©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Regulation of a Multipromoter Gene
SELECTIVE 12-O-TETRADECANOYLPHORBOL-13-ACETATE INDUCTION OF A SINGLE TRANSCRIPTION START SITE IN THE HMG-I/Y GENE (*)

Sushma Abraham Ogram (1), Raymond Reeves (1) (2)(§)

From the (1) Department of Biochemistry/Biophysics and the (2) Department of Genetics and Cell Biology, Washington State University, Pullman, Washington 99164-4660

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Proteins of the HMG-I/Y group belong to the ``high mobility group'' (HMG)() 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) .

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 -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 NFB, 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) .

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.


MATERIALS AND METHODS

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 10cells/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).


RESULTS

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.


DISCUSSION

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 -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.

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 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).

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.

  
Table: Relative CAT activity

These results are obtained from four separate experiments performed in duplicate.



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant 5-R01-AI26356 and National Science Foundation Grant DCB-8904408 (to R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L17131.

§
To whom correspondence should be addressed. Tel.: 509-335-1948; Fax: 509-335-9688; E-mail: reevesr@wsuvm1.csc.wsu.edu.

The abbreviations used are: HMG, high mobility group nonhistone protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay.

M. S. Nissen and R. Reeves, unpublished observations.


ACKNOWLEDGEMENTS

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.


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