Characterization of a proximal element in the rat preadipocyte factor-1 (Pref-1) gene promoter
Hiroshi Takemori1, Junko Doi,1 Yoshiko Katoh1, Sunil K Halder1 .* , Xing-zi Lin1, Nanao Horike1, Osamu Hatano2 and Mitsuhiro Okamoto1
1 Department of Molecular Physiological Chemistry, Osaka University Medical School, Osaka, Japan; 2Department of Anatomy, Nara Medical University, Shijo-cho, Kashihara, Nara, Japan
Preadipocyte factor-1 (Pref-1) was shown to negatively regulate adipocyte differentiation. We recently reported that ZOG, a rat homolog of Pref-1, was specifically expressed in the adrenal zona glomerulosa. Results of the investigation of Pref-1 expression in preadipocyte and in undifferentiated adrenal cortex suggested that down-regulation of Pref-1 gene was closely correlated with the differentiation process. In this study we demonstrate that an upstream region (from -76 to -47) of the rat Pref-1 gene was essential for its expression in adrenocortical carcinoma-derived H295R cells. A nucleotide sequence found in this region, GCGTGGGCGTGGGCGGGGG (Egr/GC-box), seemed to contain three elements, two early growth response (Egr) elements and one GC-box, overlapping each other. Muta- tions of four or five nucleotides in a 7-nucleotides-stretch in the midst of the Egr/GC-box eliminated the binding of
Sp1/3, abolished the activation by Egr-factor(s) and diminished the Pref-1 promoter activity. When mutations were introduced into the outside of the middle portion, the binding of Sp1/3 to the Egr/GC-box was abolished similarly. However, the decrease in the promoter activity was less than that found with the construct mutated at the middle. These results indicated that an element present at the 7-nucleotides-stretch in the midst of the Egr/GC-box might be important for the Pref-1 promoter activity, and this proximal element was possibly activated by a still- unidentified nuclear factor(s). This element would function as the promoter of the Pref-1 gene in H295R cells, but not in Hela cells.
Keywords: adrenal gland; zonation; preadipocyte; transcription.
The mammalian adrenal cortex is composed of three distinct zones: the zona glomerulosa (ZG), the zona fasciculata (ZF), and the zona reticularis (ZR). Cells in ZG secrete aldosterone, whereas those in ZF and ZR, glucocorticoid [1-4]. Previous histochemical studies iden- tified the zona intermedia (ZI) [5], a cell layer between the ZG and the ZF, which contained neither aldosterone synthase P450 (CYP11B2) nor 11ß-hydroxylase P450 (CYP11B1) [6]. The ZI was thought to contain stem cells of the adrenal cortex.
Correspondence to M. Okamoto, Department of Molecular
Physiological Chemistry, Osaka University Medical School (H-1), 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Fax: + 81 6 879 3289, Tel: + 81 6 879 3280, E-mail: mokamoto@mr-mbio.med.osaka-u.ac.jp
Abbreviations: Pref-1, Preadipocyte factor-1; ZG, zona glomerulosa; Egr, early growth response; ZF, zona fasciculata; ZR, zona reticularis; CYP11B2, aldosterone synthase P450; CYP11B1, 11ß-hydroxylase P450; ZI, zona intermedia; ZOG, zona-glomerulosa-specific gene; FA-1, fetal antigen 1; IZA, inner zone antigen; fLuc, firefly luciferase; rLuc, Renilla luciferase; PMSF, phenylmethanesulfonyl fluoride; SAD, suppression in adipocyte differentiation; ADA, adenosine deaminase; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; SOD, superoxide dismutase; 5-LO, 5-lipoxygenase; LHR, luteinizing hormone receptor.
*Present address: Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232-0146, USA.
(Received 29 August 2000, revised 23 October 2000, accepted 23 October 2000)
As one of our attempts to characterize the precise nature of the respective cortical zones, a cDNA library prepared from ZG was screened to isolate gene products specifically expressed in the ZG [7]. One clone, ZOG, that encoded a rat homolog of Pref-1 protein, a membrane protein having six tandem EGF-like repeats in its extracellular domain, was isolated. The mouse Pref-1 protein was previously reported as a specific gene product in undifferentiated preadipocytes [8]. The expression of its mRNA was downregulated during adipocyte differentiation [8-10]. The forced expression of Pref-1 mRNA in 3T3-L1 preadipocytes inhibited the adipogenesis and the cells continued to look undifferentiated [8]. When Pref-1 protein, produced in Escherichia coli [11] or purified from amniotic fluids [12,13], was added to the culture medium of preadipocytes, the adipogenesis was suppressed. In con- trast, when antisense oligonucleotides that targeted the Pref-1 mRNA were introduced into 3T3-L1 cells, the adipocyte differentiation was accelerated [14]. These results suggested that Pref-1 played a key role in adipocyte differentiation.
Our immunohistochemical study of adult rat adrenals showed specific expression of Pref-1 protein in the ZG, but neither in the ZF nor in the ZR [7]. The level of Pref-1 mRNA in ZG was fairly constant in rats under various experimental conditions, such as high Na+- or high K+-diet treatment and corticotropin treatment. When inner portions of rat adrenal glands, containing the ZF, the ZR and the medulla, are enucleated, cells having properties of ZF and ZR are regenerated from the remaining ZG within several days after the surgery [15-17]. Interestingly, the expression
of Pref-1 mRNA was significantly suppressed in the regenerating cells at the early phase, whereas inner zone antigen (IZA), a ZF and ZR-specific marker [18], appeared in those cells [7].
The investigation of fetal rat adrenals demonstrated further the negative correlation between the level of Pref-1 expression and the extent of adrenal zone differentiation. In the early embryonic stage, most of adrenocortical primor- dial cells expressed Pref-1 protein [19,20]. When the zone differentiation began in the primordium, the cells present at its inner portion ceased to express Pref-1 and concomitantly began to express CYP11B1. This suggested that Pref-1- expressing cells in the developing adrenals may represent precursors of the three zones, and the down-regulation of Pref-1 gene may be an important step in the zone differentiation.
Our present aim is to identify cis-elements responsible for the transcriptional regulation of the rat Pref-1 gene in ZG cells. We demonstrate that an upstream region, -76 to -47, of the Pref-1 gene is important for expressing the gene in adrenocortical carcinoma H295R cells. A reporter construct having this region fails to exert the promoter activity in Pref-1-nonexpressing HeLa cells. This region has
a tandem and overlapped nucleotide sequence of two Egr elements and one GC-box, an Egr/GC box, which appears to be a proper target of Sp or Egr family nuclear factors. Further investigations of the reporter constructs mutated in the Egr/GC-box, however, suggest that a nuclear factor(s) different from Sp1 or Egr may bind to a short core sequence of the Egr/GC-box and activate the Pref-1 promoter in the cell-type-specific manner.
MATERIALS AND METHODS
Materials
H295R cells and HeLa cells were generous gifts from J. I. Mason at University of Edinburgh and from H. Fukui at Tokushima University, respectively. Antisera to Sp1/3 [21,22] were gifts from G. Suske at Philipps-University. Expression vectors for Egr1/4 [23] and Egr2/3 [24] were kindly provided by C. Skerka at Bernhard-Nocht-Institute and by J. Svaren at Washington University School of Medicine, respectively.
A
B
H295R HeLa
-412 PpuMI
-196 Kpnl
-91 Ehel
BgAll GAGATCTTC
Transcription
+170 GAGATGATC
Pref-1
5’UTR
Spil -319
Sacil -140
Sacll -47
M
I
G3PDH
Translation
C
RLU (Pref-1-fLuc/TK rLuc )
-412
+170
0,0
0.5
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-319
Luc
-412
-196
Luc
-319
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-196
-140
H
-91
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-140
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47
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-91
H
H
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-47
H
H
HeLa
Luc
pGL3
H295R
-196
Egr/Egr/GC
+170
Luc
RLU (% of WT)
Egr
Egr
GC
0
50
100
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-52
Wild Type GCGTGGGCGIGGGCGGGGG
WT
HeLa
H295R
LS1
aat-c
LS 1
LS2
aatto
LS 2
LS3
aa-tc
LS 3
LS4
-aattc
LS 4
LS5
aatt
LS 5
Isolation of the rat Pref-1 promoter
A rat genomic library in NDASH II phage vectors was purchased from Stratagene (La Jolla, CA). The library (106 phages) was screened for positively hybridized plaques with 32P-labeled rat Pref-1 cDNA fragment as described before [25]. Five independent clones that covered an entire rat Pref-1 gene (from about - 10 kbp upstream of the exon 1 to +18 kbp downstream of the final exon) were isolated. A BamHI-NurI fragment containing 2.5 kb of the promoter and 175 bp of the exon 1 was subcloned into a BamHI- EcoRV site of pBluescript KS (-) (Stratagene). The resultant plasmid named pBlue-Pref-1prom (2.5K) was used for sequencing.
Construction of plasmids
To characterize the promoter, a transient expression system of luciferase reporter gene was used. Because no appro- priate restriction enzyme site was present in the 5’- untranslated region (UTR) of pBlue-Pref-1prom (2.5K), a BgIII site (see Fig. 1A) was created at a codon of translation-initiating-methionine by using a Chameleon
Double-Stranded Site-Directed Mutagenesis Kit (Stratagene) with a mutagenic oligonucleotide (5’-CGCCCCGAGATC- TTCGATCAAGCTTATC-3’) and a selection oligonucleo- tide (5’-CGGTGGCGGCAGCTCTAGAAC-3’) that targeted a unique NotI site in pBluescript KS (-) vector. Italic letters in Fig. 1B show the substituted nucleotides. To create several 5’-deletion constructs shown in Fig. 1C, p-412, p-319, p-196, p-91 and p-47, the modified pBlue-Pref-1 prom (2.5K) DNA (having the BgIII site) was digested by PpuMI, SplI, KpnI, Ehel or SacII, and the DNAs except that digested by KpnI were treated with T4 DNA polymerase to fill in protruding ends followed by digestion with Bg/II (Fig. 1B). To prepare p-140, partial digestion with SacII was used. The promoter DNA fragments were purified using an agarose gel and cloned into the pGL3 vector. To prepare p-196(ASac), p-196 plasmid was digested with SacII and the -140/-47 fragment was removed.
To disrupt the Egr/GC-box in the promoter, seven mutant constructs were prepared by site-directed mutagenesis in p-196 (Figs 2 and 3). A selection primer (5’-CGATAAG- GATCTCGCGACCGATGCCC-3’) targeted the SalI site in the pGL3 vector. Mutagenic primers, LS1 (5’-GTGCG- CGCGAGAATTCGGCGTGGGCGGGG-3’), LS2
B
A
RLU (Pref-1-fLuc/CMV-rLuc)
RLU (Pref-1-fLuc/CMV-rLuc)
-196 -140
-47
+170
Egr/Egr/GC
0.0
0.2
0.4
0.6
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1.0
0.0
0.2
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Luc
WT
WT
Luc
LS1
LS1
Luc
LS2
LS2
Luc
LS3
LS3
Luc
LS4
LS4
Luc
LS5
LS5
Luc
ASac
PSG5
ASac
pCMV
Egr-1
Egr-2
Luc
pGL3
Egr-4
pGL3
Egr-3
A
50aa
EGF-LIKE REPEATS
SP
TM
1
Gene
2
3
4
5
6
1kbp
Spli
Nr
Sc1 Ev
Sc1
E
B
B
HB
Sc1
H
Ev
H
E
Ex1 Ex2 Ex3
Ex4
Ex5
B
GGATCCACACCAATTCAGGGGAACCCTGACTGCTTCCTCTAGCCTACTCAGGGGCTTTTTCTTCCAGCCCTCAGTCACAGTTTCTGCAGAAAGCACAAGAAAAGAACATTACCCCAATTC TTTCAGCCTCAACTCTAGCCATGTGCCCATTCCCAGCTTTCTGACCTAGGGGTTCCCATTGTCTGGCTTCCAATCCCCTAAAATGGCAAAACTCTTCCAACCTGCTTTGGCAAGGTCATA TGCCACCACCCCCTACCTTGGGCTGAAGATGCCCCCCCTAGATTCAGAGTTAAAGCTCCTTGCCCTATGTGAGGAAAAGGAAGAAGCCATATATTAAAAAACATTTTCTTTCTTTCTTTA TATTTCATTTTATTTAAAGATGTTTTTCTTATGCATATAAACATTTATATGTGTGTATACTACACGTGTGTCTGGTGCTCTTGAAACTGGGTCTACAATCAGTGGTGAGCTGCCCTGTAG
360
480 GTTCTGGGAATTGAACCTGGGTCTTCTGGAAACACAATACATGCTCTTTACTGCTGAGTCGTCTATTCAACCTCCACATTAAAAAATTTTTTTTATTTACATTTTATTTTATTTTTTATT 600 720 840 960 TTTAAAACATTTATTTGATTTTTTACATTTCTTTTTTACATTAGAGACAGATGTGTGTGTGTGTGTGGTGTGTGTGTGTGTGTGTGTGTGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG AGAGAGAGAGAGAAGAGTCATGTACTGACCAGACAGGACAACAGTCTCAGTCTCTCTCCTTCCACCATGTGAGGTCTGGAGATTAAATTCAAGCTGTCAGGCTTGGTGGCAGGCACCTTT ATGGTTTACCAAATGAGCTACATCAATGACTCTATTTACATTTTATTTAAGGGGTTTCTGTGGGGATCAAAGGACAACTGCAGGAGTTAAGGATCAAACTCAAGTTTGGCTGCAAGCACC TTTACCCAGTGAGCCACCTCCCTGGCCCAGTAAATGTTATCAATTCACTTGGCTTCTCCTCCTTTTTGAGCCTCAGAATGGAAAATGGGGGCTTCCCAGCTGAGTAAAAGGCTCTGAGCC CTCGCTGGAGGGGATAGCAGGAAAAAGGGATTGCCCCAGGGACCTGGTAGAATCAATAACCCCCCCCACTCCCATCCTCTAAGCCACCCCTGGTTGAAAGCCACTGTGTGTGTGTGTGTG TGTGCGCGTGCGTGCGTGCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCTGTCTGTCTGTCTGTCTGTCTGCCTGCCTGCCTGCCTGCCTGCCTGCATTTGACAGTGAACATA TTGGCGAGACTGTCAACATGTGTGACTGTGAACAAAGATGAATCGGATTGTGTGTACTTTGGTGTAATTCGTGTGTTGGGTGAATCTGGGACAGATTTGGGGACTGGGCATAGGTGTTGC
1080 1200 1320 1440
TCTCAGGTGTGTAGCTGTACATGTTGGAAAGAATCACTGCTTTTATGTTTCAGCATGATAAGGCCCAGACTGTGATGGTGGTACCTGCGAGAGGAGGAGGGGTAGGGGGTGACGCACGAT GTCGTCGGTGTTTACCAGTGGTCTCCTGTGTTTACAGGCGGGCTGCGTAGAGGGGCGGCATAGGCAGGAGGACTCTCAGTCTTTCATAGCTGGTGCTTCCCCTGCTTGGAGCAGATCGGA CAGTGTTCCTGCTGCCCTGGTCTGTGACCTGTGCCTCGGGAGCAGGCATTAAGGTTTGCCTGTGTTGAAAGGTGTGTTTGGGTTGGGGGTCTGTGGAGCAAGTGCATCTGCACCTTCCCC AAACTCCACAATGGCAAGGCTCGTCGGAGGGCTTCGGCTTTTCGTGGTGGTTTTCGTGTGTGCATCTGTGATCATGTGCGTCTTTCACATCTTCGTGGTGTGCGTTTGGGTCCTCCTGGG CTTCTGACCCGCGCCATGTGTGCGCGGGACTCCAGCCCTAAGTGTCTTACTGTGTTTTGTGAGCGTTTTGTATTCTGGGTGCGTACGAGGGCTTTCGCTCGCCTGGCTTGGTTCCTGAGA
1920
CTTGCGCTAAGCGGGCCTGTGCGAGCGCCGCCACCTCCGCCTCCAGCCGCGTGCTGTCCTGTGTGCGGACCGCGTGCCCATCGTGCGCAGGTACCCGTCTAGCCAAAGAGTGCAGCCGCG
SAD
2280
GC box
GT box
Egr/GC box
Exon 1 GGGGGTACGACAGTACGAAAAGGCGGCGCGCGGGCCGCAGCGGCAGCTCCTCGGCAGCCGCACTTAGTAGCGTGCGCCCCGGTGCAACCCTGGCTTTCTTTCCGCTGGACACCCGTGCCC
GCbox MIATGALLR
2640
GTCCTCTTGCTCCTGCTGGCITTCGGCCACAGCACCTATGGTGAGCCCCCCCTGGCGGTCTGGCTTGCGCCCCCTCTGGGGAAGCCTGCGATGCCCCGCCGACCACCCTGCCGACCACCC VLLLLLAFGHSTY
CATTCTCCCTGTCCCGTGCGCCCCAAGTCCCGCCCGTCCCGCCTAACCCTAAGCCCCGCACTGTGCCACTCATCCTCCCTGCGCCACTCTTGCGTGTTGAGCCCAAT .. ( 800bp)
Intron 1
AAGGCTACCCCTTCATTGTCCCTATTCNTACTTTTAGGGGTCTGGGGGCTCTTTTTTTCTTTGGGGAGAAAGGTGTCTGCTGTGGACTTGGAGAATCAGGGGGATGCTGTGCTTAGCCCA TGGCTAGGAGCAGGCAAGATTGTTTTTCTGGGTAGGTATCCACAGATAAGAAATGCCTACTGTGTGCCAGGCTGGCAGGACCCAAGAGGGTCTTTGGAGCCCCTGCTGAGGGGCAGCAGG
3120
CCATCTGAGGGAAGATGGTGTTTGTAGTGTCCTTTAGGCCTTGTGGGGTGAATTGCGTACCCTTTTTCTCCTTCTCAGGGGCTGAGTGCGACCCGGCCTGTGACCCTCAGCATGGATTCT GAECDPACDPQHGF
3240
Exon 2 GIGAGGCTGACAATGTCTGCAGGTAATGGAGAAAGCTGTTTGGGGGGGCCATGGTGGGGGTAGGATCCTGTGGAGTCTTGGAGTCAGGCTGGAAGGAGGGGGTGCTTTTCCAAAAAAAAA CEADNVCR ☒
AAAAATGCGTCACAGCTTTCTTGCTTCTGCAGAAAACTGGGGGAGGGGGAGAAGAGAGAAGGAGGGGTGCAGGAAGGAGGGGGATCAGAAGAGTGCCTTGCAGGGGGAGACCACTGTAGA 3360 3480 Intron 2 CCACTGCACCAAATGGGGTTCCTGTGGGGGTCTGGTAGACCCTTGTGTTTNTGCCCCTCAGCACCTTCTCTGAGTTTTCCATTCAGGTGACTGCTGATTGTTGAAAGGAGCCACTGGCCT GGGACTCCACAAAGACCACCAAGTGTTCTCTGTTCAGCAGACAACAGTTCGAGTCTCTGGACCCCTGCTCCCCTCATGTGTCCCTCTCTCTCTCCCCTTTGTCCACACAGTGTGAGCCT 3600
CEP
3720
Exon 3 GGCTGGGAGGGCCCCCTGTGTGAGAAGTGCGTAACCTCCCCTGGCTGTGITAATGGACTCTGTGAAGAACCATGGCAGTGTGTCTGCAAGGAAGGCTGGGACGGGAAATTCTGCGAAATA
3840
Intron 3
4200 4320
DIRACTSTPC
4440
Exon 4 GCCAACAATGGGACITGCGTGGACCTCGAGAAAGGCCAGTACGAATGCTCCTGCACCCCTGGATTCTCTGGAAAGGACTGTCAGCACAAGGCTGGGCCCTGCGTGATCAATGGSTAAATA
ANNGTCVDLEKG Q Y ECSCTPGFSGKDC QHKAGPCVING ☒ ☒
TTCTCACCTCACTCTGGGGGTCTTGCCTCTCCCCCACCCCCAGATGAAAGATGTATTCCCAGCAATCTGCCGCCTCCAATCCACTCTGACTGGGCGGTGGCGGTGGTGGCGGTGGTGGTG 4560 GATTATGTAGGAATGTGTCATTTCCATAATTTTCTTTTAAAAACAAAATGGTCATGAAGGCGATTTCATATTCCCCTGAGGTCCCCAGGCTCCTATGCTGATGAATG . . ( 800bp) …
Intron4
AAAAATAATAAGGGTGGGAAGGGTGGGGGGGGTGGGGACGCAGACTGGAGCCCTAACAGTAGATCTCCCCTGGAGGACTGTGCCCCTTACTCTCCTCATCCGGGAACGAGCTCATTGAA
5040
SPC QHGGACVDDE ☒
Exon 5 GGCCGGGCCTCGCATGCTTCCTGCCTGTGCCCCCCTGGCITCTCGGGCAACTTCTGTGAGATCGTGACCAACAGCTGTACCCCTAACCCATGCGAGAACGATGGCGTCTGCACCGACATC 5160
5280
P V S N C A S G P C L N G G 2 ☒ GTGAGCITCGAGTGTCTGTGCAAGCCCCCGITCATGGGTCCCACATGCGCGAAGAAGCGCGGGACCAGCCCCGTGCAGGTCACCCACCTACCCAGCGGCTACGGGCTCACCTACCGCCTG V S F ₡ L C K ☒ P ☒ P F ☒ M ☒
5400
G P
T
C
A
K ☒
K ☒
R
G
T
S P V ☒
2 V
T H
L
P
S G
☒
L
5520
T PGVQEL P
V ☒
2
2
P
E
H
2
I
L K ☒
V
S
M ☒
K ☒
E L
A
P
L
L
T
E
T
5640
G ☒
L
₸
S L
V ☒ V ☒
L
G
T
V ☒
I
V
F
N
K ☒
C
E
A
W
S N L
Y
H
I L A L R N M ☒ K ☒ N CTGCTGITGCAGTACAACAGCGGCGAGGAGCTGGCGGTCAATATCATCTTCCCGGAGAAGATCGACATGACCACCITCAACAAGGAGGCTGGTGATGAGGATATTAAGCAGCGTGTCCC ☒ ☒ ☒
5760
L LLO Y NSGEELAVNIIF PEKI DMTTFNKEAGDEDI * CTCCCCCTCCCCCAGGCCCTTCTACATTATCGGGGITCCTCACAGCTCCCTCTATGCGCTCTATGCITCITTGTGGTGGAGITCGCTCTTGTGTGGAATCTAGTGAACGCTACGCTTACA ☒ 5880 ☒
6000 6120
6480
TITATTTTCTCGTGITGCTGTGTGACAAGCTGCCGCTAAGAACCCCTCCCTCCCTCCCTCCCTCCCTCCCTATTAATGCATGATATAATGAATAATAATAAGAATITCATCTCTAAATGA GTTAAAGAAATAAGTATGTTATTCTAAACTCTATCGAATATCAAATATAAAATCAAAAAGACCAAAAAAAAACATGGCAACAGTACCAGGGCTTGGTGCTGANGTCCCTCTCCCAGGGGT ATGGGTGTCACCCACCAGGTCCTCGTGAAACCACAAGAGTCCTGTGCTTGACCACTCACTGTGAAAAGGGCTAAACTCTTTCGTTCGTTAATTCTCACGCACACACGCACACTCACTCAC 6240 ATCAAGTCTCACACCGCAACCCTTAGATCTTTCCAATTGATTTGTGACTTTGCTGCGTGCGGTGGCTGTGTGCCAGGCAGNAAGAGACCCAGGTTGGCTGTTGGCAGAGAGGTCCGGGGG ACCTAGTCCCAACCCTGCTGCTCACTCGCCGTGAGACCCTTGGCGAGAGCCCCCCCTCCAACCCTGGGCCACTTCCCCACCCCTCCCCATAAGGGAGGGGTGCAAACACCCAAACACCAA GGTCTTCTCTAGCTCCCAAATTCANAATTAGGAACGAGGGATGTGTTCAAAATTCTTCCTGACTTCGGTAGTANGATCTGATGGAGACTTGGTCTCNTCCTGGGATGCGAAANGTTTTGG CCTATCCTGGCTGTGCACTTGATATTGAAGTCCATCCCNATANCGTTTCTCAAAATGTGGCGANCCTCCTNGGCTCACATCCAGTTTGGGAACAATTGGCCCCTTTAGGTCAAAATGTGC CATTTGGAGTTGCCTCTTGAGCAACCCACTCCCGCCCCAAGACTTCACCCCCAGGCCCCCCAGGCCCCACCCCATACCTGCCCCTCACCCTGTAGAGTAGAGATGTGCTTTCAGACTTAA GTGTACCTTGAACCACAAGTGCCCCTCCCCGGCAAACACTGAGTGAGCAGTGCAGCCCCCACCAAGGGTTTTCACGAAGCTTTAGTGAAAACTACTGCTTGTGGAGTGTTTTTCTTGTTC
TGTGGCCCAAGTGGGAAAGCACTGGCCACAGTCGTGGATGTACCTTTGTTGTGGCTGAGGTTAGGACCTCAGATCCCGGGACACCTAGCCAGCTTCTTAAGTAGGATTTTGACTTATTTT TTAAAAGAGCATCTATCTTTTTTTGCAAAACAAAACAAACCGGCAAGGACAGAAAAAAAAAAAAGTAGAAAGAAACGGGAGAGAGCCCTGTGTAGCTTCCCAGCTCTCGTTTGGGGTNTG GATGNTACACAGTCCTTGGTNGCCCCGTTAGGAATATTTTGCTTCCTAGATTCATTTGGGTAGGAACCTATGCCCTAAGCTTTAAATGTGAGCCGTGTTTACCTTGCTCTGAGTACTTGC 744 TTGCAAGTCATTGTCACCGTTGTCGTTCAATCATGATCACAGCACACACTTCATGGTCATCAACCATTGGTCATACGCGTCTCCGTTGTCACACATGATCGACCCAATCACCCCTCCAAC 7560 CTCTACCCCAGGTTCATTTCATCCAAAGCCCTGACTCAAAGAAATCTCTCCCCAGCCATTAGTCATTCATTACCTTCAGCCCGACCCCCTTCCAGCCCCACCCTGAAATCCCTCCCCCTT 7680 7800 TGGCCCCAACCTCATTCTGATGAGGGTGTGTCCCANCTCCCCAATCCCCACCACATCCCTGCCTCCTANCTCCTCCTCTTCCCTCANTCCCTCCCTCCANGCCCCTCCCTCCCTCCAANA CCCTCTCCTGCCTCCAATCANCCCCATCCTGAGGCAAACCACTCCA
6600 6720 6840 6960 7080 7200 7320
S N ☒ C E I ☒ V S C T P N P E ☒ GGGGGCGACTTCCGITGCCGCTGCCCAGCTGGATTCGTCGACAAGACCTGCAGCCGTCCGGTGAGCAACTGCGCCAGTGGCCCGTGCCTGAACGGGGGCACCTGCCTCCAGCACACCCAG GGDFRCRCPAGFVDK T C F T N ☒ ☒
S
R
F
S H
4680 4800 AGATCTTACTCCAAACTGTTACTAATGCAGCCTCAGTTTCCCCATCTGAATAGTGACTTACAATCTTTGTTCTGGCCTGAGAGACTAAGAGATGGGAGAGGGCTTAGGAGAGGGTATGGG 4920 AGGTCGGTTCTGTTCCTATTGCTCCGTCCTAGCCCAGCCCCCCCTCCCCCAGGACCCTTGACCATATCTTTGTGTTACACITCTCCCTGCCAGCACGGAGGCGCCTGCGTGGATGATGAG
3960 4080
GWEGPLCEKCVTSPGCVNGLCEEPWQCVCKEGWDGKFCEI GGTGGGCACTGAGCTATCCTACCCTCACCCCTTTTCCCTGTCCCTTCTCCCTCCCTCTCCTACACCTACATTCCAGAACCCCTAAGGCTCCCTATTTGTAGTATGTGATCTATGGTGTAT ATGCACTTCCTGCTGGGCCTCAAATGCCTCCTCAAATGTATAGACATATATATTTTGTTTACCTCAGCACTGCGGGCTCCCAGACTTGGGGTTGGGGAGGAGGGGG . . ( 1900bp) . ACCCTTTCCCTAAATGTGAGAAGNTGATGTGAGAGGGAAACAGGCCTCAGGGAATNGTAGTGGTTTCCCTGTTTTGTGAAGGGGCAGAGGCTTNTGGGATGGAGGTTGTGNTACCACANT ACAACGGTGCCAGCAAAGGGTNTAGCTGCTTNTCTGGCCTTGGCTTCCTCACTAGTGACCTTCCTGACCCTCAGCACTTCAGGTNTTCACTGCACCCTCTTACTACAAACCCCACTTGGA AATCACTAGCCGAGCCATTTAGGGTTTCTGTCACACTGGCTTNTGGAAAGAAAAGCTAAGCACTAGGTTATCCCCCCCCCCCGTTTCCCAGATATTCGGGCTTGCACCTCTACCCCCTGC
2760 2880 3000
2400 CCTTCGTGGTCCGCAACCAGAAGCCCAGCGCAGCCCCCGGAGCAGCCCCTGCACCGCCACCGCTACCCGGACCACGACCCAGGCCGCCCCGACATCATCGCGACCGGAGCCCTCCTGCGC 2520
1560 1680 1800
2040
2160
TGCGCGAGCCGCTTCCGGGCGCCGCGGCGCTGGGGGGCGGCCCCGCCAGGGGGTGGGTCACCGGGACTGGCCGGCGCCTGCCCCGTGCGCGCGAGGCGTGGGCGTGGGGGGGGGCCGCGG
120 240
ATCCTGGGCGTGCTCACCAGCCIGGITGTGCTGGGCACCGTGGCCATCGTCTITCTCAACAAGTGCGAGGCCIGGGTGTCCAACCTGCGCTACAACCACATGCITCGCAAGAAGAAGAAC
N
☒
(5’-GCGAGGCGTGAATTCGGGCGGGGC-3’), LS3
(5’-GCGAGGCGTGGGAATTCGCGG-GGCCGC-3’), LS4 (5’-GCGTGGGCGTGAATTCGGGCG-CGGG-3’), LS5 (5’-GGCGTGGGCGAATTCCGCGGGGG-3’) and LS1/4 (5’-GTGCGCGCGAGAATTCGGCGTGAATT- CGGGCCGCGGG-3’), designed to create the new EcoRI site at the Egr/GC-box, were used for preparing the LS1 to LS5 and LS1/4 mutant promoter constructs, respectively. The LS6 mutant was prepared by replacement of an 11-nt EcoRI fragment of LS1/4 with annealed oligonucleotides (5’-AATTGGGAATG-3’ and 5’-AATTCATTCCC-3’). All mutated promoter regions were incorporated back into the original pGL3 vector using KpnI/BgIII digestion.
To prepare chimeric promoter constructs containing the -91 to -65 and the -76 to -47 regions, two sets of oligonucleotides, sense-91/-65: 5’-gatcCGCCTGCCCCG- TGCGCGCGAGGCGTGG-3’; antisense-91/-65: 5’-gatc- CACGCCTCGCGCGC-ACGGGGCAGGCG-3’ and sense- 76/-47: 5’-gatcCGCGAGGCGTGGGCGTGGGCGGGG- GCCGCG-3’; antisense-76/-47 5’-gatcCGCGGCCCCCG- CCCACGCCCACGCCTCGCG-3’ were prepared. The lowercase letters show the linker sequences. The sense and the antisense oligonucleotides were mixed and introduced into the BgIII site of the pGL3 promoter vector or the pGL3 enhancer vector. The resultant constructs were named pGL3prom-91/-65, pGL3prom-76/-65, pGL3enh- 91/-65 and pGL3enh-76/-65.
Cell culture and promoter assay
H295R cells were maintained in Dulbecco’s modified Eagle’s/Ham’s F-12 medium containing 1% ITS (Becton Dickinson Labware, Lincoln Park, NJ, USA), 2% Ultroser
G (BioSepra Inc., France) and antibiotics at 37 C under an atmosphere of 5% CO2-95% air as described before [26]. Hela cells were grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and antibiotics. Transfections were performed with the Lipofectamin Plus reagent (Life Technologies, Grand Island, NY, USA) in 12-well plates. H295R cells (1 x 105 per well) and HeLa cells (5 × 104 per well) were plated at 48 h prior to addition of DNA. Pref-1 promoter constructs (0.5 µg) were mixed with 0.1 µg of pTK-rLuc plasmid (Renilla luciferase expressing vector; Promega, Madison, WI, USA), an internal standard, and the mixture was incubated with 2.5 ML of the Plus reagent (Life Technologies) in 50 µL serum free medium for 15 min. To the DNA solution 50 pL of serum free medium containing 2.5 ML of Lipofectamin (Life Technologies) was added, and the mixture was further incubated for 15 min and diluted with 400 uL of the serum free medium. Cells, washed twice with serum free media, were incubated with the DNA solution for 3 h and transferred to 1 mL of serum-containing medium. After the 48-h incubation, cells were harvested and assayed for luciferase activities by the Dual-Luciferace Reporter Assay System (Promega). For the cotransfection experiments, 0.1 µg of Pref-1 promoter constructs were cotransfected with 0.5 µg of overexpression vectors for Egr-family transcription factors.
Nuclear extracts
Nuclear extracts were prepared as described previously with some modification [27]. Briefly, cells were washed twice with ice-cold NaCl/Pi and then harvested. Subsequent steps were performed at 0-4 C. First, cells were suspended
C
SAD
GC-box
rat
-197
GGTACC
CGTCTAGCCAAAGAGTGCAGCCGCGTGCGCGAGCCG-CTTCCGGG-CGCCGCGGCGCTGGGGGGCGGCCCCGCCAGGGG-GTGGGTCA
human bovine
-204 ------ TGTGCCCGTCTAGCCAAGAAGTGCGCCCGTGTGCGCGAGCGGGCTTCT’GGGACGCCGCC GTGGICGGGGGCGGCCC TGCGAGGGGAGGGGGTCA
-206 GGTACCTGTGCCCGTCTAGCCAAGA-GTG-GCCCGCGTGCGCGAGCGGGTTTCCGGGACACCGOAGTGGIGGGGGGCGGCCCCGCGAAAGGCGGGGGTCA
Egr/GC-box
rat human bovine
-105 CCGGGA-CTGGCCGGCGCCTGCCCCGTGCGCGCG-AGGCGTGGGCGTGGG-CGGGGGCCGGGGGGGGTACGACAGTACGAAAAGG-EGGCGCGCG-GGC
-109 CAGGGAACTGGCCGGCGCCGGCCCCGTGCGCACGGAGGCGGGGGGGGGGGGCGGGGGCCTCAAGGGGG-GAGGCGGTACGAAAAGGGCGGCGCGCGCGGC
-107 CAGGGA-CTGGCCGGCGCCGGCCCCGTGCGCACGAAGGCGGGGGCGAGGGGCGGGGGCCGCG-GGGGG-GCCGCGGTACGAAAAGGGCGGCGCGCGCGGC
Transcription
rat human bovine
+10 CGCAGCGGCAGCTC-CTCGGCAGCCGCACTTA
GTAGCGTGCGCCCCGGTGCAACCCTGGCTTTCTTTCCGCTGGACACCCGTGCCCCCTTCGTGG
+10 GGCGGCGGCAGCTCGTCCGGCAGCGGCGGTGGAGAGGGCAGCGCACAGCCCGGTGCAGGTCTGGCTTTCCCCTCGCTGCGCGCCCGCGCCCCCTTTCGCA
+10 GGCGGCGGCAGCTCG ---- GCGGCGGCAGCGGAGAGCGCAGCGCGCAGCCCGGTGCAGCCCTGGCTTTCCCCTCGCCGCGCGCCCGCGCCCCCTTTCGCG
CAP
Translation
rat
+85
TCCGCAACCAGAAGCCCAGOGCAGCCECCGGAGCAG CCCCTGCACCGCCACCGCTACCCSGACCACGACCCAGGCCGCCCCGAGATG +171
human +91
TCCGCAACCAGAAGCCCAGTGCGGCGCCAGGAGCCGGACCCGCGCCCGCACCGCTCCCGGGACCGCGACCCCGGCCGCCCAGAGATG
+177
bovine +87
TCCGCAACCAGAAGCCCAGCGCGGCGCCCGGAGCAGGTCCCGCGCCCGCGCCGCTCCCGGGACCGCCCCCCCGGCCGCCCEGCGATG +173
GC-box
in 10 volumes of hypotonic buffer A composed of 10 mM Hepes (pH 7.9), 10 mM KCI, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.15 mm spermine, 0.5 mM spermidine, 0.35 M sucrose, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 14 mg.mL- aprotinin, and 10 µg.mL- leupep- tine. The cells were homogenized, and the homogenates were subjected to centrifugation at 2000 g for 10 min. The pellet containing the nuclei was resuspended in 10 volumes of buffer A. The nuclear suspension was then overlaid on buffer B (buffer A containing 0.5 M sucrose) and cen- trifuged at 2000 g for 15 min. The nuclear pellet was suspended in the same volume of extraction buffer (20 mM Hepes (pH 7.9), 330 mM NaCl, 1.5 mm MgCl2, 0.5 mM dithiothreitol, 10% glycerol, 0.5 mm PMSF, 14 mg·mL-1 aprotinin, and 10 µg.mL- leupeptine) and incubated at 4 C for 45 min. The incubation mixture was centrifuged at 10 000 g for 20 min, and the resulting supernatant was used for the gel mobility shift assay.
Gel mobility shift assay
For the gel mobility shift assay, three pairs of synthetic oligonucleotides were designed based on the -76/-47 sequence covering two Egr elements and one GC-box in the rat Pref-1 promoter. They included the following sets: WT, sense-76/-47 and antisense-76/-47 (the same oligo- nucleotides used for constructing the pGL3prom-76/-47 vector described above); LS3, 5’-gatcCGCGAGGCGTGG- GAATTCGCGGGGGCCGCG-3’ and 5’-gatcCGCGGCC- CCCGCGAATTCCCACGCCTCGCG-3’; LS1/4, 5’- gatcCGCGAGA-ATTCGGCGTGAATTCGGGCCGCG-3’ and 5’-gatcCGCGG-CCCGAATTCACGCCGAATTCTCG- CG-3’. Each set of oligomers was annealed by heating at 70 C in 1 x Klenow buffer supplied from New England Biolabs (Beverly, MA, USA), and slowly cooled to room temperature.
The oligonucleotides were 3’-end-labeled with [@-32P]dCTP using Klenow fragment in the presence of cold dATP, dTTP and dGTP. The radiolabeled DNA was purified by ethanol precipitation. For each binding assay, unlabeled competitor oligonucleotides (100 fold excess) or antisera to Sp1 or Sp3 (1 µL) was mixed with 10 µg of nuclear extracts in 20 µL of binding buffer composed of 10 mM Hepes (pH 7.5), 16 mm KCI, 0.5 mm MgCl2, 1 mM dithiothreitol, 0.4 mm EDTA, 1 µg poly(dI-dC)-poly(dI-dC). The radiolabeled duplex oligonucleotide (50000 cpm) was added to the mixture followed by incubation at room temperature for 15 min to form protein-DNA complexes. Samples were immediately analyzed on 5% polyacrylamide gels, and autoradiography was carried out by using an intensifying screen.
Northern blot analysis
Northern blot analyses were conducted as described previously with some modification [28]. Cells (70% confluent on 6-cm dish) were lysed in 0.8 mL guanidine solution (4 M guanidine thiocyanate, 50 mM Tris/HCI (pH 7.6), 1 mM EDTA, 2% sodium lauroyl sarcosinate, and 2% 2-mercaptoethanol). The lysates were mixed with phenol/chloroform, the resultant aqueous phase was taken, and the RNAs in the aqueous phase were precipitated with ethanol. The total RNA dissolved in water was separated by electrophoresis and transferred onto nylon membrane, Hybond-N+ (Amersham, Arlington Heights, IL, USA). Hybridization with radioactive probes (rat Pref-1 cDNA fragment) were performed in 50% formamide solution containing 5 × NaCl/P;/EDTA, 0.5% SDS, 5 x Denhardt solution and 0.5 mg·mL-1 carrier DNA at 42 C for 16 h. After the hybridization the membrane was washed once with 2 x NaCI/P;/EDTA containing 0.1% SDS for 20 min
A
Egr/GC-box
-70 Egr
Egr
GC -52
CGCCTGCCCCGTGCGCGCGAG
GCGTGGGCG
GGGCGGGGG
CCGCG
-91
-76
-65
-47
B
pGL3 promoter
C
pGL3 enhancer
SV40 prom
Luc
Luc
SV40 enh
RLU( fold change )
RLU( fold change )
0
2
4
6
8
0
2
4
6
8
pGL3 prom
HeLa
pGL3 enh
HeLa
H295R
H295R
-91/-65
-91/-65
-76/-47
-76/-47
at 65 C, and then twice with 0.2 x NaCl/P;/EDTA containing 0.1% SDS for 20 min.
Identification of the transcription initiation site
The 5’-rapid amplification of cDNA ends (RACE) analysis was employed to identify the transcriptional initiation site of the rat Pref-1 gene with a RACE kit (Takara, Kyoto, Japan). Briefly, total RNA (1 µg) prepared from the adrenal ZG was reverse-transcribed into cDNA with 5’-phos- phorylated RO primer (5’-CAGGGGAGGTTACGCACTTC- 3’: in the exon 3). The DNA-RNA hybrid was digested by RNaseH. The first-strand cDNA was self-ligated with T4 RNA ligase. A series of nested invert-PCR was performed to amplify the 5’ end of the rat Pref-1 cDNA by using two sets of primers (the 1st PCR: 5’-GG-TGTAGCCTGGCTGG- GAGG-3’ [in exon 3] and 5’-GGGTCACAGGCCGGGT- CGCA-3’ [in exon 2]; the 2nd PCR 5’-CC- CTGTGTGAGAAGTGCGTA-3’ [in exon 3] and 5’-CAT- AGGTGCTGTGGCCGAAAG-3’ [in exon 1]). The 1st and 2nd PCRs were carried out at 94 C, 40 s; 56 C, 40 s; and 72 C, 1 min for 30 cycles with a final extension at 72 C for 5 min. The 2nd PCR products were purified by a spin column (Qiagen, Chatsworth, CA, USA), cloned into pT7BlueR vector (Novagen, Madison, WI, USA) and sequenced.
RESULTS
Structure of rat Pref-1 gene
Screening a rat genomic library by using Pref-1 cDNA fragments as the probes, we isolated five independent clones. Restriction enzyme digestion analyses (Fig. 4A) and sequencing (Fig. 4B) of these clones revealed that the rat Pref-1 gene, about 8 kb-long, was composed of five exons and four introns. The overall organization of the gene well resembled that of the mouse gene described by Smas et al. [29]. Comparison of the nucleotide sequence of the rat Pref-1 gene with those of seven independently isolated cDNA clones [7] indicated that no alternative splicing occurred during the transcription in ZG. To determine whether the transcription initiation site of the gene in rat ZG was the same as that reported for mouse 3T3-L1 preadipocytes, we
A
probe
-76 /-47
GC-box
Egr
H295R
Hela
H295R
extract (-)
Hela
H295R
(-)
(-)
Hela
ori
complex 1-+
complex 2++
free probe
B
extract
H295R
HeLa
X50 competitor
-44 /-18
-76 /-47
GC-box Egr
-44 /-18
-76 /-47
GC-box Egr
(-)
(-)
ori
complex 1-+
complex 2-
free probe (-76 /-47)
I
C
probe
-76 /-47
GC-box
Egr
antibody
«-Sp1
«-Sp3
u-Sp1&3
tt-Sp1
«-Sp3
«-Sp1&3
«-Sp1
«-Sp3
-Sp1&3
(-)
(-)
(-)
ori
Sp1 Sp3 .-
Sp3-
free probe
performed 5’-RACE analysis on total RNA prepared from ZG. All the 11 independently isolated RACE products had the same 5’-end nucleotide residue, a guanylate, in the Cap sites, as indicated by an arrow in Fig. 4C. The Cap site has been identified as the transcription starting region of the mouse Pref-1 gene in 3T3-L1 cells [29].
The 5’-flanking region of the rat gene, shown in Fig. 4B, lacked TATA and CAAT boxes. The region from -200 to the transcription starting site was rich in G and C, its GC- content being about 90%. Three consensus Sp1 binding sites were noticed (‘GC-boxes’ in Fig. 4B). One of the three had a sequence overlapped with two Egr binding sites, which was designated herein ‘Egr/GC box’. Smas et al. identified a cis-element AAAGA and named it ‘suppression in adipocyte differentiation (SAD)’, because it was responsible for down-regulation of Pref-1 gene during adipogenesis [30]. The SAD sequence in the mouse gene was present at -181 to -177, whereas in the rat, it was present at -182 to -178.
Promoter region of rat Pref-1 gene
To identify a nucleotide sequence in the 5’-flanking region that would be involved in tissue-specific expression of the gene, we utilized two cultured cell lines in the following experiments; the first, H295R cells, constitutively express Pref-1 gene, and the second, Hela cells, do not (Fig. 1A). Several fLuc reporter plasmids with nested deletions of the 5’-flanking region were constructed and introduced into the cells (Fig. 1B,C). The promoter activity of a construct containing up to - 412 position was the highest in both the cells. Unexpectedly, the magnitude of activity in H295R cells, about 70-fold as large as that of control pGL3, was similar to that in Hela cells, about 50-fold. Reporter constructs having DNA fragments longer than -412 (up to -10 kb) also failed to show cell-type specific promoter activity (data not shown). When constructs having the
fragments shorter than -412 were introduced into HeLa cells, the reporter activities decreased concomitantly with the length of the fragments, little promoter activity being observed in the construct containing -47. In contrast, the reporter activities in H295R cells, were not affected substantially until the deletion reached to -140 position. When a region from -140 to -91 was deleted, there was a substantial decrease in the reporter activity by one third. More remarkably, the deletion of a region from -91 to -47 resulted in a drastic decrease in the reporter activity. These results suggested that a trans-activating factor(s), present in H295R cells but absent in Hela cells, could bind to the region from -91 to -47 to activate the promoter in a cell- type specific manner.
The region from -91 to -47 was divided into two overlapped oligonucleotides, those from -91 to -65 and from -76 to -47, and the respective fragment was inserted into either a pGL3 promoter vector or a pGL3 enhancer vector (Fig. 5). Because the pGL3 promoter vector contains only a promoter element, the vector inserted with a test oligonucleotide would reveal the enhancer activity of the inserted oligonucleotide. On the other hand, the construct made from the pGL3 enhancer vector with a test oligonucleotide could be used for evaluating the promoter activity of the oligonucleotide. As shown in Fig. 5B, neither oligonucleotide could act as an enhancer both in Hela cells and in H295R cells. In contrast, the oligonu- cleotide from -76 to -47 inserted into pGL3 enhancer vector could act as a promoter in H295R cells, but not in Hela cells (Fig. 5C). Its promoter activity was about seven times as high as the control. These results suggested that the region from -76 to -47 in the rat Pref-1 promoter had the cell-type-specific promoter activity, but it did not have an enhancer activity. It was noted that two Egr elements and one GC-box overlap each other in this region (GCGTGGGCGTGGGCGGGGG: Egr/GC-box) (Fig. 3A).
RLU (% of WT)
-70
Egr
Egr
GC
0
20
40
60
80
100
120
-52
Wild Type
GCGTGGGCGTGGGCGGGGG
WT
LS1
aat-c
LS1
LS1/4
aat-c
aattc
LS1/4
LS6
aat
aa — aattc
LS6
LS4
-aattc
LS4
Sp1 and Sp3 bind to the Egr/GC-box
In order to identify nuclear factors bound to the region from -76 to -47, gel mobility shift analyses were performed. The entire DNA fragment (-76/-47), a GC-box (GGGCGGGG), or an Egr-element (GCGTGGGCG) was radiolabeled, mixed with nuclear extracts prepared from H295R or Hela cells, and subjected to electrophoresis (Fig. 6A). Either nuclear extract could produce two major complexes, complexes 1 and 2, with the DNA -76/-47 or the GC-box, and one major complex, complex 1, with the Egr-element. Non-radiolabeled probes of DNA -76/-47 and GC-box efficiently, and that of Egr-element partially, competed with binding of the radioprobe -76/-47 with the nuclear extracts, but a nonradiolabeled DNA fragment -44/-18, having non- related AT-rich sequence, did not act as the competitor (Fig. 6B). Comparing these results with previous reports on Sp-family factors, nuclear factors binding to GC-rich regions, we surmised that factors involved in the formation of complexes 1 and 2 might be Spl and Sp3. To confirm this, antiserum against Sp1 or Sp3 was added to the incubation mixtures and the mixtures were subjected to the gel-shift analyses (Fig. 6C). DNA- protein complexes recognized by the antibody should appear at an upper position near the origin. When the DNA -76/-47 was used as the radioprobe, the anti-Sp1 serum shifted specifically the complex 1 to the origin, and in contrast, the anti-Sp3 serum shifted the complex 2. The treatment with both antisera abolished most of the complex 1 signal. The similar results were obtained with the GC-box as the radioprobe. With the Egr-element as the radioprobe, however, only the anti-Sp1 serum seemed to shift the complex 1. These results suggest that Sp1 and Sp3 are the major nuclear factors bound with the sequence from -76 to -47, and the GC-box and the Egr-element in the Egr/GC- box may be target sequences for Sp1-/Sp3-binding and Sp1- binding, respectively. However, it should be noted that these nuclear factors seemed to be present in H295R cells as well as in Hela cells, and therefore they might not be involved in the cell-specific expression of Pref-1 gene in H295R cells.
The core of the Egr/GC-box is essential for the cell-type-specific transcriptional activation
To further scrutinize a role of the Egr/GC-box in Pref-1 gene expression, several reporter constructs having muta- tion within the Egr/GC-box were tested for the promoter activities in H295R cells as well as in Hela cells (Fig. 2). Mutation only in the first Egr element (LS1) seemed not to influence the reporter activity in both cells. Mutation in the GC-box alone (LS5) even enhanced the activity, whereas mutation in an overlapped region of the GC-box and the 2nd Egr-element (LS4) slightly decreased the activity. Interestingly, mutation in a short stretch composed of no more than seven nucleotides at the midst of the Egr/GC box (LS2 or LS3) reduced the activity to about 40% of the control in H295R cells, but little decreased the activity in Hela cells. These results suggested that the short nucleo- tide sequence present in the core of the Egr/GC box, GGCGTGG, might be responsible for the cell-type-specific expression of the rat Pref-1 gene.
Egr family factors are not involved in the cell-type-specific promoter activity
The above results indicate the presence of a nuclear factor(s) in H295R cells, but not in HeLa cells, that binds with the short nucleotide stretch covering the two over- lapped Egr elements or at least one of the Egr elements, within the Egr/GC-box and activates the promoter of Pref-1 gene. To gain more insight into the property of this factor, the effect of overexpression in H295R cells of Egr factors, ubiquitously present nuclear factors having ability to bind Egr element, was examined (Fig. 3A,B). The Egr factors, when overexpressed with reporter constructs having Egr elements, such as Wild type, LS1, LS4 and LS5, would elevate the reporter activity, but they, when overexpressed with the constructs with disrupted Egr elements, such as LS2 and LS3, should have no influence on the reporter activity. Hence the cells were transformed with the series of mutant promoter constructs together with overexpression vectors for Egr factors. The overexpression of Egr factors did not affect the reporter activity of the control reporter, pGL3, and ASac reporter lacking the entire GC-rich region.
A
extract
H295R
-76 /-47: LS 1/4 -76 /-47: WT
HeLa
(-)
-76 /-47: LS 3
GC-box
-76/-47: LS 1/4
-76 /-47: WT
-76 /-47: LS 3
X50
GC-box
competitor
(-)
Egr
(-)
Egr (-)
ori
complex A
complex B
NS
complex C
NS
free probe (-76 /-47: LS1/4)
B
Sp1
Sp3→
Sp3-
The reporter activities of the wild-type construct and the mutant constructs LS1, LS4 and LS5, each of which had at least one intact Egr element, were significantly elevated in the presence of overexpressed Egr factors. In contrast, the reporter activity of the LS2 construct that had mutation in the overlapped portion of the two Egr elements was not influenced by the overexpressed Egr factors. Surprisingly,
A
competitor
LS 1/4
WT
LS 3
(-) X10 X30 X100 (-) X10 X30 X100 (-) X100
ori
complex A
complex B
complex C++
free probe (-76 /-47: LS1/4)
B
120
complex A
competitor
100
ALS 3
% bound
80
60
40
20
= LS 1/4
0
· WT
0
20
40
60
80
100
120
complex B
competitor
100
ALS 3
% bound
80
2
· WT
60
40
20
0
· LS 1/4
0
20
40
60
80
100
120
complex C
competitor
100
ALS 3
% bound
80
60
· WT
40
20
· LS 1/4
0
0
20
40
60
80
100
the reporter activity of the LS3 construct, also having mutation in the two Egr elements with the partially disrupted sequence in the first and the completely disrupted one in the second, was substantially elevated in the presence of overexpressed Egr factors. In other words, Egr factors could somehow bind with the mutated Egr elements in the LS3 construct and induce transcription of the reporter gene. Considering that in H295R cells, both the reporter activities of the LS2 and LS3 constructs were at the similarly low level (Fig. 2), these results suggested that Egr factor(s) recognized the sequence a little different from the core nucleotide sequence which was recognized by the putative H295R cell nuclear factor.
A specific nuclear factor binds to the core sequence of the Egr/GC-box
To confirm further the importance of the short core sequence for the promoter activity in H295R cells, other mutant constructs, LS1/4 and LS6, were tested (Fig. 7). LS1/4, having the sequence disrupted in the first Egr- element and the GC-box, produced the reporter activity almost at the same level as LS1 or LS4 construct. In contrast, LS6, having the sequence similar to LS1/4 but for two nucleotides at the center, produced much less activity than the other constructs. These results again indicate the importance of the short core sequence for the promoter activity.
Attempts were made to further characterize the nuclear factor by the gel mobility shift assays. The Egr/GC-box oligonucleotide would strongly bind with ubiquitously present Sp1 and Sp3 nuclear factors (Fig. 6), and therefore, if this oligonucleotide was used as the probe, the specific binding of the putative nuclear factor with the core sequence would fail to appear as the shifted band. Therefore, we used an oligonucleotide having the sequence of LS1/4 as the probe for the analyses. Nuclear extracts prepared from H295R cells or Hela cells were mixed with the radiolabeled LS1/4 oligonucleotide (Fig. 8A) or the radiolabeled Egr/GC-box oligonucleotide as the control (Fig. 8B). Three specific complexes, A, B and C, and two nonspecific complexes (NS) were formed by the H295R cell nuclear extracts (Fig. 8A). In contrast, the HeLa cell nuclear extracts produced very low level of complexes A and C but the level of complex B was almost the same as that in H295R cell nuclear extracts. The complex A formation by the H295R cell nuclear extracts was competed by adding the self-oligonucleotide (LS1/4) or the wild type oligonucleotide, but not by LS3 oligonucleotide, the
Fig. 9. Quantitative competitive gel mobility shift assay demon- strated that the complex A is a specific protein-DNA complex formed on the core element of the Egr/GC-box. (A) The 32P-labeled probe (-76/-47: LS1/4) were incubated with H295R nuclear extract in the presence of various concentration (0-100 fold) of unlabeled oligonucleotides, as described in Fig. 8. (B) Signals of protein-DNA complexes A, B, and C from three different experiments were quantitated using a phosphor imager, and data were given as percentages of signals obtained without competitor oligonucleotide. Complex A (upper panel), complex B (middle panel) and complex C (lower panel). Standard deviations are indicated.
GC-box or the Egr-element. The complex B- and C-formations were inhibited only by the self-oligonucleo- tide (LS1/4). In the control experiment (Fig. 8B), signals for Sp1 and Sp3 were competitively weakened by the wild type oligonucleotide, the GC-box and the Egr-element, but not by any of mutant oligonucleotides. A faint band was appeared between the signals for Sp1 and Sp3. This band was competed by only the wild type Egr/GC-box oligo- nucleotide, but not by any mutant oligonucleotides includ- ing LS1/4, LS3 (Fig. 8B) or LS2 (data not shown). These suggested that the faint band between Sp1 and Sp3 signals might not identical with the complex A, and under our experimental condition, only the complex A could represent a correlation with the results of mutation analyses of the Pref-1 promoter constructs (Figs 2 and 7).
Next, the gel shift analyses of the H295R nuclear extracts were performed in the presence of graded levels of nonradiolabeled competitors. Figure 9A shows representa- tive images of gel-shifts, and the levels of complexes are shown quantitatively in Fig. 9B. The complex A formation was inhibited by adding nonradiolabeled wild-type oligo- nucleotide and LS1/4 oligonucleotide, with the wild-type being a more efficient competitor than LS1/4. In contrast, the complex B- and C-formations were efficiently com- peted only by the LS1/4 oligonucleotide. Taken together, these results suggest that only the complex A formation may result from the binding of a nuclear factor specifically present in H295R nuclei with the short core sequence.
DISCUSSION
Pref-1 gene is expressed at a high level in 3T3-L1 preadipocytes, and its down-regulation occurs at the onset of adipocyte differentiation [8]. We found negative correlation between the level of Pref-1 gene expression and the progress of zonal differentiation in the fetal and regenerating rat adrenal cortex [7]. Recently, Smas et al. examined the mouse Pref-1 promoter and identified SAD, a cis-element AAAGA (-181 to -177), that was responsible for the down-regulation of Pref-1 gene expression [30]. When reporter constructs containing various sizes of the mouse Pref-1 promoter were introduced into 3T3-L1 cells and the stable transformants were isolated, suppression of the reporter activity during adipogenesis was observed in a SAD-dependent manner [30]. The SAD element was found in the rat Pref-1 promoter as well, from -182 to -178 (Fig. 4B,C). When reporter constructs containing the rat SAD element were tested in 3T3-L1 cells under the transient- or stable-transformation systems, the reporter activity was not suppressed during the adipocyte differ- entiation (data not shown). It is conceivable that the element -182/-178 in the rat promoter may not function as the suppressor element during adipogenesis. Recently, we (Y. Katoh, unpublished data: Gene Bank accession No: AB046762) and Fahrenkrug et al. [31] isolated and sequenced the promoter regions of human and bovine Pref-1 genes, respectively (Fig. 4C). They had elements of similar, but not identical, sequence to the mouse or rat SAD; AAGAA, from -188 to -184, in the human promoter, and AAGA, from -183 to -180, in the bovine.
Although we tried to identify cell type specific enhancer elements in the rat Pref-1 promoter, no enhancer activity
was found even in the DNA fragment containing up to 10 kb upstream of the Pref-1 promoter in either Pref-1 expressing or nonexpressing cells (data not shown). However, a domain that mediates the reporter activation both in Pref-1-expressing H295R cells and nonexpressing Hela cells was found in a GC-rich region of the rat Pref-1 gene. As for the other GC-rich promoters [32-34], the Pref- 1 promoter seemed to be activated by ubiquitous, noncell- type-specific nuclear factors such as Sp family factors. Our detailed analyses using deleted, mutated and chimeric promoters with SV40 enhancer identified a region essential for the H295R cell-specific promoter activity, the Egr/GC- box. Similar Egr/GC-boxes are present in the human and bovine promoters (Fig. 4). Sequences of two Egr-elements of the human promoter are GCGGGGCG, which could be recognized by Egr-factors with an affinity of almost the same magnitude as the case for the rat sequence, GCGTGGGCG [35-37]. The sequence of the first Egr- element of the bovine promoter is identical to that of the human, but the second element GCGAGGGCG, having a T to A substitution, would abolish the binding of Egr-factor [35]. Therefore, in the case of bovine promoter, the region of Egr/GC-box would contain only two elements, the first Egr- element and the GC-box.
An overlapping sequence having one Egr-element and one GC-box, GCGTGGGCGGGGCG, was found in mouse adenosine deaminase (ADA) promoters [38]. Disruption of the Egr-element without disturbing the GC-box abolished Zif268/Egr-1 binding and resulted in significant elevation of promoter activity in C1-1D cells. Conversely, disruption of the GC-box resulted in loss of Spl binding and great reduction of promoter activity. These results suggested that Zif268/Egr-1 seemed to down-regulate the ADA promoter by competing with Sp1 for the overlapping binding motif. A human vascular endothelial growth factor (VEGF) promoter has two sets of overlapping sequences of the Egr-element and GC-box [39]. Mutations introduced into the Egr-elements of both overlapping sequences did not affect platelet-derived growth factor (PDGF)-induced activation of the VEGF promoter in NIH3T3 cells. However, mutations introduced into all GC-boxes that abolished the binding of Spl to the promoter abrogated expression induced by PDGF. These results suggested that Sp-family factors might be major transcription factors in the VEGF promoter in PDGF-treated cells. In contrast to the cases of ADA and VEGF promoters, those of the human superoxide dismutase (SOD1) [40], 5-lipoxygenase (5-LO) [41] and the rat phenylethanolamine N-methyltransferase (PNMT) [42] were co-operatively regulated by Egr- and Sp-factors that seemed to act on the overlapping sequences.
Nuclear factors Sp1/3 and Egr(s) could bind with the Egr/GC-box in the rat Pref-1 promoter and activate the reporter gene in H295R cells. Mutations in the center of the Egr/GC-box diminished the binding of Sp1/3 (Fig. 8), abolished the activation by Egr(s) (Fig. 3) and decreased the Pref-1 promoter activity (Fig. 2). Mutations at both the 5’ and 3’ ends of the box also prevented the binding of Sp1/ 3 or the activation by Egr(s). However, this mutated promoter had almost the normal level of promoter activity (Fig. 7). Moreover, the gel-shift analysis using a mutated Egr/GC-box indicated the presence of a factor other than Sp and Egr(s) in H295R cells, that bound with the core sequence, GGCGTGG, of the Egr/GC-box (Figs 8,9).
These results suggested that the regulation of the Egr/GC-box in the case of Pref-1 promoter in H295R cells might be different from that in the cases of the ADA-, VEGF-, SOD1-, 5-LO- or PNMT-promoters.
The rat luteinizing hormone receptor (LHR) promoter has a GC-rich sequence including two canonical Sp1 binding sites, Sp12 (GGGGCGGGGCAGA) and Sp14 (GGGGTGGGGGCGG) [43]. Both Sp12 and Sp14 are the target of Spl protein and important for basal promoter activity. Interestingly, the Sp14 region is composed of noncanonical (GGGGTGGGG) and canonical (GGGCGG) Sp1 elements with a two-nucleotide overlap. Sp1 protein exclusively binds with the noncanonical 5’ element, and non-Sp1 trans-activators, with the canonical 3’ element [43]. Reporter constructs of the rat LHR promoter including both Sp12 and Sp14 regions showed no cell-type-specific activity. However, double mutations in the Sp12 and the 5’ half-site (noncanonical Spl binding site) of Spl4 regions revealed that the 3’-canonical Sp1 site of Spl4 was essential for full promoter activity in LHR- expressing MLTC-1 cells and this site served as the binding site for MLTC-1-specific nuclear factors other than Sp1 [43].
Taken together, we propose that the Pref-1 gene expression in H295R cells may be regulated by a specific factor(s) other than Egr-/Sp-family factors that recognizes the core sequence, GGCGTGG, in the Egr/GC-box. Comparing the rat core sequence with those of human and bovine (Fig. 4C), we noticed that the thymidylate residue in the core sequence could be replaced with guanylate or adenylate residue. Moreover, the fact that the LS4 and LS1/4 mutant constructs produced almost normal levels of reporter activity (Fig. 7) suggested that the last guanylate residue in the core sequence would not be essential for the Pref-1 gene promoter activity. These considerations enable us to suggest that the minimally essential core element is GGCGNG. This sequence well resembles those of various GC-rich elements including Egr- element and GC-box.
A possibility should be noted here that the Pref-1 gene promoter could be fully activated by cooperative interaction between the specific factor bound with the core element and a factor bound with the other element present outside the Egr/GC-box. It should also be added that neither 3T3-L1 cells nor Pref-1-negative adrenocortical Y-1 cells seemed to contain the specific factor found in H295R cells. The purification of the factor is now in progress in our laboratory to understand the precise molecular mechanisms underlying the cell-type-specific expression of Pref-1 gene in ZG cells.
ACKNOWLEDGEMENTS
We thank Drs J. Ian. Mason (University of Edinburgh), H. Fukui (Tokushima University), G. Suske (Philipps-University), C. Skerka (Bernhard-Nocht-Institute) and J. Svaren (Washington University) for providing us with H295R cells, Hela cells, antisera to Sp1/3, Egr1/4 expression vectors and Egr2/3 expression vectors, respectively. The authors are also grateful to Dr H. Tojo (Osaka University Medical School, Osaka, Japan) for his technical advice. A part of this work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.
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