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*
Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520; and
Department of Medicine, Divisions of Hematology and Oncology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß and
IFN-
responses, 3) mutational analysis of the G region reveals that
a complex interaction exists between the factors binding to this region
as shown by their mutual interdependence for detection in DMSA, and 4)
inhibition of expression of HMG proteins by antisense HMGI-C RNA in EL4
cells causes the loss of IFN-ß and IFN- inducibility of the
endogenous Ly-6 gene. These findings taken together suggest that, in
response to IFN treatment, an HMG protein-dependent complex involving
multiple regulatory factors is assembled and is required for IFN
inducibility of the Ly-6E gene. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß
and - are important cytokines with pleiotropic effects on cells
ranging from antiviral and antiproliferative effects to the
augmentation of the immune response (1). Most of these
effects are mediated by the products of genes whose expression is
transcriptionally activated by IFNs. Characterization of the signaling
pathways employed by IFNs for immediate induction of gene expression
has led to the discovery of the Janus kinase
(JAK)3-STAT pathway of
signal transduction. The latent transcription factors termed STATs
reside outside the nucleus in untreated cells. Upon stimulation with
IFN, they become activated by tyrosine phosphorylation catalyzed by JAK
and multimerize. Multimerized protein complexes translocate to the
nucleus where they recognize discrete cis-acting regulatory
DNA sequences to enhance the expression of target genes
(2).
These regulatory sequences are present upstream of a large number of
different genes and are termed IFN-stimulated response element (ISRE)
(3) or IFN- activation site (GAS) (4).
However, only a distinct subset of such genes is activated in response
to IFN treatment in a given cell type. How this specificity is achieved
is one of the central questions that remain in defining IFN signal
transduction pathways. An example of how specificity can be achieved in
a signaling system is demonstrated by the virus-inducible expression of
the human IFN-ß gene. In this system, viral-mediated induction of
IFN-ß gene is the result of coordinate interaction between multiple
transcription factors and the binding of high mobility group I(Y)
(HMGI(Y)) protein. HMGI(Y) is a DNA-bending factor that can facilitate
cooperative interaction between different transcription factors to
assemble an enhanceosome (5). The formation of an
enhanceosome, a higher order nucleoprotein complex involving multiple
regulatory and cell type-specific factors, provides a mechanism for
achieving specificity in extracellular signal-regulated transduction
pathways (6).
Recent reports have highlighted the importance of the IFNs in the
generation of memory CD8+ T cells (7, 8); furthermore, it was demonstrated that the expression of
Ly-6C Ag is a strong marker for the memory phenotype (7).
Like their murine counterparts, a human homologue of Ly-6 genes, the
9804 gene, is also responsive to IFNs (9). Interestingly,
this gene is also inducible by retinoic acid during differentiation of
acute promyelocytic leukemia cells (10). Another member of
the murine Ly-6 multigene family, the Ly-6A/E gene, is expressed on
hemopoietic stem cells, fibroblasts, and T and B lymphocytes. This
expression can be greatly induced by either IFN-ß or IFN- in
various tissues and cell lines. The Ly-6E Ag is found to be associated
with tyrosine kinases in T cells (11), and reduced
expression of Ly-6E in T cells causes impairment of functional
responses, as well as the inhibition of fyn tyrosine kinase activity
(12).
We have previously characterized the GAS site responsible for the IFN responsiveness of the Ly-6E gene in fibroblasts (13). Due to the physiological relevance of the IFN-mediated expression of these Ags in lymphocytes, this study was undertaken to define the molecular mechanism of IFN-mediated induction of the Ly-6E gene in T cell lines. The availability of allele-specific Abs directed against Ly-6E and Ly-6A Ags facilitated this investigation because it allowed us to take a more physiologic approach of utilizing the genomic gene deletions to identify the regulatory regions responsive to IFNs. Genomic gene deletion analysis performed in EL4 and BW5147 T cell lines resulted in the identification of multiple regulatory elements present within the Ly-6E promoter responsible for IFN inducibility.
A complex 
30-bp G region contains the previously identified GAS
site. This site was shown to bind STAT1 and to be essential for the
inducibility of the Ly-6E gene in fibroblasts in response to IFN-ß
and IFN- (13). However, in T cells used in this study,
multiple regulatory factors bind specifically to this region, and they
were identified to be Oct-1, Oct-2, and HMGI(Y) protein. Mutational
analysis of this region revealed a tight clustering of DNA-binding
sites for these proteins and their mutual interdependence for
generating DNA-protein complexes. Surprisingly, the binding of Oct
factors required intact GAS site in addition to the octamer motif.
Similarly, HMGI(Y) binding required the presence of both GAS and Oct
sites. Functional analysis using mutants of the GAS site and the
octamer sequence confirmed the necessity of the GAS site and the
contribution of the binding of Oct factors to IFN inducibility in T
cells. The role of HMGI(Y) binding for in vivo inducibility of the
endogenous Ly-6 gene was addressed by inhibiting HMGI protein synthesis
by antisense RNA and analyzing the cell surface expression of Ly-6 Ag.
Inducibility of endogenous Ly-6 gene by IFN in the
antisense-transfected clones was lost. These data suggest the
involvement of HMGI proteins in the assembly of a transcription complex
containing different factors to achieve cell type-specific IFN
response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EL4 and BW5147.3 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Murine T lymphoma cell lines EL4 and BW5147.3 were grown in modified DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FCS.
Interferon
Recombinant murine IFN- was purchased from Life Technologies
(Gaithersburg, MD). Recombinant murine IFN-ß was provided by LEE
Biomolecular (San Diego, CA).
Transfections
Stable transfection was achieved by cotransfection using 20 µg
of the test plasmid with 2 µg of the pSV2neo plasmid. Cells
(1 x 107) in PBS containing 2 mM 2-ME
were electroporated at 320 V and 500 µF with a Bio-Rad (Richmond, CA)
gene pulser. The cells were then plated in DMEM containing 10% FBS.
After 36–48 h, the cells were collected, counted, and replated in
fresh medium containing G418 in 96-well culture plates at a density of
104/well. Selection was achieved with 2 mg/ml
G418 for BW5147.3 cells and 1 mg/ml G418 for EL4 cells. After 2 wk,
individual transfectants were observed and expanded for analysis.
Transient transfection assays
BW5147 Cells were transfected by DEAE-dextran-mediated DNA
transfer (14). BW5147 cells (3 x
106) were harvested and resuspended in 1 ml
serum-free DMEM containing 10 mM HEPES (pH 7.2), DNA (10 µg),
DEAE-dextran (250 µg/ml), and chloroquine (0.1 mM). After incubation
at 37°C for 30 min, the cells were washed twice with serum-free DMEM
containing HEPES. The cells were resuspended in 10 ml complete DMEM and
split into two separate flasks, one for a control and the other for
treatment with IFN. Cells treated with IFN- received 500 U/ml of IFN
20 h after the transfection was initiated, and assays were
performed after 48 h. For luciferase assays, the cells were
collected by centrifugation and rinsed twice in PBS. The cell pellets
were resuspended in 400 µl of 1x lysis reagent (Luciferase Assay
System, Cat. No. E1500; Promega, Madison, WI), and the suspension was
incubated at room temperature for 15 min. The lysate was centrifuged
for 1 min in a microfuge to pellet cell debris. After measurement of
protein concentration by Bradford assay, appropriate aliquots of the
cell lysate were mixed with 100 µl of the luciferase substrate at
room temperature, and activity was measured in a luminometer.
Reporter constructs
The pTKLuxbu+ was obtained from J. Hambor (unpublished observations; Ref. 15). This plasmid contains the basal thymidine kinase promoter linked to the reporter gene. G-tk-Luc construct was derived by subcloning a DNA fragment containing the G sequence 5'-AAGCTTCTGCTCAGAATTTATGCATATTCCTGTAAGTGACCTCACCCATCCTAGATCT-3' into the HindIII-BglII sites of the polylinker of pTKLuxbu+ upstream of the TK promoter. The G-pBSKS+ construct was made by subcloning this DNA fragment into HincII site in the pBSKS+ cloning region. Constructs G-M2-tk-Luc and G-M3-tk-Luc were made by PCR using the G-pBSKS+ as template. The PCR products were cloned directly into the HindIII-BglII of the pTKLuxbu+. The 5'-AAGCTTCAGAATGGCGGCATATTCC-3' and 5'-AAGCTTCAGAATTTATGCATATTCCTGGCCGTGACCTCACCCATCCT-3' primers were the 5' primers used for the synthesis of these constructs, respectively. The G-M2-tk-Luc construct was made by using the above 5' primer with the 3' primer corresponding to T7 of pBSKS+.
Cytofluorometric analysis
Transfected cells were analyzed for surface expression of Ly-6E by immunofluorescence using a FACScan (Becton Dickinson, Mountain View, CA). mAb SK70.94 recognizes the Ly-6E Ag, and mAb Sca-1 (E13 161-7) recognizes both Ly-6A and Ly-6E Ags. Binding was detected with FITC-conjugated rabbit anti-mouse secondary Ab.
DNA mobility shift assay
DNA mobility shift assays (DMSA) were performed with nuclear extracts prepared according to Dignam et al. (16). Approximately 108 cells were collected by centrifugation, washed twice with PBS, resuspended in 300 µl of buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 0.4 mM PMSF, 3 µg/ml aprotinin, 1 µg/ml leupeptin, 0.5 µg/ml pepstatin, 50 mM NaF, 30 mM NaPPi, and 0.1 mM Na3VO4), and incubated on ice for 15 min. The cells were lysed by pushing five times through a 25-gauge needle (slow draw, fast push). The membranes were then pelleted in a microfuge for 30 s and resuspended in 100 µl of buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 1 mM DTT, 0.4 mM PMSF, 3 µg/ml aprotinin, 1 µg/ml leupeptin, 0.5 µg/ml pepstatin, 50 mM NaF, 30 mM NaPPi, 0.2 mM EDTA, and 0.1 mM Na3VO4). The suspension was rotated at 4°C for 30 min and then pelleted for 5 min in a microfuge. Protein concentration was determined using the Bradford assay, and extracts were stored at -70°C.
Probes were prepared from synthetic oligonucleotides or restriction
fragments and end-labeled with 32P using Klenow
DNA polymerase. Nuclear extract (protein concentration, 2 µg/µl)
was incubated with the probe in the presence of 1 µg of nonspecific
competitor poly (dI.dC):poly(dI.dC) duplex in a 10-µl binding
reaction. The binding buffer was 20 mM HEPES (pH 7.9), 4% Ficoll, 1 mM
MgCl2, 40 mM KCl, 0.1 mM EGTA, and 0.5 mM DTT.
Bound complexes were separated from free probe by electrophoresis for
2 h at 180 V on a 4% nondenaturing polyacrylamide gel in 0.25x
TBE buffer and identified by autoradiography. The sequences of the
duplex oligonucleotides used, with their consensus sequences
underlined, for competition were as follows: for the Oct-1 element,
5'-TGTCGAATGCAAATCACTAGAA-3' (17); for the
high affinity SIE element, 5'-GTCGACATTTCCCGTAAATC-3'
(18). Bacterially derived recombinant human HMGI (rhuHMGI)
was a gift from Ray Reeves (Washington State University, Pullman, WA).
Binding assays involving rhuHMGI consisted of about 15 ng of purified
protein, 2 µg of poly(dG-dC), and 1.25 µg of acetylated BSA. In
some experiments, nuclear extracts were incubated with anti-Oct-1,
anti-Oct-2, anti-HMGI, or preimmune sera for 120 min at 4°C
before addition to the binding. LT-E probe was generated by
AccI and StuI enzymatic digestion of LT 5' DNA
(19) and end-labeled by Klenow DNA polymerase by using
[-32P]dATP and
[-32P]dCTP (3000 Ci/mmol; Amersham Pharmacia
Biotech, Piscataway, NJ). Desired fragments were gel purified on an 8%
nondenaturing polyacrylamide gel and eluted by soaking in TE
buffer.
Antibodies
Anti-Oct-1 and anti-Oct-2 peptide polyclonal Abs were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-HMGI-Y was from Ray Reeves (Washington State University).
Genomic deletion constructs
The intact Ly-6E gene described in Ref.
20 was the parent construct for six mutants made in the 5'
flanking region diagrammed in Fig. 1
. A
double digest with enzymes that cleave the construct only once followed
by religation was used to generate the following constructs: Ly-6E.1,
SpeI + AvaI produces a 2000-bp deletion; Ly-6E.2,
SpeI + KpnI produces an 1150-bp deletion;
Ly-6E.3, KpnI + AvaI produces an 850-bp deletion;
Ly-6E.4, NsiI produces a 170-bp deletion and Ly-6E.5, by
partial digestion with BsaI + PflmI, produces a
130-bp deletion. Ly-6E.6 was produced by transferring KpnI +
AvaI fragment derived from a genomic clone into
KpnI + BamHI site of plasmid pBSKS. An
oligonucleotide primer spanning the sequences from -1990 to -1970
containing a 4-bp mutation,
5'-CGGATGCATATTCCCGGCCGTGACCTCACCCATCC-3',
and the T7 of pBSKS+ were used with the above
template in a PCR. The NsiI fragment generated from
digestion of this reaction was cloned into the NsiI site of
the Ly-6E.4 plasmid.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A genomic clone containing 3.2 kb of the 5' flanking sequence of
the Ly-6E gene was transfected into T cells, and cell surface
expression was analyzed by FACS (Fig. 2).
The pattern of expression of this clone is very similar to the
endogenous gene both in terms of basal levels and IFN inducibility.
Further deletion analysis of this clone was performed to identify
regulatory elements responsible for IFN inducibility (Fig. 1
). These
constructs were stably transfected into BW5147 and EL4 cells. Both of
these cell lines express the endogenous Ly-6A gene product, which can
be differentiated from the transfected Ly-6E Ag by mAbs. Stable cell
lines were established from single colonies, and expression of the
transfected Ly-6E Ag was assessed using the specific mAb SK70.94.
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and Table I show basal,
IFN-ß-inducible, and IFN--inducible levels of Ly-6E Ag on
BW5147 transfectants. Clones derived from transfection of deletion
construct Ly-6E.1, which lacks the region between -2900 and -900,
show no basal and no IFN-inducible Ly-6E expression. Transfection of
the Ly-6E.2 construct, which lacks the region from -2900 to -1760,
demonstrated basal levels of Ly-6E Ag, comparable to the amount
expressed by the wt (wild-type) Ly-6E construct. However, IFN-ß
inducibility was lost, and IFN- inducibility was significantly
reduced in these clones. Thus, in BW5147 cells, the distal region
between -2900 and -1760 contains one of the necessary elements for
IFN inducibility of Ly-6E gene.
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ß- or
IFN--inducible Ly-6E Ag was observed in these transfectants (see
Table I). Transfection of deletion constructs Ly-6E.1 and Ly-6E.2 into
EL4 cell line yielded clones that showed no basal and no IFN-inducible
Ly-6E Ag. These results confirmed the functional role of this region
between -2900 and -1760 for IFN-ß and IFN- inducibility in
both T cell lines.
The Ly-6E.3 construct, which lacks the region between -1760 and -900,
was used to derive stable transfectants in both EL4 and BW5147 cell
lines. Surprisingly, transfectants in both cell lines showed a 20-fold
higher basal level of Ly-6E Ag (Fig. 2 and Tables I and
II). Interferon inducibility could not be
assayed due to the high level of basal Ly-6E expression. Such high
basal expression suggests the presence of a negative regulator in
addition to the IFN-responsive elements in this region. Considering the
data from all the deletions suggests the possibility of at least two
distinct regions required for IFN responsiveness: one in the distal
fragment (-2900 to –1760) and the other in the middle part (–1760 to
–900) of the promoter. Further deletion analysis was done to localize
the regulatory region in the middle portion of the promoter.
Construct Ly-6E.4 lacks the region -1220 to -1050 (170 bp), and
construct Ly-6E.5 lacks the region -1341 to -1211 (130 bp). Stable
transfectants derived from Ly-6E.4 and Ly-6E.5 in BW5147 cells resulted
in normal basal expression of Ly-6E Ag; however, in both cases,
IFN-ß and IFN- inducibility was lost in all clones tested.
These two deletions overlap the G region and point toward the
regulatory importance of this region in T cells. This region was
previously shown to contain the Ly-6E GAS element to which STAT1
binding was demonstrated in BALB3T3 fibroblasts (13). To
confirm the functional importance of this region in T cells, we
generated the Ly-6E.6 construct that contains a 4-bp mutation within
the STAT1-binding site in the genomic clone (see Table III). Basal expression of Ly-6E Ag was
seen on stable transfectants from this construct, but both IFN-ß
and IFN- inducibility was absent in all clones assayed.
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The G region binds Oct transcription factors
We have identified multiple DNA binding factors in nuclear
extracts from EL4 cells that specifically recognize the G region in
DMSA (Fig. 3). The results were similar
with extracts from untreated and IFN--treated cells. Sequence
analysis revealed a region between -1230 and -1220 having the
sequence 5'-ATGCATAT-3' on the top strand, and 5'-ATGCATAA-3'on the
reverse strand (displaced by 2 bp) that is very similar to the
consensus octamer motif, 5'-ATGCAAAT-3', which is found in Ig gene
promoters and enhancers (see Table III). Fig. 3 shows that, despite the
lack of a consensus Oct motif, all the complexes detected by G probe
except factor X were specifically reduced by the consensus Oct and not
by the SIE oligonucleotide competitors. As previously shown, Oct-1 has
a lower mobility than Oct-2 in DMSA (22); hence, Oct-1 is
absent in A20-2J nuclear extracts.
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. As
expected, anti-Oct-2 Ab blocks the formation of the lowest complex
while anti-Oct-1 Ab produces an additional complex of lower
mobility, the so-called supershift (23). However,
DNA-protein complexes detected with radiolabeled Oct probe comigrate
with two complexes labeled Oct-1 and Oct-2 in Fig. 4 (data not shown).
Mutational analysis was undertaken to define the crucial DNA contacts
for the binding of these proteins. The G-M2 mutation consists of a 4-bp
change within the Oct motif; the G-M3 mutation has a 3-bp change within
the previously defined STAT1-binding site; and the G-M4 mutation
contains a single change from T to C within the GAS site. None of these
mutants was able to bind any proteins, except that G-M2 detected factor
X, the intermediate mobility complex (see Fig. 3). Furthermore, Abs had
no consistent effects on complexes detected with G-M2 probe.
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activity of the G region on a heterologous promoter in a transient
transfection analysis is demonstrated in Fig. 5. IFN- inducibility of the
G-M2-tk-Luc reporter construct is reduced by about 40% when compared
with the G-tk-Luc reporter construct, and IFN- inducibility of the
G-M3-tk-Luc reporter construct is completely lost. Together these
results suggest that the G region can confer IFN inducibility onto a
heterologous promoter and that this induction is in part mediated by
the binding of the members of the Oct family proteins. It is
interesting to note that the binding of Oct-1 and Oct-2 is dependent on
the adjacent GAS site, in addition to octamer sequence.
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HMGI proteins are small, highly charged proteins that constitute
an important component of active chromatin structure. HMGI(Y) is a
member of this group that specifically recognizes the minor groove of
double-stranded poly(dA-dT) DNA. It has been implicated in the
regulation of various genes including the lymphotoxin gene
(19). Given the complexity of interactions in the G region
and the precedence of involvement of HMGI proteins in combination with
Oct factors in the regulation of HLA-DR (24), we
hypothesized the involvement of HMG-like proteins in facilitating these
interactions. Fig. 6 depicts a DMSA
performed under conditions specific for detection of HMGI-like
proteins. In this assay nuclear extracts from EL4 cells were assayed
with G and the G-M2 probes. A specific fast migrating complex that is
characteristic of HMGI protein was seen with the G probe. Probe
containing mutations in octamer site (G-M2) was unable to detect this
complex (Fig. 6). Similarly, the GAS site mutants (G-M3 and G-M4) did
not detect this complex from nuclear extracts (data not shown). These
results suggested specific binding of an HMG-like protein in this
region.
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(lanes 2–4), a
specific retarded complex detected by LT-E probe was competed
effectively by competitor G sequence. The same complex was almost
completely inhibited in the presence of anti-HMGI(Y) Ab. Similar
specific binding was demonstrated by G region probe and recombinant
protein (Fig. 7, lanes 6–8).
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ß and - inducibility of the
endogenous Ly-6A gene in EL4 cellsTo assess the physiologic role of HMGI proteins in IFN inducibility of the Ly-6 gene in EL4 cells, we attempted to block the synthesis of HMGI proteins in these cells by expression of antisense sequences. Transfection of an antisense construct for HMGI-C cDNA has been shown to effectively inhibit HMGI-C, as well as HMGI(Y) protein synthesis previously (25). The exact reason for the effect of this antisense construct on the expression of both proteins is not known, but the high level of sequence similarity between the two is a possible explanation.
EL4 cells express very low basal levels of Ly-6A when stained with mAb
Sca-1, which recognizes a common epitope between Ly-6A and -E Ags.
Treatment with IFN-ß and IFN- induces significant expression of
this Ag. Fig. 8 shows the effect of
transfection of the HMGI-C antisense construct on the expression of
Ly-6A Ag and its IFN inducibility via FACS analysis. In 9 of the 11
clones, IFN-ß and IFN- inducibility of Ly-6A Ag was
substantially reduced. As a control for nonspecific effects of this
transfection, the expression of TCR was examined using the anti-CD3
mAb 29B in these transfectants. The level of TCR expression was similar
to the wt EL4 cells on all of these transfected clones.
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shows the effects of antisense
HMGI-C on the expression of HMGI proteins in DMSA utilizing the LT-E
probe. Parental EL4 cells demonstrate two complexes, designated R1 and
R2. In nuclear extracts obtained from an antisense transfected clone 7,
the R2 complex is absent. Fashena et al. have previously shown that
multiple retarded complexes are detected from nuclear extracts using
this probe (19). These are thought to be due to
interactions of different affinities, both as a consequence of the
propensity of these proteins to multimerize and to their ability to
recruit other proteins into these complexes. Furthermore, IFN-ß-
and IFN--induced ISGF3 and IFN- activation factor (GAF) complexes
are not affected in this clone, thereby showing no alteration in the
levels of transcription factors directly involved in the IFN signal
transduction pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production after antigenic stimulation of
CD8+ T cells, suggesting that it is a marker for
the memory phenotype (7). Since IFNs are the most
important physiologic inducers of the murine Ly-6 genes, we have been
studying the underlying molecular mechanisms. To define the regulatory regions necessary for IFN inducibility, we undertook a functional analysis of the Ly-6E promoter in two T cell lines, EL-4 and BW5147. Genomic deletion analysis identified multiple regulatory regions within the Ly-6E promoter to be necessary for IFN inducibility of this gene. These included a distal regulatory region within the Ly-6E promoter upstream of -1760 and a complex regulatory region defined as the G region around –1220 of the promoter, which contains the previously characterized GAS site functional in fibroblasts (13). Interestingly, the distal regulatory region was found to be essential for IFN inducibility in B cells while the G region was dispensable for IFN responsiveness in these cells (21). Characterization of transcription factors involved in T cell lines was done by using DNA probes derived from the G region in DMSA.
Multiple DNA-binding factors from nuclear extracts of EL-4 cells were
detected by these DNA probes. Two of these factors were identified to
be Oct-1 and Oct-2 by specific Abs, and a sequence similar to the
consensus octamer is present in this region. Mutational analysis
was conducted to confirm the specificity of these DNA-protein
interactions. Surprisingly, the binding of Oct family members requires
the GAS site in addition to the octamer sequence. A single base pair
mutation in the GAS site abolished detectable binding of all proteins
normally detected in DMSA by this probe. Reporter gene analysis
carrying these mutations (G-M2 and G-M3) confirmed the functional
importance of these elements. The G-M2 mutation caused about 40%
reduction in IFN- inducibility while the G-M3 mutation totally
abolished IFN responsiveness. These data strongly support the
involvement of Oct factors in IFN inducibility of this gene in T
cells.
Oct-1 is a ubiquitous transcription factor required for constitutive transcription of the SnRNA genes and the cell cycle-specific expression of the histone H2B gene (27). Oct-2, on the other hand, is involved in the transcriptional control of Ig genes in B lymphocytes. In T lymphocytes, Oct-2 is induced in response to antigenic stimulation and plays a role in gene expression during T cell activation (24). Although the involvement of Oct family transcription factors in IFN-regulated gene expression is unprecedented, there are examples of cooperative interactions between multiple transcription factors belonging to different families to achieve inducible gene expression. The hallmark of such nucleoprotein complexes is their dependence upon the binding of chromatin-associated HMGI proteins. HMGI proteins are sequence-specific DNA-bending proteins that facilitate cooperative assembly of enhanceosomes (28, 29, 30).
There are similarities between the IFN-mediated induction of Ly-6E gene and the established examples of enhanceosome, i.e., the need for the involvement of multiple regulatory elements, distal region and G region and the binding of multiple proteins belonging to different families of transcription factors. Furthermore Oct-2 binding to the HLA-DR promoter has been shown to be facilitated by HMGI (24). Given these considerations, we investigated the binding of HMGI proteins to regulatory elements of Ly-6E enhancer. DNA probe derived from G region was able to detect a fast-migrating complex characteristic of HMGI protein. Experiments with competitor sequences, recombinant HMGI protein, and mutational analysis confirmed the specificity of binding of HMGI in this region. Furthermore binding of HMGI was dependent on both the Oct- and STAT-binding sites.
To address the biological role of the binding of HMG proteins to the G
region, we employed a genetic approach. The previously described
antisense construct was used to inhibit the expression of HMGI(Y) and
HMGI-C proteins in EL4 cells, and IFN inducibility of the endogenous
Ly-6 gene was assayed by cell surface expression. There was complete
loss of IFN-ß responsiveness while IFN--mediated induction was
significantly reduced. The preservation of some response to IFN-
provides an internal control for the lack of any nonspecific effects of
the antisense RNA on some other aspect of the Ly-6 expression. This
experiment, when taken together with the above findings, suggests that
members of the HMGI proteins play a role in promoting interactions
between transcription factors of different families to achieve a
cell-type specificity observed in IFN inducibility of Ly-6E gene.
Further support for this model comes from our other studies
(21) where it has been shown that the G region is not
needed for IFN- or IFN-ß responses in B cell lines. IFN
inducibility in B lymphocytes is achieved by a different set of
regulatory elements present in the Ly-6E promoter.
In these studies we have not observed any inducible factor binding to the GAS site in the G region in response to IFN treatment. However, in a separate study (21), we defined a distal IFN responsive region that contains an ISRE and is bound by STAT1. This is a 56-bp region located at -2300 in the Ly-6E promoter and is functional in B cell lines A20 and M12, as well as in T cell lines BW5147 and EL4, but not in fibroblasts. Since this region is lacking in the Ly-6E.2 deletion, this would explain the loss of IFN inducibility of this construct. The necessity of both regions (distal STAT1 site and G region) for IFN response in T cells is demonstrated by the lack of inducibility of both Ly-6E.2 and Ly6-E.6 mutants. These data suggest that a cooperative interaction between the STAT1-containing complex binding to the distal ISRE site and multiple other complexes around the G region could be the basis of IFN responsiveness in T cells. On the other hand, the G region alone is capable of conferring IFN inducibility to a heterologous promoter linked to reporter luciferase construct. This result is consistent with the presence of a negative element in this region of the intact gene, which is functional only in T cells and not in B cell lines. An alternative explanation for these results is the possibility that the data from the reporter constructs are quite distinct from the physiologic regulation of the intact gene.
Presence of STAT1 in the extracts of IFN-treated EL4 T cells is
demonstrated by the SIE probe in DMSA (Fig. 9B), under the
same conditions in which the G region probe was unable to detect any
IFN-inducible complex. We believe STAT1 participation in direct binding
to the GAS site in the G region is not only possible but it is a
plausible explanation for the inducibility of the luciferase reporter
constructs. However, its presence in the DMSA when using G region
probes is probably obscured by the close proximity of multiple
DNA-binding sites within the G region and the relative abundance of the
other proteins in lymphoid cell lines, i.e., Oct, HMGI, and X, which
constitutively bind to this region. Given the complexity of these
interactions in lymphoid cells, we cannot rule out the possibility of
involvement of some hitherto unrecognized protein(s) in this regulation
as well. Furthermore, such a protein could participate via
protein-protein interactions and hence would not be detected by assays
based upon DNA binding employed in this study.
In conclusion, these data suggest that, for IFN inducibility of the Ly-6E gene, there is a requirement for complex binding of multiple transcription factors that is facilitated by the presence of HMGI proteins and that these factors are assembled in a cell type-specific manner.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alfred Bothwell, Section of Immunobiology, PO Box 208011, Yale Medical School, 310 Cedar Street, New Haven, CT 06520. E-mail address:
3 Abbreviations used in this paper: JAK, Janus kinase; HMG, high mobility group; rhuHMG, recombinant human HMG; wt, wild type; MFI, mean fluorescence intensity; DMSA, DNA mobility shift assay; GAS, IFN- activation site; ISRE, IFN-stimulated response; SIE, serum inducible element; NCE, nuclear cell extract.
Received for publication October 20, 1998. Accepted for publication May 4, 1999.
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, consists of multiple interacting polypeptide chains. Proc. Natl. Acad. Sci. USA 87:8555. regulated DNA-binding factor. EMBO J. 10:927.[Medline]
/ß and requires a single DNA element to which a tyrosine phosphorylated 91 kDa protein binds. Proc. Natl. Acad. Sci. USA 90:6806. promoter. J. Immunol. 150:4376.[Abstract]
/ß (IFN-/ß)- and IFN--mediated induction of Ly-6E gene in B cells. Blood 92:2399.This article has been cited by other articles:
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 K.-Y. Chau, N. Munshi, A. Keane-Myers, K.-W. Cheung-Chau, A. K.-F. Tai, G. Manfioletti, C. K. Dorey, D. Thanos, D. J. Zack, and S. J. Ono The Architectural Transcription Factor High Mobility Group I(Y) Participates in Photoreceptor-Specific Gene Expression J. Neurosci., October 1, 2000; 20(19): 7317 - 7324. [Abstract] [Full Text] [PDF]
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D. L. Pflugh, S. E. Maher, and A. L. M. Bothwell Ly-6I, a New Member of the Murine Ly-6 Superfamily with a Distinct Pattern of Expression J. Immunol., July 1, 2000; 165(1): 313 - 321. [Abstract] [Full Text] [PDF]
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