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Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7760
Address all correspondence and requests for reprints to: Dr. Martin L. Adamo, Department of Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760. E-mail: adamo{at}biochem.uthscsa.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The majority of transcription
occurs from two sites, termed start sites 2 and 3, which initiate
transcription at approximately 345 and approximately 245 bp upstream of
the 3' end of exon 1, respectively.
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Recently, our lab reported that an exon 1 promoter construct extending from +75 to +362, which includes start sites 3 and 4, could function as a promoter independently of the sequence upstream of +75 (8). In searching this region for potential transcription factor binding sites, we have noted perfect matches to the consensus GATA transcription factor binding site at approximately + 108 and at approximately + 183. The GATA transcription factors are characterized by two unique zinc-finger DNA-binding domains that recognize a common DNA-binding site, (A/T) GATA (A/G) (reviewed in Ref. 9). They are designated as GATA-1/Eryf1, GATA-2, GATA-3, GATA-4, GATA-5, and GATA-6.
The six GATA family members show distinct but overlapping expression patterns. GATA-1 is expressed at high levels in hematopoietic cell lineages and is also present in a nonhematopoietic tissue, the testis (10). More widely expressed GATA-2 is found in erythroid cells, endothelial cells, and embryonic brain cells (11). GATA-3 is expressed in many tissues, including embryonic brain, T cells, mast cells, and kidney (12).
The newly identified family members GATA-4/5/6 play a role in expression of lineage-specific genes in a variety tissues derived from mesoderm, including heart and gut (13). GATA-4 was also recently reported to be expressed in neuronal cell lines (14). In this study, we test the hypothesis that the putative GATA elements in exon 1 of the IGF-I gene are important for basal exon 1 promoter activity. We also have attempted to characterize the proteins that bind to these sites.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmids and site-directed mutagenesis
All IGF-I exon 1 promoter constructs were cloned upstream of the
firefly luciferase structural gene in the pGL2-Basic vector
(Promega Corp., Madison, WI). A vector consisting of the
SV40 promoter/enhancer directing luciferase transcription
(pGL2-Control, Promega Corp.) was used as a positive
control. The -1500/+319, -1000/+319, -500/+319, -500/+282,
-500/+192, -250/+319, -250/+282, -250/+192, +75/+319, +75/+282, and
+75/+192 constructs were generated by PCR-amplification of these
regions using a sense primer with a KpnI restriction site
and an antisense primer with a HindIII restriction site.
Primers were synthesized at the Center for Advanced DNA technologies
(University of Texas Health Science Center at San Antonio). The
PCR products were ligated into the same sites of pGL2-Basic.
Two perfect matches to the consensus GATA transcription factor binding
site are located in the sense strand of exon 1 at +108 and at +183,
respectively (Fig. 1
). The sequence at +108 is 5'-AGATAA-3' (GATA-A),
and the sequence at +183 is 5'-AGATAG-3' (GATA-B). The consensus GATA
sequence is 5'-(A/T)GATA(A/G)-3' (9). Each of these sequences was
mutated to 5'-CTCGCC-3' in the +75/+192 construct by using a mutagenic
sense primer for the GATA-A element and mutagenic antisense primer for
the GATA-B element. The GATA-A/GATA-B double mutation was generated
using a similar strategy. In addition, a second GATA-A mutation was
generated by mutating the sequence to 5'-GAGCGG-3'. The identities of
the mutations in the +75/+192 constructs were confirmed by DNA sequence
analysis.
A GATA-1 transcription factor expression plasmid under control of the SV40 enhancer/promoter (pXM-GATA-1) was a kind gift of Drs. Andrew Perkins and Stuart Orkin, Harvard Medical School. For cotransfection studies, the pXM plasmid lacking the GATA-1 structural gene insert was used as a baseline control.
Transfections
Transient transfection was performed using 2 µg of each DNA
and the lipofectamine plus system in Opti-MEM medium (Life Technologies/BRL, Gaithersburg, MD). Three hours after
transfection, Opti-MEM medium was replaced with 5 ml complete medium.
After 24-h incubation, lysates were prepared and assayed for luciferase
enzyme activity using the reagents and protocol supplied by
Promega Corp., with chemiluminescence measurements
performed on a model IL-A911 semiautomatic luminometer from Tropix
(Bedford, MA). Protein concentration was assayed on the lysates using
the method of Bradford (15).
Preparation of nuclear extracts and oligonucleotide labeling
Nuclear extracts were prepared from SK-N-MC cells using the high
salt extraction method (16). Protein concentrations were determined by
the method of Bradford (15). Oligonucleotides (sense and antisense
strands) corresponding to the sequences of the wild-type and mutant
GATA-A and GATA-B elements at +108 and +183 (see Table 1) were annealed and end-labeled with
-32P-ATP and T4 DNA kinase and separated from
unincorporated nucleotides using the method described in Adamo et
al. (17).
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Statistical analysis
All data are mean ± SEM for the indicated
number of observations. Statistical differences between means were
determined using one-way ANOVA in the SIMSTAT 3 package (Normand
Peladeau, Provalis Research, Montréal, Canada).
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Results |
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). Deletion of 5'-flanking/5'-UTR
sequence to -1000, -500, or to +75 resulted in higher levels of
luciferase activity (Fig. 2A). In the constructs containing 500 or 250
bp of 5'-flanking sequence, deletion of 3' exon 1 sequence to +282 or
+192 did not alter the promoter activity (Fig. 2B). However, when the
5' end of the promoter was deleted to +75, 3'-deletion to +192 lowered
the promoter activity, but still resulted in a significant promoter
activity over pGL2-Basic (Fig. 2B). A promoter construct containing
exon 1 sequence from +75 to +192 also significantly stimulated
luciferase activity, compared with pGL2-Basic in C6 and OVCAR-3 cells
(Fig. 3, A and B). This is consistent
with the data from SK-N-MC cells. Thus, a minimal IGF-I exon 1 promoter
region is located between +75 and +192, a region that includes start
site 3 but not start sites 1 or 2.
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, A and B, single or double mutations of the GATA-A and GATA-B
elements had slight or no effect on promoter activity in both C6 and
OVCAR-3 cells. However, in SK-N-MC cells, where the wild-type +75/+192
construct stimulated luciferase activity by 19-fold, mutation of the
GATA-A element increased luciferase activity to 32-fold above
pGL2-Basic, i.e. a 68% stimulation of wild-type promoter
activity (Fig. 3C). In contrast, mutation of the GATA-B element
resulted in luciferase activity that was only 4.3-fold over pGL2-Basic,
i.e. a 4.4-fold inhibition of wild-type promoter activity
(Fig. 3C). The GATA-A/GATA-B double mutation also resulted in
luciferase activity that was only 6.5-fold over pGL2-Basic,
i.e. a 2.9-fold inhibition of wild-type promoter activity
(Fig. 3C).
To distinguish between the possibilities that the GATA-A element binds
an inhibitory transcription factor(s) or that this GATA-A mutation
introduces a positive transcription factor binding site, a second
mutation, GATA-AM (2), was made in the GATA-A element and was assessed
in SK-N-MC cells. This mutation resulted in an increase of luciferase
activity to 110-fold over pGL2-Basic (Fig. 4). These data suggest that the GATA-A
element is a binding site for an endogenous inhibitory factor(s) in
SK-N-MC cells. The GATA-B sequence seems to be a binding site for an
endogenous stimulatory factor(s) in SK-N-MC cells.
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for sequence). Incubation
of nuclear extracts with a labeled oligomer containing the wild-type
GATA-A sequence resulted in two bands of reduced mobility, compared
with free probe (Fig. 5). The intensity
of these bands was abolished when 30-fold to 300-fold molar excess of
unlabeled wild-type GATA-A oligomer was used but was not altered by
100-fold molar excess of an Oct-1 factor binding site (Fig. 5). An
oligomer containing a mutation of the GATA-A sequence, which stimulated
promoter activity, or one containing a mutant GATA consensus sequence,
did not compete for binding at 30-fold and 100-fold molar excess but
were weakly competitive at 300-fold molar excess (Fig. 5). Oligomers
containing the GATA-B sequence or a GATA consensus sequence were
capable of competing for binding at 30-fold molar excess and
abolished the binding at 300-fold molar excess (Fig. 5).
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). The intensity of this band was
abolished when 30-fold to 300-fold molar excess of unlabeled wild-type
GATA-B oligomer was used, but it was not altered by 100-fold molar
excess of an Oct-1 factor binding site (Fig. 6). Oligomers containing
either a mutation of the GATA-B sequence or a mutant GATA consensus
sequence did not compete for binding at 30-fold and 100-fold molar
excess but were weakly competitive at 300-fold molar excess (Fig. 6).
Oligomers containing the GATA-A site or the GATA consensus sequences
were capable of competing for binding at 30-fold molar excess and
abolished the binding at 300-fold molar excess (Fig. 6). These data
suggest that there are distinct endogenous nuclear proteins in SK-N-MC
cells binding to the GATA-A and GATA-B elements, and this binding is
GATA sequence-specific. In contrast, no bands with reduced mobility
were detected in EMSAs using nuclear extracts from C6 or OVCAR-3 cells
and double-stranded oligonucleotides containing GATA-A element or
GATA-B element (data not shown).
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, none of the antibodies against these
three GATA proteins were capable of inhibiting or supershifting the
bands formed by GATA-A or GATA-B oligomers and nuclear extract from
SK-N-MC cells. In contrast, these antibodies were capable of
supershifting the complexes formed by 32P-labeled
double-stranded oligonucleotides containing consensus GATA binding site
and cell extracts from two hematopoietic cell lines, BB88 and ATL-16T
(data not shown).
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). This suggests that there is a
GATA-binding site(s) in the presumed cryptic pGL2-Basic promoter
region.
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). GATA-A
mutation abolished the stimulatory effect of the GATA-1 protein on
promoter activity. In contrast, when the +75/+192 construct containing
the GATA-B mutation was cotransfected with pXM-GATA-1, luciferase
activity was stimulated by 15-fold, compared with cotransfection with
pXM plasmid. Cotransfection of the +75/+192 construct containing the
GATA-A/GATA-B double mutation with pXM-GATA-1 resulted in only 2.9-fold
stimulation of luciferase activity compared with cotransfection with
pXM plasmid. As shown in Fig. 8B, the anti-GATA-1 antibody was unable
to inhibit or supershift the bands formed by GATA-A or GATA-B oligomers
and nuclear extract from SK-N-MC cells. However, it was capable of
supershifting the complexes formed by 32P-labeled
double-stranded oligonucleotides containing consensus GATA binding site
and cell extracts from BB88 cells (data not shown). These data suggest
that GATA-1 protein is capable of stimulating IGF-I exon 1 promoter
activity through the GATA-A element but is unlikely to be the
endogenous protein binding to the GATA-A and GATA-B sites in SK-N-MC
cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hall et al. (18) showed that the rat IGF-I exon 1 promoter required approximately 1700 to approximately 800 nucleotides of 5'-flanking region and 319 nucleotides of exon 1 sequence for maximum promoter activity in SK-N-MC cells. Deletion to only 131 bp of 5'-flanking region abolished the promoter activity. The same group observed similar results for the human exon 1 promoter: maximum promoter activity was detected in the construct -1633/+319 and 5'deletion to -238 led to loss of promoter activity (19). However, Jansen et al. (20) reported that, in the context of 192 bp of exon 1 sequence, only 553 bp of 5'-flanking region was required for the greatest promoter activity, and a construct containing only 212 bp of 5'-flanking sequence still contained significant promoter activity. The major difference between the results obtained by these two laboratories and our laboratory seems to be in how much 5'-flanking sequence is required for significant promoter activity. We observed significant promoter activity in the -250/+192 construct, which is consistent with the results of Jansen et al. (20). Furthermore, we detected significant promoter activity in the +75 to +192 region in SK-N-MC cells. It is not clear why the -131/+319 construct was inactive in the study of Hall et al. (18). One possibility would be the use of different clones of SK-N-MC cells.
In this study, we have characterized the function of two potential GATA elements in the IGF-I exon 1 promoter. These elements are located at +108 and +183, i.e. within the +75 to +192 region of exon 1. Mutations of these two elements had a cell-type specific effect on promoter activity. Minor effects were observed in both C6 and OVCAR-3 cells. Although the GATA-A and GATA-B elements are both perfect matches to the GATA consensus sequence, their mutants resulted in opposite effects on promoter activity in SK-N-MC cells. Mutation of the GATA-A sequence stimulated promoter activity, whereas mutation of the GATA-B sequence inhibited promoter activity. In gel mobility shift assays, we showed that unlabeled oligonucleotides containing the consensus GATA binding site competed with the 32P-labeled GATA-A and GATA-B oligonucleotides for binding by nuclear extracts from SK-N-MC cells. These data indicated that the proteins binding to the GATA elements in the IGF-I promoter region are probably related to the GATA family. Only three GATA family proteins, GATA-2, GATA-3, and GATA-4, have been reported to be expressed in neuronal cells (13). However, our supershift assays suggested that none of these three GATA family proteins bound to either the GATA-A or GATA-B sites. Therefore, it is possible that new GATA family proteins are binding to these GATA elements.
Cotransfection experiments suggested that GATA-1 protein had the ability to stimulate IGF-I promoter activity through the GATA-A element. However, luciferase activity resulting from pGL2-Basic was also elevated when it was cotransfected with the GATA-1 expression vector. Searching the sequence upstream of the luciferase gene in the pGL2-Basic plasmid, we found several perfect matches to the GATA consensus sequence. Thus, we would hypothesize that when GATA-1 protein is overexpressed in SK-N-MC cells, it is capable of binding to the GATA-A and the GATA-B elements in the IGF-I exon 1 promoter, and the cryptic GATA-binding site(s) in pGL2 plasmid backbone. Furthermore, GATA-1 protein can only stimulate promoter activity by binding to the GATA-A site and the cryptic GATA-binding site(s) in the vector. The affinity for GATA-1 protein binding is presumably different among these sites. Binding of GATA-1 factor to the GATA-A and GATA-B elements is much more favorable than binding to the cryptic GATA binding site(s)in pGL2 plasmid backbone. Thus, when expressed, GATA-1 transactivates IGF-I exon 1 promoter through the wild-type GATA-A sequence. Moreover, when the GATA-B site is mutated, there is more GATA-1 available to transactivate IGF-I exon 1 promoter through the GATA-A site. However, the possibility that GATA-1 protein stimulates IGF-I exon 1 promoter activity in an indirect manner, which may involve increased expression or stimulation of other transcription factors, should not be excluded.
Site-directed mutagenesis suggests that the GATA-A site is a binding site for an inhibitory factor(s). It has not been reported that GATA-1 protein is expressed in neuronal cells, so it is unlikely that GATA-1 protein is the transcription factor that binds to the GATA-A site in intact SK-N-MC cells. However, this study suggests that GATA-1 protein has the potential to stimulate IGF-I exon 1 promoter activity through the GATA-A site in those cell lines and tissues in which GATA-1 protein is expressed.
Site-directed mutagenesis and DNA-protein binding assays indicate that
sequences between about -10 and +50, including IGFI-FP1 at -3, are
important for basal activity in C6 and SK-N-MC cells (2, 3). C/EBP 
is the best characterized transcription factor involved in the
regulation of IGF-I promoter activity (7). This mechanism involves the
PGE2 stimulation of cAMP levels, followed by a
protein kinase A-dependent increase in the amount of nuclear
C/EBP protein that then binds to a sequence within footprint HS3D
located at approximately +192, resulting in increased exon 1 promoter
activity. C/EBP 
, LAP, and HNF-1 can stimulate IGF-I exon 1
promoter activity by binding to cis-acting elements located from +18 to
+30 of the IGF-I exon 1 sequence in Hep3B cells (21, 22). In addition,
HNF-1 can act through another site, located at approximately
-145.
HNF-3ß has a strong stimulatory effect on the IGF-I exon 1 promoter
in Hep3B cells (23). The locations of the two binding sites for
HNF-3ß are between +105 to +114 and +142 to +151. The upstream
binding site overlaps with the GATA-A element. Our study suggests that
the GATA-A element may function as a stimulatory factor-binding site in
hematopoietic cell lineages in which GATA-1 protein is expressed,
although this hypothesis remains to be tested. It is possible that a
multiprotein complex is formed between GATA protein(s), HNF-3ß and/or
other transcription factors in the region around +108. The GATA-B
sequence is included in region III detected by DNase I footprinting
using rat liver nuclear extract (5), which is metabolically regulated
during insulinopenic diabetes. The GATA-B sequence also overlaps the
beginning portion of footprint HS3D (4). However, it does not overlap
with the C/EBP binding site within HS3D. Identification of the
transcription factors that bind to these GATA elements will be a future
goal.
In summary, a minimal IGF-I exon 1 promoter region is located from +75 to +192. The GATA-A and GATA-B elements located within this minimal promoter region have a cell-type specific effect on promoter activity. Two distinct GATA transcription factor family-related proteins or protein complexes, apparently other than GATA-2, GATA-3, and GATA-4, probably bind to the GATA-A and GATA-B sites in SK-N-MC cells. They inhibit promoter activity through the GATA-A motif and stimulate promoter activity through the GATA-B motif. GATA-1 protein has the potential to stimulate IGF-I exon 1 promoter activity through the GATA-A site, but it is unlikely to be the endogenous protein binding to these GATA elements in SK-N-MC cells.
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Acknowledgments |
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Footnotes |
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Received June 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activates insulin-like
growth factor-I gene transcription in osteoblasts. Identification of a
novel cyclic AMP signaling pathway in bone. J Biol Chem 272:31793–31800-subunit
gene in MA-10 Leydig tumor cells. Mol Endocrinol 12:378–390 gene enhancer. Mol Cell Biol 11:2778–2784 and
LAP. Mol Endocrinol 8:1636–1645 activates promoter 1 of the human
insulin-like growth factor I gene via two distinct binding sites. Mol
Endocrinol 9:1488–1499This article has been cited by other articles:
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