From the Departments of Neuroscience,
Biochemistry and Molecular Biology,
§ Pediatrics, and ¶ Anesthesiology, College of
Medicine, University of Florida, Gainesville, Florida 32610
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ABSTRACT |
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Mitochondrial manganese superoxide dismutase
(Mn-SOD) is the primary cellular defense against damaging superoxide
radicals generated by aerobic metabolism and as a consequence of
inflammatory disease. Elevated expression of Mn-SOD
therefore provides a potent cytoprotective advantage during acute
inflammation. Mn-SOD contains a GC-rich and TATA/CAAT-less
promoter characteristic of a housekeeping gene. In contrast, however,
Mn-SOD expression is dramatically regulated in a variety of
cells by numerous proinflammatory mediators, including
lipopolysaccharide, tumor necrosis factor- Reactive oxygen species produced during both normal cellular
function and, most importantly, as a consequence of the inflammatory response, have been implicated in the initiation and propagation of a
variety of pathological states (1, 2). The superoxide dismutases
(SODs)1 are the primary
cellular defense that has evolved to protect cells from the deleterious
effects of oxygen free radicals (3, 4). Three forms of SOD have been
identified in eukaryotic cells: the cytoplasmic copper/zinc SOD
(Cu,Zn-SOD), the extracellular Cu,Zn-SOD, and the mitochondrial
manganese SOD (Mn-SOD). In contrast to the cytoplasmic Cu,Zn-SOD, which
is expressed constitutively in most cases, Mn-SOD expression
is highly regulated by a variety of proinflammatory mediators
(5-9).
Recent data have decisively demonstrated the critical cellular
importance of Mn-SOD in a variety of different tissues. For example,
homozygous mutant Mn-SOD mice die within 10 days of birth, exhibiting severe dilated cardiomyopathy, lipid accumulation in liver
and skeletal muscle, and metabolic acidosis, as well as decreased
enzyme activity of aconitase, succinate dehydrogenase, and cytochrome
c oxidase, which are all extremely sensitive to alterations
in the cellular redox state (10). Additionally, transgenic mice
expressing elevated levels of human Mn-SOD in lung tissue were highly
protected from hyperoxic injury after exposure to 95% oxygen and thus
survived longer than nontransgenic littermates (11). Overexpression of
Mn-SOD has also been implicated in the suppression of
tumorigenicity of human melanoma cells (12), breast cancer cells (13),
and glioma cells (14). Alterations in Mn-SOD levels have also been
associated with a number of neurodegenerative diseases, including
Parkinson's disease (15), Duchenne muscular dystrophy,
Charcot-Marie-Tooth disease, and Kennedy-Alter-Sung syndrome (16).
Mn-SOD synthesis in eukaryotic cells is up-regulated dramatically by
proinflammatory mediators including lipopolysaccharide (LPS), tumor
necrosis factor alpha (TNF- Although highly regulated, Mn-SOD contains a GC-rich
promoter lacking a TATA- and a CAAT-box. This promoter architecture was originally associated with housekeeping genes that are constitutively expressed (22). The additional layer of transcriptional regulation of
this gene differentiates it from most housekeeping genes.
Unfortunately, current knowledge about the molecular mechanisms
controlling transcriptional regulation from promoters that lack a TATA-
and CAAT-box is limited. Most of the studies addressing regulation of
TATA- or CAAT-less promoters have focused on either the initiator
element (Inr), which controls transcriptional initiation (23), or the
general transcription machinery, especially TFIID (24-26) and, most
recently, TFII-I (27). In addition, most studies have analyzed
transcription from TATA- or CAAT-less promoters by employing naked DNA
templates in vitro, a model system that may not adequately
reflect the physiological situation.
In order to explore the mechanism of the transcriptional regulation of
this unique TATA- and CAAT-less gene promoter, we first investigated
alterations in the chromatin structure of rat Mn-SOD by
mapping regions hypersensitive (HS) to cleavage by DNase I in both
control and stimulated cells. To further delineate the binding of
specific transcription factor(s) in the proximal promoter of
Mn-SOD, we used in vivo footprinting coupled with
ligation-mediated polymerase chain reaction (LMPCR) to screen the
region surrounding the prominent promoter HS site and the
transcriptional initiation site in both control and induced cells. Our
findings have provided evidence for the molecular architecture within
the promoter of this TATA and CAAT-less gene.
Cell Culture--
The L2 rat pulmonary epithelial-like cell line
(ATCC CCL 149) was grown as a monolayer in 150-mm cell culture dishes
containing Ham's modified F12K medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum, 10 µg/ml penicillin G, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37 °C in
humidified air with 5% CO2. At approximately 90%
confluence, cells were treated with 0.5 µg/ml Escherichia
coli LPS (E. coli serotype 055:B5, Sigma), 10 ng/ml
TNF- DNase I Hypersensitive Site Studies--
We employed isolated
nuclei for low resolution DNase I HS site studies and permeabilized
cells to analyze HS site 1 at high resolution. To analyze DNase I HS
sites at low resolution, nuclei were isolated essentially as described
(28). For high resolution DNase I HS site studies, L2 cells were
permeabilized in 4 ml of cold nuclear isolation buffer (60 mM KCl, 15 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 5 mM MgCl2, 0.25 M sucrose, 1 mM dithiothreitol, and 60 mM Tris-HCl, pH 8.0) containing 0.05%
lysophosphatidylcholine (Sigma) for 1 min on ice (29). Aliquots of
approximately 3 × 107 permeabilized cells/ml of
nuclear isolation buffer were digested with increasing concentrations
of DNase I (Worthington) at 37 °C for 4 min. Digestions were
terminated by dilution to a final concentration of 1% SDS, 100 mM EDTA, pH 8.0, and 50 µg/ml proteinase K. Genomic DNA
was purified by incubation at 55 °C for 3 h followed by organic
extractions and precipitation with ethanol. Samples were then treated
with 100 µg/ml RNase A, organically extracted, precipitated, and
suspended in TE (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). DNase I-digested DNA was restricted with an
appropriate enzyme and size-fractionated on either of two gel systems
to achieve the desired resolution: a low resolution 1% agarose gel
(FMC BioProducts) in TAE buffer, pH 7.8 (40 mM Tris, 3 mM NaOAc·3H2O, 1 mM EDTA, 4 mM NaOH) to resolve DNA fragments ranging from 0.5 to 15 kilobase pairs or a high resolution procedure that utilizes a 2.5%
Metaphor agarose gel (FMC BioProducts) in TAE buffer to resolve DNA
fragments ranging from 0.1 to 1 kilobase pair. The DNA was
alkaline-denatured, electrotransferred to a nylon membrane, and
cross-linked to the membrane with UV light (30). The membrane was
prehybridized in a buffer containing 0.5 M sodium
phosphate, pH 7.2, 7% SDS, 1% bovine serum albumin, and 1 mM EDTA at 65 °C for 15 min. The DNA fragments used for
probe templates were isolated by restriction digestion of various
subclones of the rat Mn-SOD genomic clone (31) and
radiolabeled using a random primer DNA labeling system (Life
Technologies, Inc.). Hybridization and autoradiography were performed
as described (8).
In Vivo Dimethyl Sulfate (DMS) Treatment--
L2 cells at 90%
confluence were washed with room temperature phosphate-buffered saline
buffer and then replaced with phosphate-buffered saline containing
0.5-0.25% DMS for 1-2 min at room temperature. The cell monolayer
was washed with 4 °C phosphate-buffered saline to quench the DMS
reaction. The cells were lysed in 67 mM EDTA, pH 8.0, 1%
SDS, and 0.6 mg/ml proteinase K and incubated overnight at room
temperature. Genomic DNA was purified as described for DNase I HS site
studies. The DNA samples were digested with BamHI, and
strand cleavage at modified guanine residues was achieved by treatment
with 1 M piperidine at 90 °C for 30 min. Naked genomic DNA was harvested and purified from cells without any DMS treatment and
digested with BamHI. The in vitro
guanine-specific reaction was performed as described (30).
LMPCR--
The LMPCR was performed as described previously (32).
The following primers were used for LMPCR. For the top strand primer sets, the following were used: A, primer 1, 5'-TTGTGCCGCTCTGTTACAAG-3' and primer 2, 5'-GTGTCGCGGTCCTCCCCTCCGTTGATG-3'; B, primer 1, 5'-ATTGTAGCTCACAGGCAGAG-3' and primer 2, 5'-GGGCCTAGTCTGAGGGTGGAGCATA-3'; C, primer 1, 5'-TGATTACGCCATGGCTCTGA-3' and primer 2, 5'-TCTGACCAGCAGCAGGGCCCTGGCTT-3'; D, primer 1, 5'-GTTAATTGCGAGGCTGGCAA-3' and primer 2, 5'-CCCTAACCTCAGGGGCAACAAAG-3'; E, primer 1, 5'-GTCGTTTTACATTTATGGTGG-3'
and primer 2, 5'-GGGTTTAGTCAGGAAAGATGAACCTGGC-3'; F, primer 1, 5'-GGAAAAACCACCCGGAAC-3' and primer 2, 5'-CAGTGGCAGAGGAAAGCTGCC-3'. For
the bottom strand primer sets, the following were used: G, primer 1, 5'-CATAGTCGTAAGGCAGGTCA-3' and primer 2, 5'-GTCAGGGAGGCTGTGCTTGTGCCG-3'; H, primer 1, 5'-GCCGAGACCAACCAAA-3' and
primer 2, 5'-GCCGCCCGACACAACATTGCTGAGG-3'; I, primer 1, 5'-CTGCTCTCCTCAGAACA-3' and primer 2, 5'-AACACGGCCGTTCGCTAGCAGCC-3'; J,
primer 1, 5'-ATCAACGGAGGGGAGGA-3' and primer 2, 5'-CGGCCCAGCTTGTAACAGAGCGGCAC-3'; K, primer 1, 5'-CGGTGTGGCTATGCT-3'
and primer 2, 5'-GCTCCACCCTCAGACTAGGCCCCGCCT-3'; L, primer 1, 5'-CTTTTCCATTCCTGGTTCTGG-3' and primer 2, 5'-CAGAGCCATGGCGTAATCAGGGGCCT-3'; M, primer 1, 5'-CATCTCAGGTTTTAGTGTGTTC-3' and primer 2, 5'-CTTTGTTGCCCCTGAGGTTAGGG-3'. The polymerase chain reaction
products were size-fractionated on a 6% denaturing polyacrylamide gel,
electrotransferred to a nylon membrane, and covalently cross-linked to
the membrane by UV irradiation. The membrane was hybridized with an M13
single-stranded probe using our isolated genomic clone as template and
promoter-specific primers for extension, washed as described for DNase
I hypersensitive site studies, and exposed to x-ray film.
In Vitro DMS Footprinting--
The rat Mn-SOD promoter from
position
Sp1 (26 pmol) and/or GKLF (170 pmol) was incubated with linearized
pCR2.1 containing the rat Mn-SOD promoter in 10 mM Tris, pH
7.5, 75 mM KCl, 1 mM dithiothreitol, 0.5 mM MgCl2, and 5 µM ZnCl2 followed by DMS treatment in vitro.
DMS-modified protein-DNA complexes and naked template DNA were cleaved,
gel-fractionated, and transferred to a nylon membrane as described
above. Two oligonucleotides, 5'-ACACGGCCGTTCGCTAGCAGCC-3' and
5'-TCTGACCAGCAGCAGGGCCCTGGCTT-3', were used as hybridization
probes as described (34) for visualization of top and bottom strand
sequences, respectively.
Transient Transfection Analysis--
Transfection efficiency was
controlled by using a batch transfection method. Cell monolayers were
grown to 70-90% confluency on 150-mm plates. Cells were transfected
with 10 µg of each expression vector using the DEAE-dextran method
(34). After 24 h, cells were trypsinized, pooled, and plated onto
four separate 100-mm tissue culture plates. Inflammatory mediators were
added to the medium of each plate 24 h later as described above.
Twenty-four hours later, total RNA was isolated from the cell
monolayers for Northern analysis as described (8). Human growth hormone
was assayed from the media of individual 100-mm plates incubated for
48 h after the addition of LPS, TNF- Construction of Expression Vectors--
A 4.5-kb
EcoRI/EagI fragment of 5' noncoding sequence was
isolated from the 17-kb Mn-SOD genomic clone (31), and the
5' overhang ends were filled in using the large fragment of E. coli DNA polymerase I (Klenow fragment). The resulting blunt end
Eco/Eag fragment was cloned into the HincII polylinker site
in a promoterless, pUC 12-based human growth hormone expression vector,
p Chromatin Structure of Mn-SOD--
The induction of
Mn-SOD mRNA by LPS, TNF-
Since de novo transcription is necessary for the induction
of Mn-SOD by LPS, TNF-
Since no differences in chromatin structure were detected between
control and induced cells, we hypothesized that stimulus-specific alterations in chromatin structure may occur within each HS site. HS
site 1 was chosen based on its proximity to the transcriptional initiation site and because this site is highly sensitive to cleavage by DNase I. In order to display the structure of the HS site in the
promoter in more detail, we digested genomic DNA with BamHI, which cleaves near HS site 1 followed by Southern analysis with probe
BH. This strategy expanded the separation of the DNase I-cleaved fragments and led to the discovery that HS site 1 was composed of three
subsites (marked with stars in Fig. 1) in control and stimulated cells. Most interestingly, a fourth subsite became strongly apparent only in the samples derived from LPS-, TNF-
To better resolve potential stimulus-dependent alterations
in chromatin structure, we developed an approach to evaluate changes within HS site 1 at higher resolution. The DNase I cleavage pattern was
analyzed by fractionation on a 2.5% Metaphor agarose gel. This gel
accurately resolves fragments differing in size by as little as 2% in
the range of 200-800 bp. In addition, we utilized control and LPS-,
TNF- In Vivo Footprinting of the Mn-SOD Promoter--
Low resolution
(Fig. 1) and high resolution DNase I HS site studies (Fig. 2) suggest
that important cis-acting regulatory elements may exist in
the promoter region of Mn-SOD in both control and stimulated
cells. We therefore employed in vivo footprinting using DMS
as a molecular probe coupled with LMPCR to resolve these cis-acting elements at single nucleotide resolution and thus
display the in vivo protein-DNA contacts. The position of
each LMPCR primer set is shown in Fig.
3A. In order to verify that
the kinetics of transcription factor binding are stable throughout the
period of induction with LPS, we tested both control and stimulated
samples at 0.5, 4, and 8 h of treatment. These experiments
demonstrated that the observed protein-DNA contacts are detectable as
early as 0.5 h and as late as 8 h after the addition of LPS
with representative examples from each time point illustrated in Figs.
3 and 4.
Fig. 3B illustrates in vivo footprinting and
LMPCR results for control and 0.5-h LPS-treated samples for the top
strand of the promoter from position Involvement of Sp1 and GKLF with Binding Sites I-V--
A
computer analysis of binding sites I-III shows a high degree of
homology with the consensus DNA binding sequence and the protein-DNA
contact pattern of Sp1 (36), thus suggesting that these sites may be
occupied by Sp1 or Sp1-like proteins. Furthermore, binding sites IV and
V contain the minimal essential DNA binding sequence,
5'-(G/A)(G/A)GG(C/T)- G(C/T)-3', of a recently cloned transcription
factor, GKLF (33). This is further substantiated by Zhang et
al. (37), who have presented data implicating a physical
interaction of Sp1 and GKLF. We therefore evaluated the interaction of
these proteins with a plasmid (pCR2.1, Invitrogen) containing the rat
Mn-SOD promoter (from
As shown in Fig. 5, either GKLF alone
(lane 1) or Sp1 alone (lane
2) is capable of binding to sites I-V. In addition, we
evaluated the cooperative effects of a constant amount of Sp1 in the
presence of increasing quantities of GKLF (lanes
3-5). These data implicate the potential cooperative
interaction of Sp1 and GKLF with our 5'-most binding sites originally
identified in vivo (Figs. 3 and 4). An analogous computer
analysis of the region harboring binding sites VI-X has identified a
number of putative candidate transcription factors. However, a
comparison of the protein-DNA contact pattern as well as the
physiological functions of these candidates has led us to conclude that
these binding sites are most likely occupied by a set of novel basal
transcription factors.
Detection of Stimulus-specific Enhanced Guanine Residues--
As
described previously, an LPS-, TNF-
As a matter of completeness, recent studies by Das et al.
(38) have reported that there is a relationship between the activation of NF- Detection of an Enhanced Cytosine Residue at +51--
The Inr
(YYA+1N(T/A)YY) (41) and downstream promoter element (DPE)
((A/G)G(A/T)CGTG) (24), typically located at approximately +30, have
been shown to be important core elements for the regulation of
TATA-less promoter genes. A computer analysis of these downstream sequences has revealed a reverse Inr-like sequence between Promoter Deletion Analysis of the Mn-SOD--
To address the
functional significance of the cis-acting elements
identified by in vivo footprinting, we used unique
restriction sites within the Mn-SOD 5'-flanking sequence to
create a series of promoter deletions in a human growth hormone (hGH)
reporter plasmid, as depicted in Fig.
8A. We evaluated promoter
function by using vectors containing various Mn-SOD promoter
fragments following transfection into L2 cells, with measurement of
both secreted hGH levels (Fig. 8B) and hGH mRNA levels
(Fig. 8C). To ensure that hGH in the medium reflected the
majority of total hGH produced in the transfected cells, we compared
hGH in the cell monolayer with secreted hGH and found that 97% was
secreted into the medium, whereas only 2-3% of total hGH was retained
in the cells (data not shown). In order to exclude the possibility that
induction of hGH is somehow due to the vector itself, we demonstrated
that LPS, TNF-
The results of multiple transfection experiments are shown in Fig. 8,
B and C. As the Mn-SOD promoter was progressively
shortened, the deletions had no effect on either basal or stimulated
hGH expression (mRNA or protein) until the Mn-SOD
promoter fragment was shortened from the SacII restriction
site to the NaeI site, at which point both basal and
stimulated expression were lost. Protein levels (Fig. 8B)
and Northern analysis (Fig. 8C) of hGH were comparable,
suggesting that the cytokine treatments did not significantly affect
translation, post-translational modification, or secretion.
Mn-SOD has characteristics similar to most housekeeping
genes, such as a GC-rich promoter, which lacks both TATA and CAAT boxes. In contrast to most housekeeping genes, however,
Mn-SOD is dramatically regulated in a variety of cell types
by numerous proinflammatory stimuli. There are a few examples of other
housekeeping genes that can be regulated or induced by nutrients or
hormones, such as the dihydrofolate reductase, HMGCoA
reductase (22), pyruvate dehydrogenase Recently, significant progress has been made on the structure and
function of TATA-less promoters (reviewed in Ref. 48). Most of these
studies involved identification of Inrs and characterization of general
transcription factor(s) by using in vitro systems. For
example, TFIID, TFII-I, YY1, or the core RNA polymerase II was found to
bind to the Inr, thus aiding in the nucleation of the preinitiation
complex (48). This preinitiation complex is thought to interact with
upstream activators, such as Sp1, and/or enhancer elements to
facilitate transcriptional initiation at TATA-less promoters.
Furthermore, Burke and Kadonaga (24) have identified a DPE,
(A/G)G(A/T)CGTG, located at +30, which was shown to be important for
the regulation of TATA-less promoter genes. Another potential core
element possibly associated with TATA-less promoters was localized to
both sides of the transcriptional initiation site of the rat catalase
gene (49).
To date, however, we have limited information on the general machinery
involved in the transcription of genes lacking both a TATA and CAAT
box. Specific transcription factors such as Sp1 and the Wilms' tumor
suppressor (WT1) have been associated with the developmental and
neoplastic down-regulation, respectively, of the TATA/CAAT-less
insulin-like growth factor-I receptor (43). Sp1 has also been
associated with the regulation of numerous TATA/CAAT-less genes
including urokinase-type plasminogen activator receptor (44),
T-cell-specific MAL (47), and human Pim-1 (45).
Unfortunately, our knowledge of the molecular architecture of an
inducible TATA/CAAT-less promoter is quite limited. We believe,
therefore, that our chromatin and in vivo footprinting
studies on the transcriptional regulation of Mn-SOD
have defined a collection of constitutive transcription factors that
may delineate the general architecture of an inducible TATA/CAAT-less promoter.
Our data on chromatin structure and in vivo DMS footprinting
are summarized in Fig. 9. HS subsites 1-1 to 1-4 are constitutive HS sites, which are present in both control and
stimulated cells. Their relative positions have been mapped to within
±50 bp and flank the location of three large cis-acting
protein binding regions (binding sites I-III, IV-VIII, and IX-X in
Figs. 3 and 4). Our in vivo footprinting results have been
compared with the vast transcription factor literature, including
existing consensus DNA binding sequences as well as available DMS
in vivo/in vitro footprinting or methylation
interference data. The above analyses led us to postulate that binding
sites I-V may be occupied by either Sp1 or GKLF. This was supported by
our in vitro footprinting studies (Fig. 5), which document
the ability of both GKLF and Sp1 to interact with these binding sites.
The in vitro DMS protection data is also summarized in Fig.
9 and illustrates the high conservation of guanine contacts as compared
with the in vivo results. It is also obvious from the data
in Fig. 5 that GKLF and Sp1 may in some manner compete or possibly
cooperate in the binding of these five sites. Attempts to identify the
proteins that interact with binding sites VI-X have led to the
conclusion that these sites are most likely occupied by a novel set of
trans-acting factors.
, and interleukin-1. To
understand the underlying regulatory mechanisms controlling Mn-SOD expression, we utilized DNase I-hypersensitive (HS)
site analysis, which revealed seven hypersensitive sites throughout the
gene. Following high resolution DNase I HS site analysis, the promoter
was found to contain five HS subsites, including a subsite that only
appears following stimulus treatment. Dimethyl sulfate in
vivo footprinting identified 10 putative constitutive protein-DNA
binding sites in the proximal Mn-SOD promoter as well as
two stimulus-specific enhanced guanine residues possibly due to
alterations in chromatin structure. In vitro footprinting
data implied that five of the binding sites may be occupied by a
combination of Sp1 and gut-enriched Krüppel-like factor. These
studies have revealed the complex promoter architecture of a highly
regulated cytoprotective gene.
INTRODUCTION
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Abstract
Introduction
References
), interleukins-1 and -6 (IL-1 and IL-6),
and interferon-
(6, 7, 9, 17-21). This up-regulation is blocked
completely by actinomycin D, suggesting that the increase in
Mn-SOD mRNA in response to LPS, TNF-
, or IL-1 may
result from an increase in the rate of transcription of
Mn-SOD (7-9, 18), results confirmed by nuclear run-on
studies (data not shown).
EXPERIMENTAL PROCEDURES
(kindly provided by the Genentech Corp.), or 2 ng/ml IL-1
(kindly provided by NCI, National Institutes of Health) for 0.5-8 h to
induce Mn-SOD expression. Untreated cells were used as controls.
467 to +32 was ligated into pCR2.1 (Invitrogen). Human Sp1
protein was purchased from Promega, and the purified gut-enriched
Krüppel-like factor (GKLF) fusion protein containing the
three-zinc finger DNA binding domain was kindly provided by Dr. Vincent
W. Yang (Johns Hopkins University) (33).
, or IL-1
. Each vector
was tested in more than four independent experiments, bringing the
number of independently transfected plates to approximately 16 for each
vector. The concentration of secreted hGH was measured using an
125I-labeled monoclonal antibody assay kit purchased from
Nichols Institute Diagnostics with a lower limit sensitivity of 0.06 ng/ml. Each experimental sample was assayed in duplicate.
GH, (Nichols Institute Diagnostics). Unique restriction enzyme
sites were utilized to delete increasing portions of the
Mn-SOD Eco/E sequence, creating the vectors illustrated in
Fig. 8A.
RESULTS
, or IL-1
may be a
consequence of de novo transcription rate, the prolongation of Mn-SOD mRNA half-life, or both. Co-treatment of
induced cells with actinomycin D indicates that de novo
transcription is necessary for stimulus-dependent gene
expression. To address the contribution of these proinflammatory
mediators to the induced steady-state message levels, nascent
transcription was evaluated in both isolated nuclei and
lysophosphatidylcholine-permeabilized cells. These nuclear run-on
studies showed that LPS, TNF-
, or IL1-
treatment increased the
rate of transcription of Mn-SOD over control by 3-9-fold
(data not shown).
, or IL-1
, we next evaluated
alterations in the chromatin structure of Mn-SOD in response
to these mediators. Nuclei were isolated from control and stimulated L2
cells, a rat pulmonary epithelial-like cell line, and then exposed to
increasing concentrations of DNase I. Purified genomic DNA was then
digested with restriction enzymes near the region of interest and size fractionated on 1% agarose gels. Fig.
1A shows a low resolution Southern analysis of DNase I-digested DNA restricted with
KpnI and indirectly end-labeled with the probe designated
PK, which abuts the 5' KpnI site. HS site 1 maps to the
promoter region of Mn-SOD, whereas HS sites 2-7 map within
the gene itself. These seven HS sites that were found in both control
and stimulated cells are shown on the restriction map of
Mn-SOD in Fig. 1A. We also evaluated regions
approximately 10 kilobase pairs 5' to HS site 1 and 3' to HS site 7 and
detected no additional hypersensitive sites near Mn-SOD. In
addition, DNase I digestion of deproteinated genomic DNA demonstrated
that all of these HS sites are specific to in vivo chromatin
structure (data not shown).
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Fig. 1.
Low resolution chromatin structure analysis
of Mn-SOD. L2 cells were treated with 0.5 µg/ml
LPS, 10 ng/ml TNF- , or 2 ng/ml IL-1
for 4 h, and the nuclei
were prepared as described under "Experimental Procedures" and
incubated with increasing concentrations of DNase I (0, 0 units/ml; 1, 100 units/ml; 2, 150 units/ml;
3, 200 units/ml; 4, 300 units/ml) at 37 °C for
4 min, and genomic DNA was isolated from each treatment. A,
10 µg of DNase I-digested genomic DNA from each sample was restricted
with KpnI and subjected to 1% agarose electrophoresis. The
probe PK (PstI-KpnI fragment) was used to
indirectly end-label the genomic restriction fragments as well as the
set of nested DNase I-cleaved fragments delineating each HS site. Probe
PK is a 0.43-kb PstI-KpnI DNA fragment that
abuts the KpnI restriction site at the 3'-end of rat
Mn-SOD. The seven HS sites are numbered on the
right. The restriction map of rat Mn-SOD shown
above illustrates the relative positions of the probe used
and the HS sites revealed. The location of the DNase I HS sites are
shown with stars and numbered 1-7. HS site 1 is located
within the promoter, and HS sites 2-7 are within Mn-SOD. The five exons of
Mn-SOD are shown as black rectangles.
The codes for the restriction endonucleases are as follows:
B, BamHI; H2, HincII;
H3, HindIII; K, KpnI;
N, NcoI; P, PstI;
R, RsaI. B, cells were treated as
described for A. DNase I-digested genomic DNA was restricted
with BamHI and subjected to electrophoresis in 1% agarose
gel. Probe BH (BamHI-HincII fragment) was used
to display an expanded view of HS site 1 with three subsites appearing
in control and stimulated cells and an inducible subsite (lowest band)
evident in LPS-, TNF-
-, or IL-1
-treated cells.
-, or
IL-1
-treated cells, as shown in Fig. 1B.
-, or IL-1
-stimulated cells permeabilized with
lysophosphatidylcholine rather than isolated nuclei in order to more
efficiently retain intact protein-DNA complexes, as has been previously
reported (35). L2 cells were treated with LPS, TNF-
, or IL-1
for
4 h, permeabilized as detailed under "Experimental Procedures," and incubated with increasing concentrations of DNase I. The DNase I-cleaved genomic DNA was then purified, digested with the
restriction endonucleases HincII and RsaI, and
size-fractionated on 2.5% Metaphor agarose gels. Hybridization with
probe HN revealed five discrete DNase I HS regions, indicated as HS
subsites 1-1 to 1-5 (Fig. 2). The HS
subsites 1-1, 1-2, 1-3, and 1-4 were found in both control and treated
cells, implying that these four HS subsites are involved in basal
expression of Mn-SOD. Most importantly, an inducible HS
site, subsite 1-5, was found only in cells treated with LPS (Fig. 2).
The same subsite was also observed following treatment with TNF-
and
IL-1
(data not shown). This suggests that the promoter region around
HS subsite 1-5 may be involved in specific alterations in chromatin
structure that accompany gene activation.
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Fig. 2.
High resolution chromatin structure analysis
of HS site 1 in the Mn-SOD promoter region using a
2.5% Metaphor agarose gel. L2 cells were treated with LPS for
4 h, permeabilized with 0.05% lysophosphatidylcholine, and
treated with increasing concentrations of DNase I (1, 133 units/ml; 2, 156 units/ml; 3, 185 units/ml;
4, 213 units/ml; 5, 240 units/ml; 6,
267 units/ml) at 37 °C for 4 min. Fifteen micrograms of purified,
DNase I-digested genomic DNA was restricted with HincII and
RsaI and size-fractionated on a 2.5% Metaphor agarose gel.
After electrotransfer, the HS regions were revealed by probe HN. HN is
a 0.32-kb HincII-NcoI DNA fragment adjacent to
the HincII restriction site approximately 0.7-kb upstream of
the transcription initiation site. The locations of the DNase I HS
subsites are numbered 1-1 to 1-5 on the map of the promoter and on the
right side of the autoradiogram. HS subsites 1-1 to 1-4 were found in control as well as treated cells. An inducible HS
subsite, 1-5, was found only in cells treated with LPS or TNF- and
IL-1
(data not shown). The transcriptional initiation site is shown
as an arrow. The codes for restriction enzymes are as
follows: H, HincII; N,
NcoI; R, RsaI.
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Fig. 3.
In vivo DMS footprinting:
identification of basal binding sites I-VI of the Mn-SOD
promoter. A, primer sets used in LMPCR. A total
of 13 primer sets were used to screen 720 bp upstream and 180 bp
downstream with respect to the transcriptional initiation site.
A, B, C, D, E,
and F are top strand primer sets, which were used to screen
bottom strand. G, H, I, J,
K, L, and M are bottom strand primer
sets, which were used to screen top strand. The sequences for primer
sets used for the shown data are under "Experimental Procedures."
The directions of each pair of arrowheads represent 5' 3'. The arrow and +1 designation illustrate the
direction and transcriptional initiation site. B, in
vivo DMS footprinting of the top strand (
286 to
166). Primer
set J was used for LMPCR. Control or LPS-treated cells (30 min)
were exposed to DMS in vivo, and DNA was isolated and
fractionated as described under "Experimental Procedures." The same
results were observed in 4-h LPS-treated samples. Lanes
G, guanine sequence derived from DMS-treated purified
genomic DNA; lanes C, guanine sequence from
in vivo DMS-treated control cells; lanes
L, guanine sequence from in vivo DMS/LPS-treated
cells. Each lane represents individual plates of cells.
Open circles represent protected guanine
residues, whereas filled circles represent
enhanced guanine residues. The arrowheads represent enhanced
adenine residues. Each bar represents an individual binding
site with Roman numeral designation. The
nucleotide positions relative to the transcriptional initiation site
are illustrated on the left. C, in
vivo DMS footprinting of the bottom strand (
258 to
134).
Primer set C was used for LMPCR. Cells were treated for 4 h with
LPS. All of the symbols are identical to those used in
B.
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Fig. 4.
In vivo DMS footprinting:
identification of basal binding sites II-X in the promoter of
Mn-SOD. A, in vivo DMS
footprinting of the top strand ( 254 to
121). Primer set I was used
for LMPCR. For in vivo samples, cells were treated with LPS
for 8 h, and identical results were obtained for 30 min and 4 h LPS-treatment. As in Fig. 3, lanes G,
C, and L reflect in vitro DMS-treated
DNA, in vivo DMS-treated control, and LPS-exposed cells,
respectively. Each lane represents an individual plate of cells.
Open circles represent protected guanine
residues, whereas filled circles represent
enhanced guanine residues. The arrowheads represent
enhanced adenine residues. Each bar represents an individual
binding site with Roman numeral designation. The
nucleotide positions relative to the transcriptional initiation site
are illustrated on the left. B, in
vivo DMS footprinting of the bottom strand (
150 to
31).
Primer set B was used for LMPCR. Cells were treated for 4 h with
LPS. All of the symbols are identical to those used in
A.
286 to
166 relative to the
transcriptional initiation site. Fig. 3C illustrates control
and 4-h LPS-treated samples for the bottom strand. As depicted in Fig.
3, numerous guanine residues exhibited altered DMS reactivity, which
appeared as either diminished or enhanced hybridization signal relative to the in vitro DMS-treated DNA lanes. We have summarized
this in vivo footprinting data by postulating the existence
of protein binding sites at obviously clustered residues and through
symmetry in the contacts and in the DNA sequence. Fig. 3, B
and C, illustrates binding sites for proteins I-VI. Fig. 4,
A and B, illustrates the protein-DNA contacts
seen in cells stimulated for 8 and 4 h of LPS on the top and
bottom strands, respectively. Binding sites II-VII are shown in Fig.
4A, while Fig. 4B illustrates binding sites VI,
VIII, IX, and X. Interestingly, in addition to the guanine contact
sites, we also observed consistently reproducible enhanced adenine
residues marked by arrowheads in Figs. 3 and 4, which are
also clustered near specific binding sites.
467 to +32) using in vitro DMS footprinting.
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Fig. 5.
In vitro DMS footprinting of
binding sites I-V. Lane G represents
protein-free guanine residue ladder. Lanes 1 and
2 show GKLF (170 pmol) and Sp1 (26 pmol) alone,
respectively. Lanes 3-5 represent increasing
amounts of GKLF (40 pmol to 1.0 nmol) with a constant amount of Sp1 (26 pmol). The corresponding in vivo binding sites I-V are
marked as Roman numerals. The protected guanine
residues are depicted as open triangles, and the
enhanced guanine residues are shown as filled
triangles.
-, or IL-1
-inducible HS
subsite 1-5 maps upstream of the constitutive HS sites in the
Mn-SOD promoter (Fig. 2). We further examined this region by
DMS in vivo footprinting and LMPCR. We treated L2 cells with LPS for 30 min, 1 h, 4 h, or 8 h; with TNF-
for
1 h or 4 h; and with IL-1
for 4 h. Fig.
6A illustrates representative
data for LPS treatment with the same results observed for TNF-
and
IL-1
(data not shown). We were able to detect enhanced guanine
residues at positions
404 and
403 on the top and bottom strand,
respectively, only following treatment with LPS (Fig.
6A).
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Fig. 6.
Detection of enhanced guanine reactivity as a
consequence of LPS, TNF- , or
IL-1
exposure. A, in
vivo DMS footprinting of the top (
410 to
393) and bottom
(
415 to
395) strands illustrates LPS-specific guanine enhancements.
Primer set K was employed for LMPCR for the top strand, and primer set
D was used for the bottom strand. Lanes G and
C are designated as in Fig. 3. Lanes L
are in vivo DMS-treated cells, previously exposed to LPS.
Filled circles represent enhanced guanine
residues. The nucleotide positions relative to the transcriptional
initiation site are illustrated on the left. Identical
results were obtained following stimulation with TNF-
or IL-1
(data not shown). B, lack of NF-
B binding on the promoter
region of Mn-SOD. Primer set K was employed for LMPCR for
the top strand (
359 to
350), and primer set D was used for the
bottom strand (
355 to
338). Lanes are designated as in Fig. 3. The
sequence of the putative NF-
B binding site perfectly matches its
consensus DNA binding sequence, GGG(G/A)(C/A/T)T(T/C)(T/C)CC (40).
However, we did not find any protein bound to this putative NF-
B
binding site in vivo on either DNA strand of the
Mn-SOD promoter. The same results were observed for TNF-
-
or IL-1
-treated cells (data not shown).
B and the elevated steady-state levels of Mn-SOD
mRNA by TNF-
or IL-1 in lung adenocarcinoma (A549) cells. In
contrast, Borrello and Demple (39) have recently presented equally
convincing data demonstrating that Mn-SOD gene transcription
can occur independently of NF-
B activation. To address the
importance of NF-
B in the regulation of Mn-SOD gene
expression, we identified a putative NF-
B binding site from
353 to
344 by computer analysis, which displays a perfect identity to the
proposed consensus sequence (40). However, as shown in Fig.
6B, we did not detect any alteration in guanine reactivity
to DMS in vivo on either strand for this putative NF-
B
binding site in LPS-treated cells. The same results were obtained for
both the TNF-
- and IL-1
-treated samples (data not shown).
40 and
34 as well as a putative DPE sequence between +56 and +62 of the
Mn-SOD promoter. As a result of the reported significance of
these elements to gene transcription, in vivo footprinting was performed to further examine the region downstream to the transcription initiation site. Interestingly, we observed an enhanced adenine residue at position
38 on the bottom strand (Fig.
4B) within the Inr-like sequence and an enhanced cytosine
residue at position +51 (Fig. 7), also on
the bottom strand just upstream of the putative DPE site. Furthermore,
treatment with LPS (Fig. 7) dramatically increases the intensity of
methylation by DMS at this cytosine residue, which obviously coincides
with elevated gene transcription.
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Fig. 7.
Identification of an enhanced cytosine
residue at +51-position on the bottom strand of the Mn-SOD
gene. Primer set A was used for LMPCR. Lanes
G, C, and L show in vitro
DMS-treated DNA, in vivo DMS-treated control, and
LPS-exposed (30 min) cells, respectively. Each C and
L lane represents an individual plate of cells.
The enhanced cytosine residue is marked by a star. The
nucleotide positions relative to the transcriptional initiation site
are illustrated on the left.
, and IL-1
had no effect on a vector containing the
minimal thymidine kinase promoter (TKGH) (data not shown) as compared
with the results with a 4.5-kb fragment of the Mn-SOD
promoter (Eco/E, Fig. 8).
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Fig. 8.
Promoter deletion analysis of the
Mn-SOD gene. A, schematic
representation of the 5'-flanking sequence of the rat
Mn-SOD-specific promoter deletions. The first exon is
depicted by a box. The numbers indicate the
positions of the restriction enzyme sites relative to the
transcriptional initiation site. Placement of the deletion fragments 5'
to the human growth hormone reporter gene in the promoterless human
growth hormone vector (p GH) is indicated. B, levels of
hGH expressed from rat lung epithelial cells after transfection with
vectors containing portions of the 5'-flanking sequence of
Mn-SOD gene. The height of each bar represents
the mean of 64-118 transfected plates of cells. Each vector was
studied in 4-14 independent transfection experiments. The
error bars represent the S.E. For each vector,
the effects of treatment on hGH expression compared with basal
expression were statistically analyzed by analysis of variance or
Student's t test. A p value of <0.05 was
considered significant and is indicated by the asterisk.
C, 20 µg of RNA was size-fractionated on a 1% denaturing
agarose gel, transferred to a nylon membrane, and UV-cross-linked.
Membranes were hybridized with 32P-labeled human hGH, and
rat cathepsin B (loading control) cDNA probes were made
by random primer extension. Membranes were also hybridized with a
32P-labeled rat Mn-SOD cDNA probe to assess
simultaneous induction of the endogenous Mn-SOD.
DISCUSSION
(42), and insulin-like
growth factor-I receptor (43). GC-rich promoters lacking both a TATA
and CAAT box have also been associated with other inducible and
tissue-specific genes, such as the urokinase-type plasminogen activator
receptor (44), Pim-1 (45), CD7 (46), and
MAL genes (47).
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Fig. 9.
Summary of the chromatin structure and
in vivo/in vitro DMS footprinting
studies. A, Mn-SOD promoter sequence is
depicted from position 419 to +62 relative to the transcriptional
initiation site (+1). HSS1-1 to HSS1-5 represent
HS subsites 1-1 to 1-5 within HS site 1 defined by the high resolution
DNase I HS site studies. The position of each HS site was defined by
the fragment's migration relative to molecular markers within an
accuracy of ±50 base pairs. The results of in vivo and
in vitro DMS footprinting data are marked by
circles and triangles, respectively. The
protected guanine residues are marked as open
circles or triangles; the enhanced guanine
residues are marked as filled circles or
triangles. The arrows represent enhanced adenine
residues, with the star designating the enhanced cytosine
residue at position +51. Each bar represents an individual
binding site with Roman numeral designation. The
Inr-like and DPE-like sequences are boxed. The positions of
the restriction sites used for promoter deletion analysis are also
depicted. The rat Mn-SOD promoter sequence differing from
the published sequence (52) is underlined. B,
model of the in vivo architecture of the Mn-SOD
promoter. The top portion of B
illustrates the presence of 10 basal binding sites and a nucleosome(s)
associated with the boundary 5' to binding site I. The
bottom portion depicts our interpretation of the
induced state of the Mn-SOD promoter. The alteration of
chromatin structure associated with the boundary 5' to binding site I
is due to a displacement of a nucleosome(s) upon stimulus treatment. In
addition, we postulate that, based on our DNase I analysis, a more open
and accessible chromatin structure is evident following stimulation.
The spacing between each binding site is approximately scaled. The
thin arrow represents the basal expression, and
the thick arrow represents the induced expression
of Mn-SOD.
The Inr (YYA+1N(T/A)YY) (41) and DPE ((A/G)G(A/T)CGTG) (24)
located at approximately +30 were shown to be important for the
regulation of TATA-less promoter genes. Using computer analysis, we
located a reverse Inr-like sequence (40 to
34) and a putative DPE
sequence (+56 to +62) in the Mn-SOD promoter. Even with the reported significance of these elements in other genes, we did not
observe any protected or enhanced guanine residues on either strand as
far 3' as +180 bp. However, we did observe both an enhanced adenine
residue at position
38 within the Inr-like sequence (Fig. 4B) and an enhanced cytosine residue at position +51
upstream to the putative DPE sequence (Fig. 7), as summarized in Fig.
9. DMS-dependent methylation of cytosine residues has been
associated with single-stranded DNA (50) and may correlate with an
altered DNA structure near the start of transcription. Furthermore, the intensity of this enhanced cytosine residue was significantly stronger
in the LPS-treated cells compared with control cells, where the level
of transcription is dramatically elevated. We therefore believe that
these results may define protein or structural alterations specific to
Inr- and DPE-like elements, with further confirmation requiring
alternative molecular probes or mutagenesis studies.
In addition to the 10 basal binding sites identified by in vivo footprinting, we also detected two stimulus-specific enhancements as shown in Fig. 6A. These sites also coincide with the position of the inducible hypersensitive site (HSS1-5, Fig. 2) as summarized in Fig. 9. Although computer analysis of this region revealed an identity with the NF-IL-6 consensus DNA binding sequence, 5'-(A/C)TTNCNN(A/C)A (51), reported methylation interference data (51) for NF-IL-6 is not completely consistent with our guanine enhancements seen in vivo.
In order to assess the importance of these basal binding sites as well
as the enhanced guanine residues, we have further evaluated their
functional importance using promoter deletion analysis coupled with
transient transfection in L2 cells (Fig. 8). A detailed analysis of the
promoter including specific deletion of the
stimulus-dependent enhanced residues as well as removal of
the putative NF-B sequence has revealed that these regions are not
functionally required for TNF-
-, IL-1
-, or
LPS-dependent induction of Mn-SOD. Moreover, we
have also identified a cytokine- and LPS-specific intronic enhancer
element that maps to HS site 2, which also functions independently of
both the enhanced guanine residues and the putative NF-
B sequence in
the promoter region, based on functional experiments (data not shown).
In Fig. 9B, we propose a model based on our chromatin structure and in vivo DMS footprinting studies. A strong 5' boundary for HS site 1 is observed in control cells (Fig. 1), whereas following stimulation, the boundary is replaced with an additional HS site (subsite 1-5, Fig. 2) as well as two stimulus-dependent enhanced guanine residues (Fig. 6A). Our current hypothesis, regarding the molecular mechanism that leads to stimulus-specific enhancement of DMS reactivity at these guanine residues, relates to their possible involvement in the observed alterations in chromatin structure (Fig. 2). Specifically, we are proposing that these enhancements result from or are a consequence of the displacement of a nucleosome in stimulus-treated cells based on the appearance of the HS subsite 1-5 at the 5' boundary of HS site 1, as shown in Fig. 9B. It is possible, therefore, that the enhanced guanine residues detected in vivo reflect a chromatin structure that allows for proper access of the promoter by the transcription factors involved in enhancer activity. This could result from either the binding of a transcription factor or through changes in DNA structure that result in an enhancement of DMS reactivity, a situation that is important in vivo but may not be necessary in a transient transfection with plasmids due to lack of chromatin structure.
As the primary defense against free radical-mediated damage, Mn-SOD
serves an important cytoprotective role during both normal cell
function and inflammation associated with a wide variety of
pathological disease states. Our chromatin structure, in
vivo and in vitro footprinting studies, and our data
surrounding the Inr and DPE-like elements have revealed the complex
architecture of a highly regulated, GC-rich, and TATA/CAAT-less
promoter in which 10 basal binding sites were identified. An
understanding of the regulation of Mn-SOD at the molecular
level is not only of great clinical significance but can also serve as
a model system for the elucidation of the regulatory mechanisms
important for regulated TATA/CAAT-less promoters.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1 HL39593 (to H. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Neuroscience, College of Medicine, P.O. Box 100244, JHMHC, University of Florida, Gainesville, FL 32610. Tel.: 352-392-9239; Fax: 352-392-6511; E-mail: hnick{at}biochem.med.ufl.edu.
The abbreviations used are: SOD, superoxide dismutase; LPS, lipopolysaccharide; TNF, tumor necrosis factor; IL, interleukin; LMPCR, ligation-mediated polymerase chain reaction; DMS, dimethyl sulfate; GKLF, gut-enriched Krüppel-like factor; kb, kilobase(s); bp, base pair(s); Inr, initiator element; hGH, human growth hormone.
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REFERENCES |
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