample was always analyzed
in triplicate, and the average value was then normalized relative to mouse
GAPDH mRNA values. The sequences of the real-time PCR primers were
as follows (m, mouse; h, human): m GM-CSF, 59-ATGCCTGTCACGTTGAATGAAG-39 and 59-GCGGGTCTGCACACATGTTA-39; m IL-3, 59-CTGCCTACATCTGCGAATGACT-39 and 59-CAGATCGTTAAGGTGGACCATG-39; m IL-4, 59-ACAGGAGAAGGGACGCCAT-39 and 59-ACCTTGGAAGCCCTACAGA-39; m IFN-g, 59-TCAAGTGGCATAGATGTGGAA-39
and 59-TGAGGTAGAAAGAGATAATCTGG-39; m GATA-3, 59-TTATCAAGCCCAAGCGAAG-39 and 59-CCATTAGCGTTCCTCCTCCA-39; m Tbet, 59-CATGCCAGGGAACCGCTTA-39 and 59-GACGATCATCTGGGTCACATT-39; m GAPDH, 59-TGGTGAAGCAGGCATCTGAG-39 and 59TGTTGAAGTCGCAGGAGACAAC-39; h GM-CSF, 59-CACTGCTGCTGAGATGAATGAAA-39 and 59-GTCTGTAGGCAGGTCGGCTC-39; h IL3, 59-GGACTTCAACAACCTCAATGGG-39 and 59-TTGAATGCCTCCAGGTTTGG-39; h GAPDH, 59-AACAGCGACACCCACTCCTC-39 and 59CATACCAGGAAATGAGCTTGACAA-39.
Chromatin immunoprecipitation assays
Chromatin immunoprecipitation (ChIP) assays were performed essentially
as described before (2, 11) using cells fixed for 10 min in 0.33 M formaldehyde (1%), before addition of four volumes of PBS containing 0.125
M glycine and sonication of the washed cells in buffer containing 0.2%
SDS. Sonicated chromatin was diluted with buffer to a final concentration
of 0.1% SDS before the addition of Abs. For each ChIP we assayed spe-
cific Abs in parallel with normal rabbit IgG to confirm the specificity of
each precipitation presented in this study (data not shown). ChIPs used the
following Abs: anti-acetyl lysine 9 histone H3 (Upstate 06-942); anti-RNA
pol II CTD repeat phospho serine 5 (Abcam Ab5131); anti-RNA pol II
CTD repeat phospho serine 2 (Abcam Ab5095); anti–dimethyl-lysine 4
histone H3 (Upstate 07-030); anti–trimethyl-lysine 27 histone H3 (Upstate
07-449); anti–trimethyl-lysine 9 histone H3 (Abcam Ab8898); normal rabbit
IgG (Upstate 12-370). The purified immunoprecipitated DNA was analyzed
by real-time PCR against a standard curve of sonicated mouse C42 DNA. The
sequence of the real-time PCR primers used in Figs. 3, 4, and 5 were as
follows: region a, 59-CACACAGCACTCCCGTGATC-39 and 59-CCTGGAGCATAAGTGCCCAAGAG-39; region b, 59-GCCACCAGCGGAAATACAACC-39 and 59-CAAGAACCTGGGCCTCAGTCA-39; region c, 59-CCACAAAGGAGGTTTCCCCTAA-39 and 59-AGACCTGGTCCCCTGTAAGATG-39; region d, 59-ATGGAGGTTCCATGTCAGATAAAGAT-39 and 59AACAGCCTCCCGCCTTATATG-39; region e, 59-CCAAGTGCAGAAAGATCCACCTA-39 and 59-GGGCAACAGAGTGAGACTCTTGT-39; region f,
59-TGAAGTTCGGCCTGGTTGAG-39 and 59-GGGAGACATGCCACAGGATTA-39; region g, 59-GCTTGCCACCTCCTCTTCAC-39 and 59-TTGCAGGCATCAGTCACTAACA-39; region h, 59-AGACCCCAAGCTCAACTCTACCT-39 and 59-CCTCACACCAGAAGGATTCCAT-39; region I, 59GGAGCCCCTGAGTCAGCAT-39 and 59-CATGACACAGGCAGGCATTC39; region l, 59-TGTCGGTTCTTGGAAAGGTTCA-39 and 59-TGTGGAATCTCCTGGCCCTTA-39; region m, 59-ATGGCAGTCACATGAGCTCCTT39 and 59-TGAAGTGACCCCCACTTTACCA-39; region n, 59-CACAGGTGGCTATCCTCTGGAA-39 and 59-CCTGAGAATCTCTGAATCCCCA-39;
mouse Chr1, 59-TGCTCCACAGTGTCCATGTACA-39 and 59-AGCAATTTCATGGGTGAGAGAAG-39; mouse CD2 Promoter, 59-CTCTCTCCTTCCCCATCTCTACCT-39 and 59-CAACCTGAACCACGTGTCTTTC-39; mouse
TBP Promoter, 59-TGCAGTCAAGAGCGCAACTG-39 and 59-CACCGCTACCGGACTCGAT-39; mouse CSF1R (21.5 kb region), 59-CACGCCGGCTGAGTGTCT-39 and 59-TCCACGTAGATGGTGTCAGCAT-39; mouse HoxC13
promoter, 59-CGCCACCCTGGGCTATG-39 and 59-TTCTGCTGCAGGTTCACGTT-39. The Chr1 probe represents a nonconserved, nonrepetitive,
nontranscribed sequence from an extensive gene desert region of mouse
chromosome 1.
DNA methylation analysis
Genomic DNA was purified from transgenic mouse thymus or T-blast cells
stimulated with 20 ng/ml PMA and 2 mM calcium ionophore A12387 for
0–4 h. DNA (5 mg) was digested with SacI, PstI, or PshAI and HindIII,
with or without the methylation sensitive restriction enzymes SmaI, FauI,
or HpaII or its methylation insensitive isoschizomer MspI. DNA was
subjected to electrophoresis on 1.8% agarose gels and transferred to
Genescreen+ (PerkinElmer, Wellesley, MA) membranes using an alkaline
transfer procedure. Membranes were probed with DNA fragments corresponding to a 652-bp PstI fragment from the human GM-CSF promoter
and 59 coding region or a 1.1 kb SacI fragment spanning the human IL-3
GM-CSF promoter and first 400 bp of coding sequence.
Detection of noncoding RNA
Noncoding RNAs (ncRNAs) were detected by a “hot-start” reverse transcription approach, using thermostable reverse transcriptase and biotinylated
strand-specific primers. Two micrograms of RNA from unstimulated
or stimulated T lymphoblasts was combined with 1 ml 10 mM deoxynucleotide triphosphates (dATP, dCTP, dGTP, and TTP), 40 U RNaseOUT
(Invitrogen), 1 ml 0.04 mM biotinylated mouse GAPDH loading control
primer, and 1 ml of 2-mM locus-specific biotinylated primer in a final volume
of 15 ml in Thermo-X Reverse Transcriptase buffer (Invitrogen). Mineral oil
(15 ml) was layered on top of the mix and then heated at 55˚C for 20 min to
remove unspecific annealing. Thermo-X Reverse Transcriptase (200 U; Invitrogen) made up in 5 ml Thermo-X buffer was next added to the reaction
and incubated at 55˚C for 1 h, and then heated at 95˚C for 10 min to deactivate
the enzyme activity. Washed Dynabeads M-280 Streptavidin (15 ml; Invitrogen) were added to each 20-ml sample and incubated for 2 h at room
temperature with continuous rotation. Using a magnet, each sample was then
washed with 100 ml 10 mM Tris, 1 mM EDTA, 2 M NaCl, resuspended in
50 ml 0.15 M NaOH, and incubated at 37˚C for 10 min to hydrolyze RNA.
The beads were washed twice in 100 ml 10 mM Tris, 1 mM EDTA, pH 7.4
buffer (TE), resuspended in a final volume of 80 ml TE, heated at 95˚C for
15 min to release the cDNA from the magnetic beads, and spun for 1 min at
13,000 rpm. The supernatants containing the released cDNAs were transferred to fresh tubes, and specific products were assayed by real-time PCR.
Data from each amplification was analyzed with the aid of a standard curve of
a progressive dilution of sonicated C42 genomic DNA. Each value
was expressed relative to mouse GAPDH mRNA. The samples were checked
for DNA contamination by testing a nonreverse transcriptase-treated sample
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Human peripheral blood was obtained from healthy volunteers with informed consent and the approval of the regional ethics committee.
Mononuclear cells were first purified by gradient centrifugation using
Lymphoprep (Axis-Shield, Oslo, Norway) according to the manufacturer’s
instructions.
CD4-positive cells were purified using cleavable anti-CD4 multisort
magnetic beads (Miltenyi Biotec). After removal of the anti-CD4 beads,
cells were fractionated into CD45RA-positive naive Th cells and CD45RAnegative memory Th cells using anti-CD45RA magnetic beads and columns, according to the manufacturer’s instructions (Miltenyi Biotec).
Transformed naive memory Th cells were prepared by stimulating CD4
positive/CD45RA-negative T cells for 3 d with 2 mg/ml PHA, followed by
several days culture in 20 U/ml human IL-2.
3045
3046
Results
Creation of a transgenic model to study the human IL-3/GM-CSF
locus
To study the full course of T cell developmental regulation, we
created a transgenic mouse line (C42) containing a 130-kb AgeI
genomic DNA segment spanning the intact human IL-3/GM-CSF
locus (Fig. 1A). To establish a mature C42 T cell model that is
fully competent to express IL-3 and GM-CSF, we prepared cultures of T blast cells from splenic T cells. This was achieved by
transient activation of splenocytes with CD3 and CD28 Abs for 3
d, to induce blast cell transformation, followed by culture of the
actively proliferating cells for at least 2 additional days in the
absence of stimulation. This process involves significant chromatin remodeling during the transformation of quiescent T cells
with compact nuclei to transcriptionally active blast cells with
decondensed nuclei (22). C42 T blast cells were then stimulated
for 4 h with phorbol ester (PMA) and calcium ionophore A23187
(I) to directly activate TCR signaling pathways. Mouse and human
IL-3 and GM-CSF mRNA expression was assayed by real-time
PCR and expressed relative to mouse GAPDH expression, after
correction for transgene copy number. Fig. 1B reveals that the
human IL-3 and GM-CSF transgenes are highly inducible and are
in fact expressed at levels 2- to 3-fold higher than their mouse
counterparts.
To confirm that C42 was a representative example of a transgenic
mouse model, we derived two additional independent lines of mice
containing one or two copies of the same BAC fragment (lines
B38 and D48) and assayed inducible GM-CSF expression in
splenic T blast cells. As shown in Fig. 1C, lines B38 and D48
expressed a level of human GM-CSF expression per gene copy
that was essentially identical to line C42. To also confirm that each
copy of the transgene was intact and integrated at a single genomic
locus, we performed Southern blot DNA hybridization analysis of
Eco RI-digested C42 genomic DNA using 59 and 39 end probes
(Fig. 1D). This revealed the presence of the predicted 7.4 kb 39/59
junction fragment as a strong band detected with both probes and
single weaker bands representing the site of integration that were
different for the 2 probes. Based on these analyses, we concluded
that line C42 T blast cells provided a reliable model for gene
regulation studies.
The IL-3/GM-CSF locus is inactive in the thymus where it lacks
tissue-specific DHSs
As the first stage in an analysis of the developmental regulation of
the IL-3/GM-CSF locus, we assayed for constitutive and inducible
DHSs in both the thymus and T blast cells (Fig. 2B). In the C42
T cells, we saw the same complement of DHSs that we had previously described in human peripheral blood T cells (Fig. 2A),
including the inducible DHSs at the IL-3 and GM-CSF enhancers
(1, 3), and the constitutive DHSs at +2.9 kb and +4.2/+4.9 kb that
represent the insulator. In this analysis, it can be seen that the 24.5 kb
enhancer DHS actually has two components: a pre-existing weak
constitutive DHS and an inducible DHS immediately downstream
that represents the enhancer. In contrast, with the exception of the
ubiquitous DHSs at the insulator, no DHSs were detected on the
transgene in the thymus. Subsequent analyses shown below in
Fig. 10C reveal that the +4.9 kb DHS is also absent in the thymus.
Because the thymus comprises ∼85% CD4/CD8 double-positive
(DP) thymocytes, this featureless chromatin pattern can be assumed to be representative of the DP population.
To determine whether the locus is also transcriptionally silent in
the thymus, we purified DP cells plus the more mature CD4 and
CD8 single positive (SP) cells and assayed for human and mouse
IL-3 and GM-CSF expression (Fig. 2C). This analysis revealed that
the locus in DP cells was completely resistant to activation by
direct induction of the calcium and kinase signaling pathways that
normally induce these genes. In contrast, the locus had begun to
acquire transcriptional competence in the CD4 and CD8 SP cells,
albeit at levels lower than in splenic T blast cells. The locus was
FIGURE 1. Creation of a transgenic model of the human IL-3/GM-CSF
locus. A, Map of a 130 kb AgeI BAC DNA fragment used to generate line
C42 transgenic mice. Enhancers are indicated by the boxes labeled as E. B,
Real-time PCR assay of inducible human and mouse IL-3 and GM-CSF
mRNA expression in T blast cells prepared from C42 T blast cells. T blast
cells were stimulated for 4 h with 20 ng/ml PMA and 2 mM calcium
ionophore A12387. Data are expressed as mRNA expression relative to
mouse GAPDH mRNA, and are corrected for gene copy number. Shown
beneath each column are the approximate levels of induction relative to the
low basal levels in nonstimulated cells. Each value represents the average
of seven independent assays. The error bars are the SEs. C, Analysis of
human GM-CSF expression in activated T blast cells from three independent lines of transgenic mice containing 1, 2, or 6 copies of the 130
kb transgene. Cells were stimulated and assayed, and the data were plotted,
as in B. D, Southern blot DNA hybridization analysis of EcoRI-digested
C42 genomic DNA using probes recognizing the 59 or 39 ends of the
transgene. The arrow marks the predicted 7.4 kb 39/59 junction fragment
arising from tandemly repeated transgenes, and the asterisks indicate the 59
and 39 site of insertion fragments.
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and amplifying for a nontranscribed control region in mouse chromosome 1
(Chr1). Biontinylated primers used to detect each region shown in Fig. 5B for
the sense (S) and anti-sense (A) transcripts were as follows: 1S, 59-TCCTTCTTCCTCTCAGCCTCCA-39; 1A, 59-TTCCTACCCTGTGCCCATCTG-39;
2S, 59-CAGAGCAGACCCTCCATCCCA-39; 2A 59-TCTGTGGCTTGCCTGGCTCCA-39; 3S, 59-AGGGAGGAATGGAGGGAC-39; 3A, 59-CTGGCAGGACTGTCGCCTCT-39; 4S, 59-GGGTGAGGAATAAGTTAGATAGCATAGGTA-39; 4A, 59-TTGTTCTCACACTGCCCT-39; 5S, 59-TGGAAAACAGGAAAAAGGG-39; 5A, 59-GTGGAAGGAGGGGCAG-39; 6S, 59CCTGGAGTGGGAGGATT-39; 6A, 59-AGGAGAAGATGGGAAAGCGTATATACTC-39; 7S, 59-ACAACCAGCCATGGCAG-39; 7A, 59-TCTACACGCCCATCTGTCCTGTC-39; mouse GAPDH mRNA, 59-GCAGCCCTGGTGACCAGGCGCCCAATACGG-39; IL-3 Intron, 59-CCACCACCCCTCACTGCTGC-39. Real-time PCR primers used to amplify each region
shown in Fig. 5B were as follows: 1, 59-CTCATAGAGGGCGTGCTCAGTT-39 and 59-AAAGCCCCTACCCACA-39; 2, 59-GATGGTGCAGGCTGGATGA-39 and 59-AGACAGTTGGAGAATCCCCATTT-39; 3, 59-GAAGGAGACCCCCAGGATGA-39 and 59-CTGCCAAGGATATCTGGGTAGTTG-39; 4, 59-CTGCCACCCCCATTTCC-39 and 59-TCAGAACGGCTCCTGGTACCT-39; 5, 59-GCCTTCCTCTGTCACCACGTA-39 and 59-TGTCAGCCTCTGACGCTTTTC-39; 6, 59-CTGAAAAACTTGGAAATGCAGAAA-39 and 59-GGTGAGGCTTGTTGTGCCTT-39; 7, 59-GCTTAGATCCAGGGAGGTGGT-39 and 59-AGCCAGGGTCTCTCTCAGAAGTT-39; mouse
GAPDH mRNA, 59-ACGGCCGCATCTTCTTGTGC-39 and 59-AAATCCGTTCACACCGACCTT-39; IL-3 Exon 1, 59-ACGAAGGACCAGAACAAGACAGAGT-39 and 59-TGTCTGGGTCATGGGAGCTT-39.
REGULATION OF THE IL-3/GM-CSF LOCUS IN T CELLS
The Journal of Immunology
significantly less active in CD8 SP cells than in CD4 SP cells,
although this might be in part because the CD8 SP population
contains some cells that are immature precursors of DP cells (23,
24). Data from CD4/CD8 double-negative cells are not presented
in this paper because they represent a heterogeneous population of
cells. Parallel assays of mouse IL-3 and GM-CSF gene expression
revealed a similar pattern of gene expression with essentially
undetectable expression in DP cells and low expression in SP cells
(Fig. 2C).
The IL-3/GM-CSF locus is expressed at an intermediate level
in freshly isolated T lymphocytes
To determine whether any differences in the regulation of the IL-3/
GM-CSF locus were maintained in mature CD4 and CD8 positive
T cells, we purified and assayed these populations directly from the
spleen. In contrast to the SP thymocytes, the human IL-3 and GMCSF transgenes were expressed at essentially the same levels in
these two populations (Fig. 2C). It was notable, however, that the
levels of GM-CSF and IL-3 expression were still substantially
lower in freshly isolated, nonproliferating splenic T cells, partic-
ularly for IL-3, than in proliferating T cells that had undergone
blast cell transformation (Fig. 1B).
To examine the basis for the 6-fold lower level of GM-CSF expression and 40-fold lower level of IL-3 expression in freshly isolated
splenic T cells compared with T blast cells, we performed a FACS
analysis of intracellular cytokine expression in both populations (Fig.
3). Before stimulation, neither population exhibited any significant
intracellular GM-CSF or IL-3 staining. After stimulation, we observed two predominant patterns of IL-3 and GM-CSF expression in
T blast cells. Approximately half of the cells expressed GM-CSF,
and one quarter expressed both GM-CSF and IL-3. This analysis
also revealed that approximately one-third of the cells expressed
GM-CSF but not IL-3, whereas only 1.4% of cells expressed IL-3
but not GM-CSF. Even this low value of 1.4% may be an overestimate, because the levels of expression were not very different to
background. This analysis demonstrates that the IL-3 and GM-CSF
genes are not in fact uniformly expressed within a population of
activated T cells, but may be activated in a stochastic fashion, similar
to what has been observed for the IL-4 and IL-13 genes in T cells
(20). These observations most likely account for why the IL-3 gene
appears to be expressed at a lower level than the GM-CSF gene when
the bulk population is examined.
The pattern of IL-3 and GM-CSF expression was different in the
freshly isolated spleen T cells (Fig. 3). After stimulation, 6% of
cells expressed GM-CSF, but just 0.4% of cells expressed IL-3,
and only 0.1% expressed both IL-3 and GM-CSF. This finding
demonstrates that there is a much lower probability of either gene
being expressed in freshly isolated T cells than in T blast cells.
These values can largely account for the much lower overall levels
of cytokine gene expression seen in the spleen compared with the
T blast cells.
The inactivity of the IL-3/GM-CSF locus in the thymus is not
due to repressive histone or DNA modifications
Gene silencing is typically maintained by histone modifications
such as trimethyl lysine 9 on histone H3 (3MeK9) when repression
involves the heterochromatin protein HP1, or trimethyl lysine 27 on
histone H3 (3MeK27) when repression involves Polycomb group
proteins. To determine whether these modifications can account for
the silent state of the locus, we performed chromatin immunoprecipitation ChIP assays of C42 transgenic thymocytes before and
after stimulation. However, we found no evidence for either of
these two commonly encountered repressive marks in the IL-3 or
GM-CSF genes, promoters, or enhancers in the thymus (Fig. 4).
The levels detected were in fact not significantly different to those
found in the CD2 gene, which is transcriptionally active in the
thymus (Fig. 3), and were close to background IgG control levels
(data not shown). In contrast, the 3MeK9 modification was readily
detectable within a nontranscribed gene desert region of mouse
chromosome 1 (Chr1), and the 3MeK27 modification was present
within the mouse HoxC13 promoter, which is a known Polycomb
target (25).
Gene silencing can also be maintained by DNA methylation,
particularly in regions such as CG island promoters where a high
density of CG elements exists. However, the IL-3 and GM-CSF
genes and promoters have only a relatively low density of CG
elements, meaning that DNA methylation may not exert a strong
influence. For example, the 2114 to +35 bp region that defines the
GM-CSF promoter contains just one CG, and this exists at a FauI
site within the Sp1 site (1). To examine this issue more closely, we
used the DNA methylation sensitive restriction enzymes FauI,
HpaII, and SmaI (whose recognition sites all include CG dinucleotides which, when methylated, prevent restriction digestion by
these enzymes) in Southern blot DNA hybridization analysis to
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FIGURE 2. Chromatin structure and mRNA expression in the human
IL-3/GM-CSF locus in C42 thymocytes and mature T cells. A, Summary of
the locations of known constitutive (gray arrows) and inducible (black
arrows) DHSs. The black bar indicates the location of the probe used to
detect DHSs downstream of the indicated SpeI in the analysis displayed in
B. The GM-CSF enhancer is labeled as GM E, the IL-3 enhancer is labeled
as 24.5 kb E, and the promoters are labeled as Pr. B, DHS analysis of
permeabilized C42 thymocytes and T blast cells, before (2) and after (+)
stimulation as in Fig. 1B. C, Relative expression of human and mouse GMCSF and IL-3 mRNA assayed and expressed as in Fig. 1B. CD4/CD8 DP
and SP cells were purified from the thymus, and CD4 and CD8 positive
cells were purified from freshly isolated spleen. The values for the thymic
cells represent the average of three or four independent analyses. The
values for the splenic T cells represent the average of six or seven independent analyses. The error bars depicted are the SEs.
3047
3048
assess levels of DNA methylation within the IL-3 gene promoter,
and the GM-CSF gene and promoter (Fig. 5). In the IL-3 promoter
there are just seven CG elements within the ∼350 bp upstream of
the transcription start that defines the promoter region. Of these, the
three CGs located within the core promoter region containing the
Sp1 and TATA elements are each recognized by either FauI or
HpaII and SmaI.
Within the GM-CSF gene there exist two HpaII sites, and we
examined cleavage at these sites within a HindIII–PshAI fragment
using a 59 end probe. Both of these HpaII sites largely resisted
cleavage, and were therefore predominantly methylated, in both the
thymus and the T blast cells, with overall cleavage levels of ∼10–
20%. This indicates that the GM-CSF gene is highly methylated at
these intragenic sites in both the inactive and the active locus. Because the phenomenon of transcription-induced DNA demethylation has been reported elsewhere (26), we also examined DNA
methylation at these enzyme sites after up to 4 h of stimulation with
PMA/I. However, within the bulk population, we saw no significant
differences in levels of DNA methylation after stimulation (Fig. 5A).
We next examined levels of DNA cleavage at 3 FauI sites within
a 652-bp PstI fragment spanning the GM-CSF promoter in the same
DNA samples as used in the preceding analysis. We used a DNA
probe spanning the entire region that can potentially detect a ladder
of four products of 207 bp or less from complete digestion plus five
products of 262–539 bp from partial FauI cleavage. In both the
thymus and the T blast cells, we observed a modest level of FauI
cleavage at each site (Fig. 5B). The pattern of FauI sensitivity,
which represents the level of DNA methylation, did not change
substantially after stimulation. Although some bands were stronger in the T blast DNA analyses, each FauI site appeared to remain
predominantly methylated, because most of the digestion products
were either partially digested or not digested at all by the methylation sensitive enzymes. In a parallel unpublished analysis, we
also performed a more localized analysis of just the FauI site
within the Sp1 site of the GM-CSF promoter, and this confirmed
that this Sp1 site is predominantly methylated (∼80–90%) in
T blast cells (data not shown).
At the IL-3 promoter, we examined sensitivity to HpaII, SmaI,
and FauI at four HpaII sites, one of which is also a SmaI site, and
three FauI sites that exist within a 1099-bp SacI fragment. MspI is
a methylation-insensitive isoschizomer of HpaII, so cleavage with
FIGURE 4. Chromatin immunoprecipitation assays of repressive histone
H3 modifications in the human IL-3/GM-CSF locus in C42 thymocytes. A
and B, Thymocytes assayed before (nil) and after (PMA/I) stimulation as
in Fig. 1B; 3MeK9-H3 (A) and 3MeK27-H3 (B) levels are expressed relative to a gene desert region of mouse chromosome 1 (Chr1) and are
corrected for copy number. The CD2 and TBP promoter (pr) regions were
used as controls for active loci, and the HoxC13 promoter represents
a known Polycomb target. Values represent the averages of two independent experiments with error bars indicating the SD. C, Map of the
human IL-3/GM-CSF locus indicating locations of primer sets used in the
above assays, depicted as black boxes with corresponding labels as lower
case letters. Parallel control IgG precipitations gave values comparable to
background levels (not shown).
MspI reveals the pattern expected if HpaII cuts to completion when
all CGs within these sites are unmethylated. However, HpaII and
SmaI both gave the same simple pattern with a low level of
cleavage at just the SmaI/HpaII site (Fig. 5C), and no cleavage,
implying ∼100% methylation at the other three HpaII sites under
all conditions examined. The SmaI site is located within a 60-bp
G/C-rich region that also contains an Sp1 site, the TATA element,
and two FauI sites at positions 671 and 711 in the SacI sequence.
Although the pattern obtained with FauI is more complex, calculation of the band sizes suggested that there was primarily a low
level of FauI cleavage at position 361, generating bands of 361
and 738 bp, somewhat less cleavage at position 711, and no significant evidence of cleavage at position 671. However, once again
it was clear that the degree of cleavage was not substantially
different between the thymus, T blasts, and freshly isolated CD4
positive splenic T cells, and did not change after stimulation.
From these analyses we can conclude that DNA methylation is
unlikely to account for the vast differences in levels of gene expression that we observe between the thymus and T blast cells.
Because we have been unable to identify any specific mechanism of
repression, we conclude that the silent state of the locus in the thymus
is likely to be caused primarily by a deficiency in the pathways required for the specific activation of the locus in mature cells.
Histone modifications are acquired throughout the IL-3/
GM-CSF locus in activated T cells
The lack of repressive histone marks within the IL-3/GM-CSF locus
in thymocytes prompted us to search for differences in the distributions of specific active chromatin marks in thymocytes and
T blast cells. To this end, we performed ChIP assays for histone H3
di-methylation of lysine 4 (2MeK4) and acetylation of lysine 9
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FIGURE 3. FACS analyses of cytokine expression in individual T cells.
Freshly isolated purified splenic T cells and cultured T blast cells were
either left unstimulated or stimulated for 4 h as in Fig.1B. Cells were
treated with brefeldin A, permeabilized, and stained with Abs to human
IL-3 and GM-CSF.
REGULATION OF THE IL-3/GM-CSF LOCUS IN T CELLS
The Journal of Immunology
3049
The regulatory elements in the IL-3/GM-CSF locus recruit
RNA Pol II and give rise to noncoding transcripts
FIGURE 5. Analysis of DNA methylation in the human IL-3/GM-CSF
locus using methylation-sensitive restriction enzymes. Genomic DNA purified from C42 thymus, CD4+ spleen T cells, or T blast cells stimulated with
PMA/I for 024 h, was digested to completion and analyzed by Southern blot
hybridization. Maps indicating the relative positions of restriction enzyme
sites are presented below each panel, with fragment sizes indicated at the
side. A, DNA was digested with HindIII and PshAI, producing a 2.4-kb
fragment spanning the GM-CSF promoter and 59 coding region. Digestions
were performed with or without the methylation sensitive enzyme HpaII.
The membrane was probed with a 59 652 bp PstI fragment (indicated by the
black bar). B, DNA was digested with PstI in the presence or absence of the
methylation sensitive enzyme FauI. The membranes were probed with the
full length 652 bp PstI fragment of the GM-CSF promoter. C, DNA was
digested with SacI in the presence or absence of the methylation sensitive
enzymes FauI, SmaI, or HpaII, or the methylation insensitive isoschizomer
of HpaII, MspI. The membrane was probed with a full length 1.1 kb SacI
fragment spanning the IL-3 promoter.
(AcK9; Fig. 6). 2MeK4 can often be found at regulatory elements
as either a precursor to or a remnant from active transcription, and it
has been suggested that this mark represents a priming event in
hematopoietic cells (27). For example, the IL-2 gene carries the
2MeK4 modification in T cells before activation, and 2MeK4 levels
We have previously demonstrated that transcriptional activation of
the GM-CSF locus is a complex process accompanied by localized
nucleosome disruption and extensive nucleosome mobilization not
only in the coding region, enhancer and promoter, but also in the
regions flanking the GM-CSF enhancer (2, 4). At other loci there
is considerable precedent for extensive non coding transcription
originating from enhancer or promoter elements (29–36), and
such a mechanism could theoretically account for both histone
modifications and nucleosome movements in the IL-3/GM-CSF
locus. We therefore performed ChIP assays for the actively engaged serine 5-phosphorylated form of RNA polymerase II (Pol II
S5P) in T cells and thymocytes and for the elongating serine 2phosphorylated form of RNA polymerase II (Pol II S2P) which
had a similar distribution in T cells (Fig. 7). In T cells we found
the expected high inducible levels of Pol II S5P and S2P in the
coding regions. In addition, we found inducible Pol II S5P and
S2P not only within the enhancers but also within the insulator
downstream of the IL-3 gene. The levels of Pol II S2P were actually higher at the +2.9 kb DHS than within the IL-3 gene itself.
This implies that IL-3 gene transcription terminates far downstream of the polyA signal sequence and proceeds at least as far
as the insulator. It is also unlikely that CTCF is directly responsible for recruitment of Pol II S5P within the insulator because levels remained low before activation when CTCF is
already bound (11).
In contrast to the T cells, there was essentially no recruitment of
Pol II S5P within the locus in activated thymocytes. This suggests
that the block in IL-3 and GM-CSF expression in the thymus is
indeed occurring at the level of transcription. If there is no Pol II
S5P recruited, then there is unlikely to be any Pol II S2P either.
To determine whether actual transcripts are being produced from
sites of polymerase recruitment, and to determine the directionality
of transcription, we performed strand-specific cDNA synthesis and
real-time PCR of transcripts upstream and downstream of the IL-3
gene (Fig. 8). Because we initially observed high, nonspecific
background levels of cDNA arising from the RNA self-priming
activity of reverse transcriptase, we performed all cDNA synthesis
reactions at high temperature (55˚C) using thermostable reverse
transcriptase and strand-specific biotinylated primers.
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do not change after induction of transcription (28). Active gene
promoters and enhancers almost universally also recruit histone
acetyl transferases such as CBP and p300 that acetylate histone H3
lysine 9. As a positive control for ChIP assays, we used the CD2
gene, which is expressed in both thymocytes and T cells. As negative controls, we used the Chr1 region that was shown above (Fig.
4A) to be associated with repressive modifications, plus a region
upstream of the inactive CSF1 receptor gene (CSF1R).
In a direct comparison with assays performed in T cells, we found
that the IL-3/GM-CSF locus was relatively devoid of both AcK9 and
2MeK4 in the thymus (Fig. 6), consistent with the lack of DHSs.
This finding suggests that the locus has low levels of active modifications in the thymus and may not be primed to respond to
pathways that induce transcription factors such as NFAT and AP-1
that would otherwise activate transcription. In T blast cells before
stimulation, AcK9 levels remained low, but increased throughout
the locus after stimulation, with levels being the highest in the
transcribed regions (Fig. 6A). In contrast, significant levels of
2MeK4 were detected in T blast cells, even before stimulation, at
the constitutive DHSs located 4.5 and 4.1 kb upstream of the IL-3
gene, and also in the GM-CSF gene itself (Fig. 6B). Throughout the
locus, 2MeK4 levels increased after stimulation.
3050
REGULATION OF THE IL-3/GM-CSF LOCUS IN T CELLS
+4.9 kb CTCF binding sites. This raises the possibility that the
inducible +4.5 kb DHS functions as an inducible ncRNA promoter.
Preliminary studies suggest that this element can in fact function as
an inducible sense strand ncRNA promoter (data not shown). The
fact that probes 6 and 7 also detected antisense transcripts suggests
that the polymerases bound at the GM-CSF enhancer are traveling
upstream from this enhancer at least as far as the insulator.
The key elements defining the insulator are the two high-affinity
CTCF sites located at +2.9 and +4.2 kb. Hence, it is significant that
we detected only low levels of transcripts from both strands between
these two sites with probe 5, barely above the limits of detection and
much lower than for the flanking sequences. This was in marked
contrast to the fact that we were able to detect significant accumulation of Pol II S5P and S2P in the vicinity of the +2.9 and +4.5 kb
DHSs (probes f and g; Fig. 7 and data not shown). This suggests that
the two high-affinity CTCF sites have a tendency to trap and/or
accumulate polymerases transcribing toward these elements.
The IL-3 gene is preferentially expressed by Th2 cells
FIGURE 6. Chromatin immunoprecipitation assays of activating histone
H3 modifications in the human IL-3/GM-CSF locus in C42 thymocytes and
T blast cells. A and B, Cells were assayed before (nil) and after (PMA/I)
stimulation as in Fig. 1B. AcK9-H3 (A) and 2MeK4-H3 (B) levels are expressed relative to the active TBP promoter. The CD2 promoter was used an
active control locus. The mouse Chr1 region and a region 1.5 kb upstream
of the mouse CSF1 receptor gene were used as inactive control loci. Values
represent the averages of two to four independent analyses. The error bars
indicate the SEs. C, Map of the human IL-3/GM-CSF locus indicating
locations of primer sets used in the above assays (depicted as black boxes
labeled with corresponding lower case letters). Arrows indicate DHSs as
defined in Fig. 2A. For the sake of better resolution of the basal levels of
signal, these data have not been corrected for copy number.
Fig. 8 shows that IL-3/GM-CSF cis-regulatory elements are indeed a source of ncRNA transcription, which is also strictly inducible. These transcripts were not found in either activated or
nonactivated thymocytes (data not shown). In activated T cells, the
highest levels of ncRNA transcripts were found on the sense strand
immediately downstream of the IL-3 gene, and this may represent
unprocessed IL-3 mRNA transcripts (region 4, Fig. 8). This is likely
because primer set four detects transcripts within a region 152–277
bp downstream of the polyA addition signal, which might include
some normal IL-3 mRNAs that have not yet terminated. These
levels were ∼5% of the levels detected for mature IL-3 mRNA
transcripts (data not shown), and ∼10-fold higher than the levels
detected for unspliced IL-3 intron RNA (Fig. 8). Substantial levels
of noncoding transcripts were also detected upstream, but not
downstream, of the 24.5 kb IL-3 enhancer. The most abundant of
these noncoding transcripts appeared to arise from polymerases
traveling both upstream and downstream from the 25.5 kb DHS, or
traveling upstream from the -4.5 kb enhancer.
At a lower but still significant level, we also detected transcripts of
both DNA strands downstream of the insulator. Because the levels of
sense strand transcripts detected with probes 6 and 7 were higher
than for probe 5, it is likely that these transcripts are initiating in the
region encompassing the +4.5 kb inducible DHS plus the +4.2 and
FIGURE 7. Recruitment of RNA Pol II to regulatory elements within the
human IL-3/GM-CSF locus. Chromatin immunoprecipitation assays of
phosphorylated RNA Polymerase II Ser 5 (Pol II S5P) and phosphorylated
RNA Polymerase II Ser 2 (Pol II S2P) in the human IL-3/GM-CSF locus in
C42 thymocytes and T blast cells. Thymocytes and T blast cells were assayed
before (nil) and after (PMA/I) stimulation as in Fig. 1B. Values are expressed
relative to a gene desert region of mouse chromosome 1 (Chr1) and are not
corrected for copy number. Results shown are the averages of two independent experiments with the SD depicted as error bars. Displayed at the
bottom is a map of the human IL-3/GM-CSF locus indicating locations of the
primer sets employed here (depicted as black boxes with corresponding
lower case letter). Arrows indicate DHSs as defined in Fig. 2A.
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Because the IL-3/GM-CSF locus is located directly adjacent to the
Th2 cytokine gene cluster, and because its regulation is also directed in part by GATA elements, it was of great interest to determine whether these genes were also preferentially expressed by
Th2 cells. To this end, we purified CD4 positive T cells from line
C42 spleens and used well-defined differentiation-inducing
cocktails including either IL-12 to direct them down the Th1
The Journal of Immunology
pathway or IL-4 to direct down the Th2 pathway. To verify that
a faithful course of differentiation ensued, we confirmed that the
Th1 cells preferentially expressed T-Bet and IFN-g mRNAs,
whereas the Th2 cells preferentially expressed GATA-3 and IL-4
mRNAs (Fig. 9A).
Analyses of human and mouse GM-CSF mRNA expression
revealed essentially identical levels in Th1 and Th2 cells (Fig. 9B).
Furthermore, extensive DHS analysis over the IL-3 and GM-CSF
locus in Th1 and Th2 cell types did not reveal any major differences in patterns, with the exception of slightly enhanced chromatin remodeling over the GATA elements within the GM-CSF
enhancer in Th2 cells (data not shown). This would suggest that,
unlike the classical Th1 and Th2 cytokine gene loci, there are no
regulatory elements in the IL-3/GM-CSF locus specifically dedicated to driving Th1- or Th2-specific mechanisms of gene expression. The human IL-3 gene was, however, expressed at levels
∼3-fold higher in Th2 cells than in Th1 cells. The mouse IL-3
gene was also more highly expressed in Th2 cells; although the
difference was more modest, it was statistically significant
(p = 0.00014). Hence, the evolution of the IL-3/GM-CSF/IL-5
gene family seems to have resulted in the establishment of discrete
sets of regulatory elements controlling these three genes, which
allow the IL-3 and IL-5 gene to be more highly expressed in Th2
cells, whereas no such bias exists for the GM-CSF gene, which is
located in between these two genes.
The IL-3 gene maintains a DHS imprint in human memory
helper T cells
The above studies in the mouse revealed a progressive upregulation
of the IL-3/GM-CSF locus during differentiation from immature
thymocytes to T blast cells, but have not identified the precise stage
at which the locus becomes fully transcriptionally competent.
Because it is known that memory T cells are able to mount a more
efficient response than naive T cells (38), we investigated whether
the IL-3 and GM-CSF genes might undergo further maturation and
maintain an active conformation subsequent to this transition. For
this purpose, we returned to the natural setting of the human IL-3/
GM-CSF locus, and we fractionated T cells from human peripheral blood. We first purified CD4-positive Th cells and then used
C45RA Ab magnetic beads to further fractionate these cells into
subsets that roughly correspond to CD45RA positive naive T cells
and CD45RA negative memory T cells (38). To determine
whether the IL-3/GM-CSF locus was a target for more efficient
induction in memory T cells, we used a brief 2 h stimulation of the
cells. Significantly, the memory T cells responded by expressing
levels of both IL-3 and GM-CSF mRNA that were 4-fold higher
than in naive T cells (Fig. 10A). Furthermore, intermediate levels
of mRNA expression were detected in naive T cells that had been
transiently activated with PHA to stimulate surface receptors, and
then cultured for several days, before being restimulated with
PMA and ionophore (Fig. 10A).
To determine whether there was a chromatin structure basis for
the more efficient upregulation of this locus in memory cells, we
also examined the DHSs surrounding the IL-3 gene (Fig. 10D) in
the two distinct populations of cells. As a reference point, we also
prepared unfractionated T blast cells from human blood. Somewhat similar to memory T cells, T blast cells have also undergone
a transient period of prior stimulation. Remarkably, the naive
T cells had a blank pattern of DHSs that was identical to the inactive state observed in the transgenic thymus (Fig. 10B, 10C). In
stark contrast, the memory T cells maintained the pattern that was
known to be present in human T blast cells (Fig. 10B, 10C).
Owing to the technical limitations of working with small numbers
of primary cells, we have so far been unable to determine what
specific chromatin or transcription factor modifications maintain
the primed state in memory T cells. However, these data provide
a clear indication that memory T cells can maintain a reorganized
chromatin structure that is likely to influence the ability of effector
genes to respond efficiently and appropriately to stimuli.
Discussion
In this study, we defined a developmental pathway whereby the IL3/GM-CSF locus is progressively activated at the levels of both
chromatin structure and modification, and transcriptionally during
the course of T cell development. The pattern of regulation of the
IL-3/GM-CSF locus in the thymus bears similarities with other T
cell-derived cytokines in that it is totally shut down at the levels of
both chromatin modification and transcription in CD4/CD8 double
positive thymocytes. Other cytokine genes such as IL-2 and IL-4
are also downregulated at this stage of T cell development (12,
13). The picture emerging from these findings is that the ability to
express cytokine genes is not acquired until after thymocytes have
passed through critical maturation checkpoints. Otherwise, these
genes could be turned on by the very process by which the thymus
selects thymocytes with functional TCRs. If unchecked, this could
in turn initiate an inappropriate proinflammatory response within
the thymus. It is now apparent that thymic development progresses
in a manner which ensures that this does not occur. However, we
have been unable to identify any specific repressive epigenetic
mechanism that maintains the IL-3/GM-CSF locus in a silent state
in the thymus. Rather, there is an absence of the transcription
factors, histone modifications, and chromatin remodeling required
to establish a transcriptionally active locus.
Others have shown that induction of the transcription factors
NFAT and AP-1 is suppressed in immature thymocytes at the stages
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FIGURE 8. Detection of ncRNAs in the IL-3 locus in C42 T blast cells.
ncRNAs were reverse transcribed and detected as described in Materials
and Methods. Data are expressed as mRNA expression relative to mouse
GAPDH mRNA, without correction for copy number. Each value represents the average of two to three independent analyses. The error bars
indicate the SE. The direction of transcription of each RNA species is
expressed relative to direction of transcription of IL-3 mRNA, whereby the
sense strand is labeled as S, and the anti-sense strand is labeled as A.
Displayed below is a map of the human IL-3/GM-CSF locus indicating
locations of primer sets used in the above assays, depicted as black boxes
labeled with the corresponding numbers. Vertical arrows indicate DHSs as
defined in Fig. 2A. Curved gray and black arrows are a schematic representations of the most abundant ncRNAs species detected in the corresponding regions after PMA/I induction. Black curved arrows represent
highly abundant ncRNA species, and gray curved arrows represent more
moderately abundant ncRNAs.
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at which cells undergo TCR selection (12, 13, 38). These observations suggested that suppression of cytokine genes in thymocytes might be a direct consequence of suppression of the
genes and factors that normally respond to TCR signaling pathways. However, our finding that even the constitutive DHSs upstream of the IL-3 gene are absent in DP thymocytes indicates that
the basis for the deficiency in DP cells is more complex than just
a defect in TCR signaling pathways. This suggests that additional
transcription factors and epigenetic modifications are also required
to prime the locus before it is able to respond to TCR engagement.
In T blast cells we found that the IL-3/GM-CSF locus had
undergone a transition from the nonresponsive silent state to an
active primed state that was marked, in part, by the appearance of
2MeK4 histone H3 modifications within the constitutive DHSs that
flank the 24.5 kb IL-3 enhancer. Further evidence for locus
priming upstream of the IL-3 gene came from our studies of human memory T cells. We obtained compelling evidence that human memory Th cells maintain a remodeled chromatin structure
in the form of constitutive DHSs upstream of the IL-3 gene.
Significantly, these DHSs were completely absent in naive Th
cells, suggesting that they have not yet been primed for efficient
transcriptional activation of the IL-3 gene. This form of active
priming in memory T cells equated with a 4-fold enhanced ability
to induce IL-3 and GM-CSF expression in response to a short burst
of stimulation of TCR signaling pathways.
Analyses of cytokine expression at the single cell level by FACS
revealed that the IL-3 and GM-CSF genes may be regulated in
a probabilistic manner, in much the same as the IL-4 and IL-13
genes (20). Hence, even in fully activated T blast cells, the IL-3
and GM-CSF genes are not necessarily coexpressed, or even expressed at all within a population of T cells. Furthermore, the
transition from a resting T lymphocyte in the spleen to a T blast
cell in culture was marked by a 10- to 60-fold increase in the
probability of activating the GM-CSF or IL-3 genes, respectively.
However, we are unable to determine from this type of analysis
whether there is any difference in the underlying chromatin
structure between the cells that do or do not express IL-3 or GMCSF, which might account for this phenomenon.
Mature splenic CD4- and CD8-positive T cells expressed essentially identical inducible levels of IL-3 and GM-CSF mRNA.
Stimulated Th1 and Th2 cells also expressed identical levels of
GM-CSF, whereas Th2 cells expressed 3-fold more human IL-3
mRNA than Th1 cells. It is unclear as to whether IL-3 plays any
significant role in the specific functions of Th2 cells. The differences in IL-3 expression levels may be attributed to the fact that
Th2 cells have upregulated GATA-3 expression (18, 19), which
would increase the activity of GATA elements located in the IL-3
promoter and 24.5 kb enhancer (1). Moreover, it was previously
shown that GATA-3 plays a major role in the regulation of the
IL-3 gene in human T cells, and that there is a strong correlation
between IL-3 and GATA-3 mRNA expression levels in human
T cell clones (39). This finding indicates that the IL-3 gene has an
expression pattern similar to the Th2 genes, but not as extremely
polarized. However, it remains puzzling why there is no reciprocal
relative increase in GM-CSF expression in Th2 cells, because
GATA elements play a critical role in the GM-CSF enhancer in
myeloid cells, which express GATA-1 or GATA-2 (2).
Others have proposed that ncRNA transcription directed by
enhancers or promoters may be one of the mechanisms that
activates gene loci and establishes active chromatin domains (29–
36), while also silencing cryptic promoters (33). In the human
b-globin locus, zones of chromatin accessibility are closely correlated with zones of ncRNA transcription (30). ncRNA transcription seems to be responsible for directing the methylation, but
not necessarily the acetylation of the underlying chromatin (32,
33), and does not have to encompass the actual coding sequences
to activate a target gene (32). In both TCR and immunoglobulin
genes, ncRNA transcription is believed to play a critical role in
promoting gene rearrangements by increasing chromatin accessibility (33–35). In the chicken lysozyme locus, ncRNA transcription involves a novel mechanism of gene activation whereby RNA
polymerase II repositions a nucleosome over a CTCF site and
suppresses a silencer (36).
In this study, we observed that transcriptional activation of the
IL-3/GM-CSF locus in T blast cells is also accompanied by extensive inducible ncRNA transcription that appears to originate
from the enhancer elements. Inducible ncRNA transcripts were
detected proceeding far upstream of the IL-3 enhancer and far
downstream of the IL-3 gene. In parallel, both 2MeK4 H3 and
AcK9 H3 were introduced in activated cells at least as far as 10 kb
upstream and 4.5 kb downstream of the IL-3 gene in activated
cells. This raises the possibility that the extensive spread of active
histone modifications, and our previously observed nucleosome
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FIGURE 9. mRNA expression in Th1 and Th2 cells. Quantitative realtime PCR analysis of gene expression assayed before (nil) and after (P/I)
stimulation as in Fig. 1B for mouse Th1 and Th2 marker genes (A) and for
human (h) and mouse (m) GM-CSF and IL-3 mRNA (B). Polarized CD4
positive T cell subtypes were prepared from line C42 splenic T cells. Values
are expressed relative to mouse GAPDH, and the error bars depicted are the
SDs from two or three separate RNA extractions and cDNA syntheses. A
Student t test confirmed that the difference in mouse IL-3 expression between
Th1 and Th2 cells was statistically significant with p = 0.00014.
REGULATION OF THE IL-3/GM-CSF LOCUS IN T CELLS
The Journal of Immunology
3053
mobilization in this locus (2, 4), may both be a consequence of
ncRNA transcription. For example, the inducible 2MeK4 H3
modifications may have been deposited as a direct consequence of
ncRNA and mRNA transcription. Elongating RNA polymerase is
known to mediate methylation of H3 K4, and trimethyl K4 H3 is
a definitive hallmark of recently transcribed genes (40, 41).
Conversely, some genes are specifically marked by 2MeK4, and
not trimethyl K4, and this is thought to represent a poised state
that primes hematopoietic genes in precursor cells (27). The
constitutively methylated IL-3 24.5 and 24.1 kb DHSs may
represent this very type of 2MeK4 H3 modified poised state.
Methylated K4 H3 is able to recruit various chromatin remodeling
and modifying complexes, thereby providing a mechanism to
maintain a primed active state within the IL-3 locus (41). Future
studies will be required to determine the identities of the transcription factors that are required to recruit chromatin modifying
factors and to initiate formation of the 24.1 and 21.5 kb DHSs in
the IL-3 locus
The constitutive DHSs found at the IL-3 +2.9 and +4.2 kb CTCF
sites within the insulator in the thymus represent the earliest
chromatin modifications that can be detected in the IL-3/GM-CSF
locus. However, there were relatively low levels of histone modifications, and there was no evidence of ncRNA transcription within
the insulator either in the thymus or in T blast cells before stimulation. We observed the acquisition of active chromatin marks
within the insulator only after stimulation, and this was accompanied by the recruitment of active RNA polymerase and the
detection of ncRNA transcripts proceeding toward or away from
this region. The origin of these transcripts is complex, but they
appear to arise variously from IL-3 mRNA transcripts proceeding
downstream from the polyA addition signal, from transcripts
traveling upstream from the downstream GM-CSF enhancer, and
perhaps also from the inducible IL-3 +4.5 kb DHS downstream
of the +4.2 kb CTCF site. Intriguingly, there are reports that CTCF
itself also interacts with RNA polymerase II (42). However, we
observed that RNA polymerase only enters the insulator region
after stimulation, whereas CTCF is recruited before stimulation
(11). Furthermore, we obtained evidence suggesting that RNA
polymerase was not recruited directly by CTCF de novo, but may
simply be trapped there by CTCF, because only a small proportion of flanking transcripts traversed the +2.9 and +4.2 kb
CTCF sites and continued into the intervening region. This
finding is in agreement with the view that CTCF elements mark
the boundaries of active and repressive chromatin domains (43,
44), but is not consistent with the observation that CTCF and
Cohesin complexes are also found within the introns of actively
transcribed genes (45). Hence, it is probably best to regard
CTCF-dependent insulators as elements that help to maintain
correct communication and looping between enhancers and
promoters (46) and, which in at least some cases, can retard the
progress of elongating polymerases.
Acknowledgments
We thank D. Brooke for assistance creating transgenic mice, F. Ponchel for
advice regarding fractionation of naive and memory T cells, A. Arpanahi for
assistance designing PCR primers, D. Kioussis for constructive comments,
C. Bonifer for providing considerable input and support, L. Straszynski and
G. Doody for help with flow cytometry, M. Hoogenkamp and F. CaleroNieto for their assistance in the design of ncRNA analyses, and H. Tagoh
and P. Lefevre for sharing Abs.
Disclosures
The authors have no financial conflicts of interest.
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FIGURE 10. An epigenetic imprint is maintained in memory T cells. A, Human IL-3 and GM-CSF mRNA expression was assayed in populations of CD4
positive T cells purified from human peripheral blood. Naive T cells represent CD4 positive, CD45RA positive cells, and memory T cells represent CD4
positive, CD45RA negative cells. Transformed naive T cells were prepared by transient stimulation with PHA. Cells were stimulated for 2 h with 20 ng/ml
PMA and 2 mM calcium ionophore A12387. Data are expressed as mRNA expression relative to human GAPDH mRNA. Each analysis was performed in
duplicate, and this panel displays one of three representative experiments. Error bars indicate the SD. B, C, and D, DHS analysis in C42 mouse thymocytes,
human naive and memory T cells as defined in A, and unfractionated human T blast cells. B, DHSs upstream of the IL-3 gene mapped from an EcoRI site
with probe B. C, DHSs downstream of the IL-3 gene mapped from a BamHI site with probe C, as summarized by the map in D. Note that the weak CTCF
site located at +4.9 kb fails to form a DHS in both the thymus and in naive T cells.
3054
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