From the
The human HMG-I/Y gene, encoding the non-histone
``high mobility group'' proteins HMG-I and HMG-Y, is
transcriptionally activated in human K562 erythroleukemia cells by
treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA
treatment induces differentiation of K562 cells within 2-4 days
after treatment. In this report, we show that transcriptional
activation of the HMG-I/Y gene is dependent on protein
synthesis and is an early event (2 h after induction) in the
TPA-mediated differentiation process. Of the four functional
transcription start sites present in the gene, only one (start site 2)
is preferentially induced upon TPA treatment. This is the first report,
to our knowledge, of the preferential utilization of a specific
transcription start site in response to a particular stimulus in a gene
that contains multiple promoters. This indicates that each start site
in the gene has the potential to be independently regulated instead of
being coordinately controlled as shown in a number of other genes. In
addition, sequences upstream of the inducible start site, which
contains a TPA-responsive element, mediates TPA inducibility through
AP1 (or an AP1-like) transcription factor. The HMG-I/Y proteins
function as key regulators of gene expression and play a significant
role in chromatin structural changes as well. The cloning and sequence
analyses previously reported indicated the structure of the HMG-I/Y gene to be highly complex and predicted its expression to be
tightly regulated. The results presented here confirm and extend these
earlier findings.
Proteins of the HMG-I/Y group belong to the ``high mobility
group'' (HMG)
In the last few years, considerable
interest has been generated in the role of HMG-I/Y proteins as
architectural transcription factors
(18) . A number of
laboratories, including ours, have shown the HMG-I/Y proteins to
function as either positive or negative accessory transcription factors
on different promoters. For example, HMG-I/Y proteins are required
in vivo for the inducible transcription of genes such as
As part of our ongoing investigation of the structure and biological
function of the HMG-I/Y proteins, we earlier cloned, sequenced, and
characterized the human HMG-I/Y gene
(11) . The
structure and transcriptional regulation of the gene is among the most
complex so far reported for any mammalian chromatin protein. It is
comprised of eight exons separated by seven introns, giving rise to
several different transcripts by an elaborate initiation and processing
regime, which includes four different transcription start sites and
extensive alternative mRNA splicing in both the 5`-untranslated region
and in the protein coding region of the gene
(2, 11) .
Nevertheless, the many different species of mRNA produced from the gene
are translated into only two major isoform proteins, HMG-I and HMG-Y.
While the alternative splicing that occurs in the coding region results
in isoform protein formation, the function and regulation of the
complex transcription and splicing events in the 5`-untranslated region
remains unknown. No noticeable TATA or CAAT box sequences are present
in and around the different transcription start sites of the gene;
instead, these promoter sequences are highly GC rich. Nevertheless,
upstream of each of the four transcription start sites is a number of
conserved sequences for potential binding of inducible transcription
factors, which suggests that the HMG-I/Y gene has the capability of
being induced by different stimuli
(29) . Indeed, we have
previously shown that HMG-I/Y gene expression is induced upon
TPA treatment of HUT-78 cells, a human T cell line
(11) .
Though the complex structure of the gene predicts it to be tightly
regulated, the mechanism of its regulation is not completely
understood. One of our main objectives is to understand the mechanism
of regulation of the HMG-I/Y gene in a cellular system in
which it is inducible. The studies reported here were performed in
TPA-treated K562 cells, a human erythroleukemic cell line. K562 cells,
depending on the stimulus used, can be induced in culture to
differentiate into either erythrocytic, myelocytic, or megakaryocytic
progeny cells
(30) . For example, hemin treatment causes
erythroid differentiation of K562 cells
(31) , while TPA induces
the same cells to undergo megakaryocytic differentiation
(30) .
Since HMG-I/Y proteins have been suggested to play important roles in
regulating chromatin structure and gene transcription, both of which
are important in the cellular differentiation process, we were
interested to determine whether TPA induces HMG-I/Y gene
expression in K562 cells and, if so, to also determine the molecular
mechanism of such an induction. We report here that TPA treatment
induces HMG-I/Y mRNA expression within a few hours of treatment and
that the regulation is at the transcriptional level. Importantly, we
also report that of the four different potential start sites present in
the gene, only one is specifically used to induce HMG-I/Y mRNA
transcripts in TPA-treated cells. Evidence is also presented that
demonstrates that the AP1 (or an AP1-like) transcription factor
mediates TPA inducibility. To our knowledge, this is the first report
of the preferential utilization of a single specific transcription
start site in a promoter that contains multiple transcription start
sites in response to an inducer of cellular differentiation.
Here, we demonstrate that the HMG-I/Y gene
transcription products are inducible in K562 cells as a delayed early
response
(37) within a few hours (2-4 h) after the
addition of TPA (Fig. 1), which is an inducer of differentiation
in K562 cells
(30) . The HMG-I/Y mRNA induction does not,
however, appear to be continuous following TPA treatment since a slow
decline is observed after 12 h of TPA treatment (data not shown). A
decrease in HMG-I/Y protein levels during differentiation of mouse
teratocarcinoma cells has been reported earlier
(42) . Therefore,
HMG-I/Y induction appears to be an early event in the
TPA-induced differentiation process of K562 cells. This response of the
HMG-I/Y gene is specific to the TPA stimulus since no
induction of the HMG-I/Y gene products was seen when K562
cells were treated with other agents such as
The interesting finding in this report is that only one of
the two major HMG-I/Y transcripts normally found in human cells is
induced by
In this work, we also show that
fragments as short as 60 bases upstream of the second exon (second
transcription start site) can confer TPA inducibility (Fig. 3),
corroborating the results of the primer extension analyses, which show
that the second start site is inducible (Fig. 2, B and
C). Removal of both transcription start sites from the
constructs resulted in the loss of promoter activity (data not shown).
The cis-acting TGAC sequence (TPA-responsive element) is also present
in the HMG-I/Y promoter, upstream of the second transcription start
site as part of a site that resembles a consensus AP1 binding
site
(40, 41) . We provide evidence indicating that TPA
induces a nuclear factor, which is similar, if not identical to, AP1 in
that its binding can be specifically competed by an authentic AP1
consensus oligonucleotide or by the sequence present in the HMG-I/Y promoter that resembles the AP1 binding site. These results
strongly suggest that the TPA inducibility of the HMG-I/Y gene
products is indeed mediated, in part, through AP1, which, in a number
of cases, has been shown to be involved in TPA
inducibility
(41) . AP1 alone, however, may not be the only
transcription factor that is involved in the TPA inducibility of the
HMG-I/Y gene since TPA induction of the HMG-I/Y gene
is blocked by cycloheximide, whereas the TPA-induced activation of AP1
is not blocked by cycloheximide. This suggests that in addition to AP1,
there may be other factors involved in the complete TPA-dependent
induction of the HMG-I/Y gene.
In summary, in this report
we provide evidence for the following:
(1) TPA, a specific
differentiation stimulus for K562 cells, induces HMG-I/Y mRNA
expression soon after treatment, suggesting that this may be an early
molecular event leading to the megakaryocytic differentiation pathway;
(2) a specific HMG-I/Y mRNA transcript is induced in response to
TPA, which indicates that the multiple transcription start sites are
independently controlled;
(3) sequences upstream of the second
transcription start site contain TPA-inducible elements;
(4) AP1
or an AP1-like factor mediates the TPA inducibility of the HMG-I/Y gene. Although the regulation of the HMG-I/Y gene
expression is obviously complex, the data presented here clearly
demonstrate that it is precisely controlled by specific stimuli. We are
in the process of further investigating the mechanism of HMG-I/Y gene regulation in other cell types to more clearly define how the
expression of this multifunctional cellular protein is controlled by
specific environmental and other factors.
These results are
obtained from four separate experiments performed in duplicate.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Laurel Holth, Mark Nissen, and Drs. Debra
Hoover, Denise Wingett, and Glenn Cantor for many helpful discussions
during the course of this work and for comments on the manuscript. We
also acknowledge the helpful suggestions provided by Drs. Gokul Das,
Susan Lobo Ruppert, and Heidi Wang. In addition, we thank David Hill
for help with the figures.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
chromosomal proteins
(1-3). The family is composed primarily of HMG-I and HMG-Y, two
isoforms of the same protein generated by alternative
splicing
(2) , and another closely related but less abundant
protein, HMG-I-C/HMG-I`
(4) . The HMG-I/Y proteins are
non-histone chromatin proteins that bind DNA. These proteins differ
from the other HMG proteins
(5) in their ability to
preferentially bind to the narrow minor groove of AT-rich sequences
in vitro(6, 7, 8, 9) by the
recognition of DNA structure rather than nucleotide
sequences
(10) . Three homologous DNA binding domains have been
identified and characterized in these proteins
(10, 11) .
The HMG-I/Y proteins have been immunolocalized to AT-rich G/Q and C
bands of mammalian metaphase chromosomes
(12) , which suggests an
important role in chromatin structural changes during the cell
cycle
(13) . The cell cycle-dependent p34
kinase(s) has been shown to phosphorylate the DNA binding domains
both in vitro and in vivo(14, 15) .
Phosphorylation of the DNA binding domains reduces the in vitro DNA binding affinity of the proteins
(14, 15) . Such
changes may modulate the regulatory functions of HMG-I/Y proteins in a
cell cycle-dependent manner. More recently, these proteins have been
demonstrated to interact in vitro with specific regions of DNA
on the surface of isolated nucleosomes
(16) , probably as a
consequence of recognition of altered structures formed by the DNA
while it is folded on the core particle
surface
(16) .
(
)
Finally, HMG-I/Y has been
shown to recognize altered DNA structures found in supercoiled plasmids
and to induce both bending and supercoiling in DNA
substrates
(17) . Together, these observations provide persuasive
support for the proposal that the HMG-I/Y proteins play important
in vivo roles in chromatin structure and
function
(9, 13) .
-interferon
(19) , tumor necrosis factor-
(20) ,
E-selectin (21, 22), and interleukin-2 receptor
(23) . In these
cases, the HMG-I/Y proteins are thought to interact with other
transcription factors such as NF
B, ATF2, or Elf-1, thereby forming
complexes on specific promoters and interacting with the basal
transcription machinery to induce transcription. Consistent with their
role as in vivo transcription factors, numerous reports have
also documented a striking correlation between elevated expression of
the HMG-I/Y gene products and neoplastic cell transformation
and metastatic tumor
progression
(24, 25, 26, 27, 28) .
Cell Culture, RNA Isolation, and Northern
Hybridization
The human K562 erythroleukemic cell line
(ATCC, Ccl243) was maintained in culture at a density of 5
10
cells/ml in RPMI medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum (Atlanta Biologicals, Norcross,
GA) and antibiotics (100 µg/ml each of penicillin-G and
streptomycin obtained from Sigma). Cells at a density of
5
10
cells/ml were stimulated as indicated for different
times with 96 ng/ml TPA, also called phorbol 12-myristate 13-acetate
(Sigma). Cycloheximide (Sigma) was added at a concentration of 10
µg/ml where indicated. Approximately 2
10
cells
were collected at the indicated times after stimulation, and RNA was
isolated using the guanidinium isothiocyanate/cesium chloride
method
(32) . The RNA pellet was resuspended and quantified, and
20 µg of total RNA was used for Northern hybridization analysis as
previously described
(33) . The probe used for HMG-I/Y mRNA was
the cDNA clone 7C
(2) . Equivalent loading of RNA was determined
by detection of glyceraldehyde-3-phosphate dehydrogenase mRNA levels
using the cDNA obtained from ATCC.
Primer Extension Analysis
20 µg of
total RNA isolated as described above was used for primer extension
analysis. About 100 ng of a synthetic oligonucleotide 39-mer
corresponding to the antisense strand of the HMG-I cDNA nucleotides
42-81
(2) was end labeled with
[-
P]ATP and T4 polynucleotide kinase (Life
Technologies, Inc.)
(32) . About 5
10
cpm of
this labeled primer was used for hybridization analysis as previously
described by Johnson et al.(2) . Quantitation of
autoradiograms was determined by densitometric analysis of
autoradiographs using LKB XL Laser Densitometer (LKB AB, Sweden).
Transient Expression Assays
The plasmid
pCAT-basic (obtained from Promega) was the vector used to determine
promoter activity. The plasmid 480 contained sequences from
nucleotide -22 to +415 of the HMG-I/Y gene cloned
into pCAT-basic between the HindIII and SalI
restriction enzyme sites
(11) . The
180 construct contained
sequences from nucleotide +223 to +415 of the HMG-I/Y gene cloned into the HindIII and XbaI
restriction enzyme sites of pCAT-basic. The plasmids were
electroporated into K562 cells. 1
10
cells/0.5 ml
were used per electroporation. The total DNA concentration used was 30
µg/electroporation, which included 15 µg of test DNA along with
salmon sperm DNA used as carrier DNA. Electroporation was performed at
960 microfarads and 0.25 kV using an IBI electroporator. Cells
electroporated with each test plasmid were pooled to normalize for
electroporation efficiency and were harvested 48 h after
electroporation. 8 h prior to cell harvest, the cells were aliquoted,
and 1 aliquot of cells was stimulated with 96 ng/ml TPA while the other
aliquot was left unstimulated. Chloramphenicol acetyltransferase (CAT)
activity was assayed using [
H]chloramphenicol and
butryl-CoA as described by Ausubel et al.(32) . Equal
amounts of total cellular protein measured by the method of Bradford
(34) was used to determine the CAT activity in extracts of
TPA-stimulated and unstimulated transfected cells.
Probes and Oligonucleotides
Probe 1
contains sequences from nucleotides -173 to +47 (see
Fig. 3A). Probe 2 contains sequences from nucleotides
+223 to +415 (see Fig. 3A). Probe 3AP1/3AP1C
duplex oligonucleotide contains a trimer of the sequence
5`-CTGTGACACATA-3` in tandem. AP1 consensus duplex oligonucleotide
obtained from Promega contains the sequence
5`-CGCTTGATGAGTCAGCCGGAA-3`. Non-self-duplex oligonucleotide probe
contains the sequence 5`-CGAAATATTTCG-3`.
Figure 3:
Panel
A, sequence of the 5`-noncoding region of the human
HMG-I/Y gene analyzed for TPA induction. AP1* is the
putative AP1 site present with a one-base mismatch (AP1* sequence
= TGACACA, AP1 consensus = TGA(G/C)T(C/A)A). PanelB, schematic representation of the CAT constructs
analyzed for TPA induction. TRE/AP1* is the TPA-responsive
element present in the HMG-I/Y promoter present as part of the
AP1 core consensus with a one-base mismatch.
Electrophoretic Mobility Shift Assays
(EMSA)
K562 cell nuclear extracts were prepared from cells
that were untreated and TPA-treated for 10 h, as described by Dignam
et al.
(35) . Double-stranded oligonucleotides were
prepared by annealing complementary single-stranded oligonucleotides,
by heating to 95 °C for 2 min in a buffer containing 10 mM
Tris-Cl (pH 7.5), 50 mM NaCl and 10 mM
MgCl, and slowly cooling to room temperature (>1 h). The
DNA fragments contained in probes 1 and 2 were gel purified and end
labeled using the 3`-fill-in reaction with
[
P]dATP (DuPont NEN) and Klenow enzyme
(Life Technologies, Inc.)
(32) . The end-labeled probes were gel
purified as described by Ausubel et al.
(32) . The
duplex oligonucleotides were end labeled with
[
P]ATP (DuPont NEN) and T4 polynucleotide
kinase (Life Technologies, Inc.) and purified over NENSORB columns
(DuPont NEN). In vitro binding reactions were performed in
20-µl volumes as described by Jin et al.
(36) . The
reaction mixtures contained 2 µg of poly(dI-dC) (Sigma), 5
mM dithiothreitol, 2 µg of bovine serum albumin, 0.5
Dignam buffer D
(35) (1
Dignam buffer D: 20
mM Hepes, pH 7.9, 20% (v/v) glycerol, 100 mM KCl, 0.2
mM EDTA, 0.5 mM dithiothreitol, 0.5 mM
phenylmethanesulfonyl fluoride), 2.5-10 µg of K562 nuclear
extract, and 15-20
10
cpm of
P-labeled probe. Approximately 200-fold molar excess of
the specific competitor oligonucleotide was used. The nuclear extract
was preincubated for 20 min on ice with poly(dI-dC) to reduce
nonspecific binding. The
P-labeled probe was then added,
and incubation continued on ice for 20 min. In the competition
experiments, the cold competitor was added 5 min prior to the addition
of the probe. The reactions were loaded onto a 5% polyacrylamide gel
(acrylamide/N,N`-methylenebisacrylamide weight ratio,
29:1), which had been prerun at 220 V for 45 min at 4 °C. The gel
buffer system used was 0.5
TBE (1
TBE:89 mM
Tris base, 89 mM boric acid, 2 mM EDTA).
TPA Induces Protein Synthesis-dependent
Transcription of HMG-I/Y mRNA
In this study, we used K562
cells, a human erythroleukemic cell line in which TPA treatment has
been shown to induce megakaryocytic differentiation
(30) . As
shown in the Northern blot of Fig. 1A, TPA induces
HMG-I/Y mRNA expression as early as 2 h, with the peak of induction
between 8-12 h, after initiation of TPA treatment. The cellular
levels of HMG-I/Y mRNA slowly decrease after 12 h of treatment (data
not shown). The blot was stripped and reprobed with
glyceraldehyde-3-phosphate dehydrogenase to confirm equivalent loading
of the RNA (Fig. 1B). The peak of HMG-I/Y mRNA induction
was approximately 5-7-fold. Since the mRNA induction was seen as
early as 2 h, we asked if protein synthesis was necessary for the
observed induction. Cycloheximide, a protein synthesis inhibitor, was
therefore added to cell cultures at the start of TPA treatment, and
exposure to the drug continued for either 2 or 4 h; its effects on
HMG-I/Y mRNA induction were then determined. Fig. 1A,
lanes3 and 5, show there is no induction of
the HMG-I/Y mRNA in the presence of cycloheximide, indicating that the
induction requires protein synthesis. These results are in agreement
with earlier observations made by Lanahan et al.(37) and confirm that the HMG-I/Y gene is a
``delayed early response gene.'' We have previously shown
that the HMG-I/Y mRNA is very stable in all phases of the cell cycle
(t > 6 h) (38). We also found that TPA
treatment does not change the t
of the HMG-I/Y
mRNA (data not shown), indicating that the early transcript induction
we observe here is due to increased gene transcription.
Figure 1:
HMG-I/Y gene
expression induced by TPA. PanelA, Northern blot of
total RNA (20 µg) showing HMG-I/Y mRNA induction in growing K562
cells treated for various times with 96 ng/ml TPA. Where indicated, 10
µg/ml cycloheximide (CHX) was added. PanelB, Northern blot shown in panelA was
stripped and reprobed with glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA to confirm equivalent
loading.
TPA Induction of a Specific HMG-I/Y mRNA
Transcript
The gene structure diagrammed in
Fig. 2A indicates the HMG-I/Y gene is quite
complex
(11) . The occurrence of alternative splicing and the
utilization of multiple transcription start sites predicts the gene to
be tightly regulated. We were interested in examining whether specific
transcription start sites are used in response to certain stimuli, such
as TPA. To address this, we performed primer extension analyses on the
RNA isolated from uninduced and TPA-induced K562 cells. Primer
extension analyses performed earlier in K562 cells defined two major
start sites for the abundant mRNA species
(2) . The common
transcription start site for the most abundant HMG-I/Y mRNA species
(represented by cDNA clones 2B and 7C cDNA
(2) ) is located at
the 5`-end of exon 2 and is referred to as start site 2 (at nucleotide
280) (Fig. 2A) (11). The common start site for the next
most abundant transcripts (represented by cDNA clones 1A and
10A
(2) ) is at the 5`-end of exon 1 (start site 1, at nucleotide
1) (Fig. 2A)
(11) . We show here that of the two
major transcript start sites utilized in K562 cells, transcription from
the start site for the 2B, 7C mRNAs (start site 2) is induced by TPA
treatment to 5-7-fold, whereas that for the 1A, 10A
transcripts (start site 1) remains constitutive (Fig. 2, B and C). Also seen here is the staggering of transcription
initiation (smallarrows, Fig. 2B) at
start site 2, which correlates with increased mRNA expression, a
phenomenon not uncommon in promoters that lack TATA and CAAT
sequences
(39) . These results show for the first time the
preferential utilization of a specific mRNA start site in response to
TPA treatment in a gene with multiple functional promoters. They also
unambiguously demonstrate that the regulation of HMG-I/Y gene
expression can involve the production of specific transcripts in
response to particular environmental stimuli.
Figure 2:
Panel A, diagram of the human
HMG-I/Y gene showing patterns of transcription start sites and
alternative splicing. RomannumeralsI-VIII are the 8 exons present. Nucleotide 1 indicates the start of exon
1, and the arrows show putative transcription start sites that
correspond to previously cloned cDNAs coding for HMG-I/Y mRNAs. The
solidlines connecting the various exons indicate
different alternative splicing patterns that result in the different
cDNA clones isolated to date. ORF, open reading frame.
PanelB, primer extension analysis of total RNA (20
µg) from K562 cells left untreated or induced with TPA for the
indicated times. <1 and <2 represent start
sites 1 and 2, respectively. The same primer used for the primer
extension analysis was used to sequence the 1A and 2B cDNA clones
(lanesGATC). * shown on each sequencing ladder
corresponds to the 5`-ends of the HMG-I/Y cDNA clones, 1A and 2B,
respectively. PanelC, densitometric scan of the
primer extension products shown in panelB.
represents the primer extension products using start site 1.
represents the primer extension products using start site
2.
Promoter/Enhancer Regions of the HMG-I/Y
Gene
To further demonstrate that sequences upstream of the
first and second transcription start sites do indeed encode promoter
activity, DNA fragments of the HMG-I/Y gene containing these
sequences were fused to the CAT reporter gene as described under
``Materials and Methods.'' The sequence of the upstream
promoter region analyzed here is shown in Fig. 3A. A
schematic representation of the two resulting hybrid gene constructs
used in this study, 480 and
180, is shown in
Fig. 3B. The
480 construct contained sequences from
-22 to +415, which included both transcription start sites
present at the 5`-end of exons 1 and 2. The shorter
180 construct
contained sequences from +223 to +415, in which the first
transcription start site was eliminated, and only 60 bases upstream of
the second transcription start site were retained. The two reporter
constructs were transfected individually into K562 cells that were then
treated, or not, with TPA and CAT activity subsequently assayed. The
results of these transfections are shown in . Both
constructs exhibited promoter activity. However, the
180
construct, which contains start 2, retained promoter elements that
conferred the greatest TPA inducibility. The uninduced CAT activity in
cells transfected with the
180 construct is barely over
background, while with TPA stimulation of the CAT activity is increased
5 times. In contrast, there is much less of a TPA induction of CAT
activity (
1.8-fold) in cells transfected with the 480 construct,
which contains both start sites 1 and 2. Since primer extension
analyses indicate that start site 1 is constitutively expressed in K562
cells (Fig. 2B), the slight TPA induction observed with
the
480 construct is probably dependent on start site 2, whose
expression is partially modulated by the presence of inhibitory
sequences elsewhere in this longer promoter fragment
(20) . In
any event, these transfection results clearly indicate that the major
TPA-inducible element in the HMG-I/Y gene is present within 60
bases upstream of the second transcription start site. This finding is
entirely consistent with the primer extension results shown in
Fig. 2
, B and C, which demonstrates that in
treated K562 cells it is the second transcription start site that is
inducible with TPA.
TPA Induces Binding of a Specific Nuclear Factor
Upstream of the Second Transcription Start Site
The primer
extension analyses and the CAT assay results indicate the presence of a
TPA-responsive element upstream of the second transcription start site.
Indeed, analyses of this region shows the presence of a putative
TPA-responsive element sequence (TGAC), which is part of a
``consensus binding site'' for the transcription factor AP1
(underlined in
Fig. 3A)
(40, 41) . To determine whether
TPA induced specific DNA-protein complex formation, EMSAs were
performed on fragments that contained either sequences upstream of the
first or second transcription start sites. Probe 1 contained sequences
upstream of the first transcription start site from -173 to
+47 (Fig. 3, A and B). Nucleotide
sequences +223 to +415 upstream of the second transcription
start site were contained in probe 2. The results of the EMSA performed
on the two probes are shown in Fig. 4, A and B,
respectively. No additional protein-DNA complexes were observed using
probe 1 when nuclear extracts from uninduced versus induced
cells were used (Fig. 4A, lanes2 and
3versus4 and 5). The intensity of
the constitutive complexes, however, appears to be slightly increased
upon TPA treatment. This seems to be a generalized increase as it is
seen when probes 2 and 3AP1/3AP1C are also used (see below). The
increase in intensity is probably due to the stabilization of these
constitutive complexes upon TPA induction with no apparent effect on
transcription from start site 1, which remains constitutive. On the
other hand, EMSA using probe 2 showed an additional retarded band
(shown by an *) when nuclear extracts from induced cells were used and
compared with nuclear extracts from uninduced cells
(Fig. 4B, lanes4 and 5versus2 and 3). The TPA-induced
complexes on probe 2 are specific as they can be competed with
unlabeled probe 2 but not by heterologous DNA sequences (shown below).
These results demonstrate that sequences upstream of the second
transcription start site are involved in the binding of a specific
nuclear factor in response to TPA.
Figure 4:
TPA-induced nuclear factor binding.
Nuclear extracts (2.5 or 5 µg) prepared from K562 cells left
untreated or TPA treated for 10 h were used for EMSA. Panel A,
probe 1 containing nucleotides -173 to +47, which includes
sequences upstream of the first transcription start site, was
P labeled and used for EMSA. PanelB,
probe 2 containing nucleotide +223 to +415, which includes
sequences upstream of the second transcription start site, was
P labeled and used for EMSA. * indicates the additional
complex formed on probe 2 when TPA-treated extracts were
used.
TPA-induced Nuclear Factor Binding Is Competed with
AP1 Consensus Sequence
As noted, there is a site in the
HMG-I/Y promoter upstream of the second transcription start
site that resembles (with one base mismatch) the AP1 consensus binding
site (nucleotides 246-252 in Fig. 3A). TPA induces
AP1 binding activity in a variety of cells, and TPA-induced gene
expression is often shown to be mediated through AP1 binding to its
cognate promoter sequence(s)
(40, 41) . Therefore, it
seemed likely that the putative AP1 binding site present in the
HMG-I/Y promoter mediates the formation of the TPA-induced
complex seen in Fig. 4B. To test this, we made a
synthetic 36-mer oligonucleotide duplex DNA, a trimer of this AP1-like
sequence (referred to as ``3AP1/3AP1C,'' as described under
``Materials and Methods'') and used it as a competitor in
EMSAs with probe 2. In addition, an authentic AP1 consensus DNA duplex
was also used as a competitor in these experiments. The results are
shown in Fig. 5A. A 200-fold molar excess of both the
3AP1/3AP1C oligonucleotide and the authentic AP1 consensus
oligonucleotide were able to specifically compete the TPA-induced
complex (* in lane4, Fig. 5A), while
unlabeled probe 2 DNA itself competed all of the complexes (lanes6 and 7versuslane4, and lane5versuslane4). Unrelated, heterologous DNA fragments of comparable
size and concentration did not compete the TPA-induced complex (data
not shown). These results show that the TPA-induced complex is formed
on a site that is specifically competed by the authentic AP1 consensus
sequence, indicating that the TPA-induced complex contains the AP1
factor or a factor with AP1-like binding characteristics.
Figure 5:
TPA-induced nuclear factor binding is
competed with AP1 consensus sequence. 5 µg of nuclear extract from
K562 cells left untreated or TPA treated for 10 h were used for EMSA.
PanelA, P-labeled probe 2 was used in
EMSA. 200-fold molar excess of unlabeled probe 2 was used as
self-competitor. Similarly, 200-fold molar excess of either the
unlabeled 3AP1/3AP1C duplex oligonucleotide or the unlabeled AP1
consensus oligonucleotide was used as competitors as described under
``Materials and Methods.'' PanelB,
P-labeled 3AP1/3AP1C duplex oligonucleotide was used as
probe in EMSA. 200-fold molar excess of unlabeled 3AP1/3AP1C duplex
oligonucleotide or 200/600-fold molar excess of unlabeled AP1 consensus
probe or 200-fold unlabeled non-self- (nonspecific) duplex
oligonucleotide probe was used as competitor. * indicates the
additional complex formed when TPA-treated extracts were used.
PanelC, TPA enhances AP1 binding activity.
P-Labeled AP1 consensus oligonucleotide was used as probe
in EMSA. 200-fold molar excess of unlabeled probe was used as
competitor.
We were
also interested to know if there is a difference in DNA-protein complex
formation with nuclear extracts from uninduced and TPA-induced cells
when the 3AP1/3AP1C oligonucleotide itself is used as a probe in EMSA.
In uninduced extracts, there are a number of complexes formed on the
3AP1/3AP1C probe (Fig. 5B, lane2).
However, when TPA-induced extracts were used, an additional complex is
formed (*) (Fig. 5B, lane7versuslane2). This new complex is specific and can be
competed with excess unlabeled 3AP1/3AP1C probe
(Fig. 5B, lanes3 and 8), as
well as with the AP1 consensus probe (Fig. 5B, lanes4, 5, 9, and 10). 3-fold
higher amounts of the AP1 consensus oligonucleotide (which contained
only one factor binding site) were required for the competition in
these assays when compared with the synthetic 3AP1/3AP1C probe, which
contains three putative AP1 binding sites. A nonspecific heterologous
oligonucleotide was unable to compete the specific complex (compare
lanes6 and 11). These results clearly
indicate that the putative AP1 binding site upstream of transcription
start site 2 is involved in the TPA-induced DNA-protein complex
formation. And finally, to verify that TPA treatment actually enhanced
AP1 binding activity in K562 cells, we also used the authentic AP1
consensus oligonucleotide as a probe in EMSA. Fig. 5C,
lane4versuslane2 shows
greatly enhanced complex formation on the authentic AP1 probe when
TPA-induced extracts were used, indicating that the TPA-induced
extracts indeed contained, as expected, significantly increased AP1
binding activity. Together, these results unambiguously indicate the
involvement of AP1, or an AP1-like factor, in the TPA induction of a
specific HMG-I/Y mRNA transcript from a single start site in a gene
with multiple functional promoters.
-interferon (data not
shown). In addition, we did not observe the induction of the
HMG-I/Y gene products in TPA-treated human U937 cells, a
promyelocytic leukemic cell line, which upon TPA treatment
differentiates into myelocytic cells (data not shown)
(43) .
These observations suggest that the TPA induction of the HMG-I/Y gene products seen here may be restricted to certain cell types or
certain cellular differentiation pathways. Cellular differentiation
involves a cascade of events controlled by differential gene
expression, which leads to the differentiated phenotype. Here, we show
the early induction of an important cellular protein (HMG-I/Y), which
has key functions both at the level of chromatin structure and gene
regulation, in response to a specific differentiation stimulus (TPA).
At this time, however, we do not have proof for a direct involvement of
HMG-I/Y proteins in K562 cellular differentiation, as its expression
does not precisely correlate with the later stages of megakaryocytic
differentiation. Nevertheless, it seems likely that HMG-I/Y proteins
play an important role in the early events, leading to differentiation
by regulating the expression of genes, such as
sis/PDGF(36, 44) , that are known to
be directly correlated with the TPA-induced differentiation of K562
cells.
5-7-fold following TPA treatment. As described
earlier, the HMG-I/Y gene has multiple functional
transcription start sites but lacks TATA and CAAT box
sequences
(11) . The lack of TATA and CAAT boxes in a number of
different promoters often correlates with the presence of multiple
transcription start sites. These findings have been reported for a
large number of genes, many of them being oncogenes, such as the human
ets-1(45) , human ets-2(46) , human
fgr(47) , and human c-src(48) . Some of
the other genes also included in this category are the rat thyroid
hormone receptor
gene
(49) , rat class 3 aldehyde
dehydrogenase gene
(50) , and 3-hydroxy-3-methylglutaryl-coenzyme
A reductase
(39) . So far, studies of all of these genes reveal
that the multiple transcription start sites are always coordinately
controlled without the preferential utilization of any specific start
site
(50) . Therefore, the TPA induction of a specific HMG-I/Y
transcript reported here is, to our knowledge, the first report where
each transcription start site within a gene appears to be independently
controlled. The HMG-I/Y gene, with its complex and multiple
promoters, therefore has the potential to be tightly regulated in
response to specific differentiation-inducing and/or other
environmental factors. Abrogation of this tight control could lead to
the aberrant expression of the HMG-I/Y gene products, which
has recently been correlated with neoplastic cell transformation and
metastatic tumor progression (6, 24-27). The preferential
synthesis of a specific mRNA transcript under these conditions could be
important for its cellular function. For example, the specific
transcript could be more efficiently translated in a given cell type,
since it is known that the 5`-untranslated regions of genes have
important roles in translational control
(51) . Another
possibility is that under certain conditions, or in certain cell types,
the specific transcript might be more stable than the others. We deem
this latter possibility unlikely, however, since the half-life of the
HMG-I/Y mRNA is >6 h and is not changed by TPA treatment
(38) (data not shown).
/EMBL Data Bank with accession number(s) L17131.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.