Structural and Functional Analysis the Insulin-Like Growth Factor I Receptor Gene Promoter

Haim Werner, Mark A. Bach, Jr., and Derek LeRoith

Bethel

Stannard,

Charles

of

T. Roberts,

Section on Molecular and Cellular Physiology Diabetes Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892

INTRODUCTION

The insulin-like growth factor I receptor (IGF-I-R) gene is expressed in most body tissues. The levels of IGF-I-R mRNA, however, are regulated by a number of physiological conditions (development, differentiation, and hormonal milieu) as well as in certain pathological states (diabetes and tumors). To understand the molecular mechanisms which control the transcription of the IGF-I-R gene, we have cloned the promoter of the rat receptor gene and have characterized its activity by transient expression assays. Different fragments of the 5’-flanking region (subcloned upstream of a luciferase reporter gene) were transfected into buffalo rat liver 3A cells (a cell line with a low number of IGF-I binding sites) and Chinese hamster ovary cells (a cell line with a higher number of cell-surface receptors). In both cell lines, most of the promoter activity was located in the proximal 416 base pairs of 5’-flanking region. However, further dissection of this proximal fragment revealed a cell type-specific pattern of promoter activity. Thus, in buffalo rat liver 3A cells, subfragments of this region each contributed to total activity, suggesting that contiguous c&-elements can act together to activate transcription. In Chinese hamster ovary cells, on the other hand, subfragments of the proximal promoter region partially substituted for the proximal 416 base pairs of 5’-flanking region. Coexpression studies using an IGF-I-R promoter reporter construct together with an Spl expression vector (under the control of an ADH promoter) were performed in SL2 cells, a Drosophila cell line which lacks endogenous Spl. The results obtained showed that Spl can frans-activate the IGF-I-R promoter in viva. Transient transfection assays were complemented with gel-retardation assays and DNase I footprinting experiments, which showed that transcription factor Spl is potentially an important regulator of IGF-I-R gene expression. (Molecular Endocrinology 6: 1545-1558, 1992) 088&8809/92/l 545-l 558$03.00/O Molecular Endocmology Copynght 0 1992 by The Endocme

The insulin-like growth factor I receptor (IGF-I-R) is a membrane glycoprotein which mediates most of the biological actions of IGF-I and IGF-II (1, 2). Molecular cloning and analysis of a human IGF-I-R cDNA revealed that the receptor mRNA encodes a precursor polypeptide which is proteolytically cleaved to give an extracellular a-subunit, involved in ligand binding, and a tfans-

membraneP-subunit containing a tyrosine kinase domain (3). The mature receptor is a heterotetramer composed of two (Y- and two P-subunits linked by disulfide

bridges (3). We have previously shown that, in rat tissues, high levels of IGF-I-R mRNA are present at fetal and early postnatal stages and decrease to low levels at adulthood (4). The expression of the IGF-I-R gene is also regulated by nutritional status (5) as well as in certain disease states such as diabetes (6). Thus, fasting in rats was associated with an increase in IGF-I-R mRNA and binding in lung, stomach, kidney, and heart (5). Likewise, the levels of IGF-I-R mRNA in kidneys of streptozocin-diabetic rats were significantly increased over controls (6). High levels of IGF-I-R mRNA have also been detected in many tumors-among others, breast and brain tumors-and in several tumor-derived ceil lines (7-9). In a human melanoma cell line, the IGFI-R mediates a strong motility response to IGF-I, IGF-II, and insulin, and therefore may be one of the determinants initiating the metastatic process (10). Moreover, overexpression of the human IGF-I-R cDNA in NIH-3T3 fibroblasts resulted in a ligand-dependent neoplastic transformation which included the formation of tumors in nude mice (11). The expression of the IGF-I-R gene is hormonally regulated. For example, physiological concentrations of estradiol were shown to sensitize MCF-7 cells to the mitogenic effects of IGF-I by increasing IGF-I-R mRNA levels and IGF-I binding (12). Progestins, on the other hand, decreased the levels of IGF-I-R mRNA in T47D human breast cancer cells, potentially through an autocrine pathway involving enhanced IGF-II secretion

Society

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MOL 1546

END0.1992

Vo16No.10

of regulatory gel-retardation

(13). Furthermore, TPA-induced neurite outgrowth in a human neuroblastoma cell line (SH-SY5Y) was associated with increased IGF-I binding and IGF-I-R mRNA levels (14). FSH and GH have also been shown to regulate the levels of IGF-I-R mRNA (15, 16). To investigate the molecular mechanisms which control the transcription of the IGF-I-R gene in both physiological and pathological states, we have previously cloned and characterized the proximal 5’-flanking region of the rat IGF-I-R gene (17). Sequence analyses revealed that this region lacks TATA and CCAAT boxes and is very GC-rich; in this regard it is similar to the promoter of the insulin receptor gene (18-21). Primer extension and nuclease protection assays, however, showed that, unlike the insulin receptor gene, transcription of the IGF-I-R gene is initiated at a single site contained within an initiator element. This start site defines a very long 5’-untranslated region of approximately 940 base pairs (bp). The initiator motif has been previously shown to be present in TATA-less, CCAATless, non-GC-rich, developmentally regulated promoters, including those for various Drosophila homeotic genes such as Ultrabithorax and Antennapedia (22, 23) and in genes that are regulated during mammalian immunodifferentiation, such as the terminal deoxynucleotidyl transferase gene (24). In addition to the initiator element, potential consensus binding sites for the transcription factors Spl , ETF, GCF, and AP-2 are present in the proximal 5’-flanking region (25-28). To better understand the structure-function relationships in the IGF-I-R gene promoter, we have now determined the promoter activity of different 5’-flanking fragments by means of transient transfection assays. In addition, we have analyzed the potential interactions

I

I

-24

-20

I

I

-16

B 1

28 R\.l1 I

Localization of Promoter Activity within Flanking Region of the IGF-I-R Gene

P I

the 5’-

To map the promoter activity of the IGF-I-R 5’-flanking region, Rsal (nucleotides -2350 to +640) and Alul (-416 to +232) genomic fragments were fused to the luciferase reporter gene in the promoterless expression vector pOLUC and used to transfect buffalo rat liver 3A (BRLSA) and Chinese hamster ovary (CHO) cells (Fig. 1). The rationale for using these cell lines was that BRL3A expresses a low number of receptors (-760 binding sites per cell), whereas CHO cells have approximately 10 times more IGF-I receptors (-7800 sites per cell). The results obtained, expressed as a percentage of the maximal activity resulting from transfecting the same cells with pSV2LUC, indicated that, in both BRLBA and CHO cells, there were no significant differences in promoter activity between the -2350/+640 fragment and the -416/+232 fragment (21.8 + 1.7% vs. 25.4 + 2.2% in BRLSA; 12.4 + 1.9% vs. 10.8 f 1 .O% in CHO), suggesting that essentially all of the promoter activity is contained in the proximal 5’-flanking region (Fig. 2). As a control for assay background, we measured the activity of the antisense fragments, which was only approximately 0.02-2.8% of the activity exhibited by pSV2LUC (Fig. 2). To determine the contribution of the 5’-untranslated regions contained in the -2350/+640 and -416/+232 fragments to the overall promoter activity, we sub-

I

- 0.8

using

RESULTS

I

- 1.2

proteins with the IGF-I-R promoter assays and DNase I footprinting.

I

I

I

I

-04

0

0.4

0.8

1.2

HDhl r----s Al”1 I

Hphl

Kb

/

zN + Al”, I

Fig. 1. Schematic Representation of the Rat IGF-I-f? 5’-Flanking and 5’-Untranslated

2 HWl I Regions

This diagram is based upon the sequences of overlapping genomic and cDNA clones isolated from rat genomic (17) and SV40transformed rat granulosa cell cDNA libraries (4) respectively. The coding region is shown in black, the 5’untranslated region is dotted, and the 5’-flanking region is open. The transcription initiation site is denoted by an arrow at the initiator (INIT) element. R, Rsal; B, BarnHI; P, WI; H, HindIll; Hp, Hphl; A, Alul; S, Smal. The genomic fragments shown at the bottom of the figure were subcloned upstream of a firefly luciferase reporter gene and their promoter activity measured in transient transfection assays.

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IGF-I-R

Gene

Promoter

1547

CONSTRUCT

LUCIFERASE

ACTIVITY

CHO 0 36 2 0 1%

21 84

I>,

455

pi

pi+21

27

+ 1 71 o/u

0 08

2 0 02%

25 41

+ 2 23B

281

? 026%

21 63 t

2 66”Y”

899

e 087%

f 301

LUC

+211

LUC

0 64 + 0 13%

0 48

-271

LUC

0 45

055*017%

+ :I 09%

t 0 05%

Fig. 2. Promoter Activity of 5’-Flanking and 5’-Untranslated Fragments of the IGF-I-R Gene AM (-416 to +232), Rsal (-2350 to +640), and Hphl (-455 to +30) genomic fragments, and a 52-bp double stranded oligomer (-27 to +21) were subcloned in the promoterless pOLUC vector upstream of a firefly luciferase cDNA. All of the above fragments were subcloned in both sense and antisense orientations, with the exception of the Hphl fragment, which was subcloned only in the sense orientation. The constructs obtained were employed to transiently transfect BRL3A and CHO cells, and the luciferase activity generated was measured with a luminometer. The values obtained were normalized by dividing them by the P-galactosidase activity generated by cotransfecting with a fi-galactosidase expression vector. In each experiment, a luciferase vector containing an SV-40 enhancer/promoter (pSV2LUC) was included, and the activity generated by this construct was given a value of 100%. Similar results were obtained when the light values obtained were expressed as fold induction over pOLUC. Results are means + SEM of three to five independent experiments. In each exoeriment, each plasmid was assayed in duplicate or triplicate. The arrow in each construct denotes the site of transcription initiation.

cloned an Hphl fragment (-455 to +30) containing a minimal portion of 5’-untranslated sequence into pOLUC and transfected it into both BRLBA and CHO cells. The promoter activity of this fragment was not significantly different from that of the -416/+232 fragment (21.6 + 2.6% vs. 25.4 + 2.2% in BRL3A; 8.9 f 0.8% vs. 10.8 of: 1.0% in CHO cells) (Fig. 2). Thus, these results suggest that essentially all of the promoter activity is contained in the 5’-flanking region, and there is no significant contribution of these particular 5’untranslated sequences to transcriptional activity. To establish whether the initiator element itself exhibited any promoter activity, we inserted a 52-bp doublestranded synthetic oligonucleotide corresponding to bp -27 to +21 into the BamHl site of pOLUC. The luciferase activity exhibited by BRL3A and CHO cells transfected with the initiator construct (both sense and antisense orientations) was not significantly different from the basal luciferase activity observed when these cells were transfected with pOLUC (0.36 f 0.1-0.83 f 0.18%) (Fig. 2). To more precisely map the location of promoter elements in the proximal 5’-flanking region, approxi-

mately lOO- to 200-bp restriction fragments were inserted immediately upstream of the initiator element (Figs. 3 and 4). When transfected into BRL3A and CHO cells, all of the fragments analyzed exhibited some promoter activity, although important differences were seen between the two cell types. Thus, in BRLBA cells, the combined luciferase activity of all of the proximal subfragments (-494/-331, 6.5%; -331 I-1 35, 9.01%; and -1351-26, 2.36%; 6.5 + 9.01 + 2.36 = 17.87%) was roughly equivalent to that of the -455/+30 fragment (21.63%). On the other hand, in CHO ceils, each subfragment had between 70-91% of the luciferase activity of the -455/+30 fragment (Fig. 4). When the results of three independent experiments in CHO cells were combined, no statistically significant differences were observed between the activities of the different fragments. To establish whether promoter activity was influenced by the number of putative binding sites for various transcription factors, we made a construct containing two copies of the -4941-331 fragment in tandem array. Each fragment contains four potential Spl sites, one consensus binding site for the transcription

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MOL 1548

ENDO.

1992

Vo16No.10

-fjoo I -5’

-400 I

-200 I

1 I

200 I

Flanking-5’

400 I

600

800

I

I

loco I

Untranslated+

-

Ava I

Pvu II I Ah I I

I I

SPl

1

4

F?lE

7 Sau3A

Hae’lll Ava I

c

z 1 Hae Ill

I t ‘Barn

HI“

H k

I “Barn

Hi”

i; +

Fig. 3. Schematic Representation of the Proximal Promoter Region of the Rat IGF-I-R Gene This figure shows the various restriction fragments which were derived from the region extending from -494 to +185 with respect to the transcription start site. The promoter activity of the following 5’-flanking fragments, -494/-331, -331/-135, and -135/-26, was analyzed by subcloning them immediately upstream of an initiator element in a luciferase vector. The following fragments, -416/-331, -331/-135, -135/-26, and -29/+185, were end-labeled and their protein binding activity studied by means of gel-retardation assays. In addition, the promoter and protein-binding activities of the initiator element itself were studied using a double-stranded synthetic oligonucleotide (-27 to +21) flanked by artificial BarnHI sites, which were employed for subcloning into the BamHl site of pOLUC. Also shown in the figure are potential binding sites for the transcription factors Spl , AP2, ETF, and GCF.

factor ETF, and a putative site for the inhibitory factor GCF (Fig. 3). The luciferase activity of p(-494/-331, INR)LUC in both BRLBA and CHO cells was, however, not significantly different from that of p(-494/ -331,-494/-331 ,INR)LUC. Similarly, no statistically significant differences were observed between constructs containing either one or two copies of the above fragment in the antisense orientation: p(-331/ -494,INR)LUC and p(-331/-494,-331/-494,1NR) LUC (Fig. 4). The promoter activities of all the constructs employed in these experiments were, in general, not dependent upon the orientation of the 5’-flanking fragment, suggesting that these regions represent enhancer-like domains. The only exception was the -331/-135 fragment, whose activity in BRLBA was orientation-dependent (9.0 + 1.3% in the sense orientation vs. 3.6 f 0.8% in the antisense orientation, P < 0.05) (Fig. 4). Determination of Transcription IGF-I-R Promoter-Luciferase

Initiation Sites of Fusion mRNAs

The results of primer extension assays using mRNAs prepared from CHO cells which were transfected with p(-2350/+64O)LUC or with p(-416/+232)LUC constructs are shown in Fig. 5. As in vivo (17) transcription of the IGF-I-R promoter-luciferase fusion mRNA starts at a single major site within the initiator element. A

minor extended band which was approximately 2-3 bases shorter than the major band was also seen. Trans-Activation

of IGF-I-R Promoter

by Spl

The trans-activation of the IGF-I-R promoter by transcription factor Spl was assessed by coexpression studies using the reporter plasmid p(-416/+232)LUC and an Spl expression vector driven by an alcohol dehydrogenase promoter (pPadh.Spl-in) (29). For this purpose we employed SL2 cells (Schneider line 2), a Drosophila cell line which lacks endogenous Spl (29). The results obtained showed that Spl Vans-activated IGF-I-R promoter activity by approximately 7- to 8-fold, whereas cotransfection of an Spl expression vector in which Spl cDNA translation is out of frame (pPadh.Splout) had no effect (Table 1). Gel-Retardation

Assays

To study the protein-binding activity of the IGF-I-R promoter, gel-retardation assays were performed using crude nuclear extracts from CHO cells and 3zP-labeled restriction fragments. Incubation of increasing amounts of nuclear extract with 40 pg (0.7 fmol) -416/-331 fragment resulted in the appearance of three major retarded bands on a native polyacrylamide gel (Fig. 6). To more accurately locate the sequences involved in protein binding, double-stranded synthetic oligonucle-

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IGF-I-R

Gene

Promoter

1549

LUCIFERASE ACTIVITY I% 0‘ pS”2L”CI

89

72

t 09%

L 08"n

,100",~1

133%1

69210%

t32%1

30205%

114%1

142%)

64+05%

(72%;

36~08%

117%1

4 7 -r 1 7%

153°K

23iO5%

Ill%1

62

+ 06%

i70%1

119%1

83

+ 1 0%

193”/01

9 0 2

4 1 z

1 3%

1 8%

Fig. 4. Promoter Activity of Subfragments of the 5’-Flanking Region in Conjunction with the Initiator 5’-Flanking fragments (in both sense and antisense orientations) were subcloned just upstream of an oligomer encoding the initiator (black bar) in a luciferase vector and used to transfect BRL3A and CHO cells. The results are expressed as percentage of the maximal activity generated by pSV2LUC. The values shown in parenthesis are percentages from the luciferase activity generated by a construct containing the proximal 455 bp of 5’-flanking and 30 bp of 5’-untranslated region. For further details, see legend to Fig. 2.

otides encoding specific consensus regulatory regions were used as competitors in binding assays. A 13-bp oligomer encoding the consensus ETF sequence (-371 to -359) (26) was unable to abolish complex formation, even at a 1400-fold molar excess (Fig. 6). However, an extended 36-bp oligomer which included the ETF sequence and two overlapping SPl sites (-386 to -351) was able to almost totally inhibit complex formation when added at the same concentration. Similarly, formation of DNA-protein complexes was totally abolished when the binding reaction was performed in the presence of a 14-fold molar excess of an Spl oligonucleotide (Fig. 6). When crude nuclear extracts from CHO cells were incubated with 0.3 fmol of a 32P-labeled AvalSau3A restriction fragment extending from -331 to -135, a major and a minor retarded band was seen (Fig. 7). Competition with a specific 16-mer double-stranded oligonucleotide corresponding to the putative IGF-I-R AP-2 binding site (-172 to -157) did not prevent complex formation; however, total inhibition was seen with a 3000-fold molar excess of an oligomer corresponding to a consensus AP-2 site (30). Similarly, total inhibition

occurred with a 300-fold molar excess of Spl oligomer. A DNA fragment extending from -135 to -26 produced one shifted band, the formation of which was inhibited by excess unlabeled probe but not by excess Spl or AP-2 oligomers (data not shown). Gel-retardation assays were also performed using crude nuclear extracts from rat neonate brain and adult liver. The rationale for using these sources of nuclear proteins was that neonate brain expresses very high levels of IGF-I-R mRNA, whereas receptor transcripts in adult liver are almost undetectable (4). Incubation of 1 pg brain nuclear extract with the labeled -416/-331 fragment resulted in the formation of three retarded bands whose pattern of migration was similar to that seen with CHO extracts (Fig. 8). When the reaction was performed in the presence of a 25- to 250-fold molar excess of the unlabeled DNA fragment, the formation of these complexes was prevented. Unrelated DNA fragments of similar size were unable to inhibit these retarded bands (data not shown). On the other hand, incubation of the 3zP-labeled -416/-331 DNA fragment with 1 pg crude nuclear extract from adult liver did not generate any retarded bands (Fig. 8). To eliminate the

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MOL 1550

ENDO.

1992

Vo16No.10

fective, whereas an Spl oligomer was effective in inhibiting complex formation (Fig. 9). Consistent with previous reports (32), fragments containing the initiator region itself but devoid of 5’-flanking sequences (-29/+185 or -27/+21) did not exhibit protein binding activity as determined by gel-retardation assays (data not shown). DNase I Footprinting Fig. 5. Analysis of Transcription Initiation of IGF-I-R PromoterLuciferase Fusion mRNAs by Primer Extension CHO cells were transfected with p(-416/+232)LUC(l) or p(-2350/+64O)LUC (2) and poly(A)+ mRNA was prepared. A 22-base oligonucleotide complementary to the 5’-end of the rat IGF-I-R cDNA clone 1 (4) was employed as primer. Also shown is a primer extension using poly(A)+ mRNA from rat neonate brain. The arrow on the left indicates the major extended band corresponding to the transcription initiation site. A minor start site was seen in close proximity to the major one. The sequence reaction shown in the four right lanes was generated using the 22-base oligomer as primer and p(-416/ 232)LUC plasmid as DNA template.

Table

1. Trans-Activation Constructs

of the IGF-I-R

transfected

1. p(-416/+232)LUC (5 fig) 2. p(-416/+232)LUC (5 pg) + pPadh.Spl-in (2 Kg) 3. p(-416/+232)LUC (5 fig) + pPadhSp1 -in (15 pg) 4. p(-416/+232)LUC (5 llg) + pPadh.Spl-out (15 pg)

Promoter

by Spl

Luciferase activrty (arbrtraty units)

Fold increase over control

1.65 + 0.28 1.66 + 0.08

1 .oo 1.01

12.12

+ 1.27

7.34

2.32

+ 0.11

1.41

Five micrograms of the reporter plasmid p(-416/+232)LUC were cotransfected into Drosophila Schneider line 2 cells with 2 or 15 fig of the Spl expression vector pPadhSp1in or 15 fig of the out-of-frame Spl expression vector pPadh.Spl-out. The total amount of transfected DNA was kept constant by adding pG4Z DNA up to a total of 20 pg. Forty-eight hours after transfection, cells were harvested and lysed as indicated in Materials and Methods, and luciferase activity and protein levels were determined. No significant differences in protein levels were seen between the different groups. The values of luciferase activity shown (normalized per protein) are means + SEM (n = 3). In each experiment, each treatment was studied in duplicate or triplicate, and the experiment was repeated three times.

possibility that the lack of binding was due to inactive liver extract, we incubated the extract with a labeled 21 -bp repeat oligomer encoding the malic enzyme promoter element-l, which was previously shown to bind to liver extracts (31). Upon electrophoresis, a clear band shift was seen, indicating that the liver extract was active (data not shown). In addition, competition experiments with brain nuclear extracts and specific oligonucleotides showed that, similar to the results obtained with CHO nuclear extracts, an ETF oligomer was inef-

Analysis

To further localize potential nuclear protein-DNA interactions, DNase I footprinting was performed with three overlapping 32P-labeled fragments which encompassed the region from -416 to +I 15. Using purified Spl protein at concentrations ranging from 2-10 ng, footprints were seen in the areas corresponding to five out of six potential Spl sites in the proximal 5’-flanking region. Thus, the following consensus Spl sites were protected by Spl protein: -3991-392, -3781-372, -3741-366, -2591-251, and -1931-l 87. The putative Spl site at -341/-336 was not protected by Spl protein. Similar footprints were observed with crude nuclear extracts from neonate brain (5 pg) but not with adult liver, in at least two of the Spl sites: -399/-392 (Fig. 10) and -193/-l 87 (data not shown). The Spl/ ETF site at -378/-359 was only slightly footprinted with a brain extract. Incubation of recombinant AP-2 protein (Promega, Madison, WI) with 32P-labeled -4161 -135 probe, followed by DNase I digestion, did not generate any footprint (data not shown).

DISCUSSION Structural analysis of the 5’-flanking region of the IGFI-R gene (17) revealed that the IGF-I receptor promoter shares certain features with the promoters of the insulin receptor and other housekeeping genes (i.e. GC-rich 5’-flanking region, lack of TATA or CCAAT boxes), while sharing certain other characteristics with a family of genes that are highly regulated during development and differentiation (i.e. transcription initiation from a single site contained within an initiator element). Transient transfection assays using CHO and BRL3A cells, whose IGF binding levels differ by lo-fold, revealed no significant differences in promoter activity between the -2350/+640 and the -416/+232 regions, indicating that most of the promoter activity is located in the proximal 5’-flanking region. The orientation of the initiator element in the sense orientation was crucial in order to generate high levels of expression. However, the initiator itself was devoid of any promoter activity, either in the sense or in the antisense orientation. These results are consistent with those of Smale and Baltimore (24), which showed that the initiator, by itself, is able to direct only minimal levels of transcription initiation from a single internal nucleotide position. Since this element acts in vivo in concert with a TATA box or upstream elements to direct specific

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IGF-I-R

Gene

1551

Promoter

-331

-416 B. CHO

extract

-

0.3

1

3

7.2

7.2

Competitor

_

_

_

-

-

ETF

Molar

_

_

-

-

-

1400

Excess

(ug)

7.2

ETF/Spl

7.2

7.2

7.2

SPl

SPl

SP)

14

140

14al

1400

PROBES 5'-GCCCTGCCGCCGC-3'

ETF oligo ETF/SPl

SPl

3’-CGGGACGGCGGCG-5’

oligo

5'-GCCCGCAGCCCGCCCGCCCTGCCGCCGCCCCCTTGG-3' 3'-CGGGCGTCGGGCGGGCGGGACGGCGGCGGGGGAACC-5'

oligo

5'-GATCGATCGGGGCGGGGCGATC-3' 3’-CTAGCTAGCCCCGCCCCGCTAG-5’

Fig. 6. Mapping of Protein Binding Sites in the -416/-331 Region by Gel-Retardation Assays A, Schematic diagram of putative binding sites in the DNA fragment extending from -416 to -331. Four potential Spl sites are indicated by open boxes; also shown is a consensus ETF site (26; black bar) which overlaps with two Spl sites. The locations of these sites are indicated. B, Gel-retardation assays were performed by incubating increasing amounts of crude nuclear extract from CHO cells with 32P-labeled -416/-331 DNA, in the absence or presence of 14- to 1400-fold molar excess of an ETF oligomer, an ETF/Spl oligomer (directed against the two Spl sites which overlap with the ETF site), or an Spl oligomer.

transcription initiation, we subcloned short V-flanking fragments immediately upstream of the initiator in a luciferase vector. All of the cloned fragments (-494/-331, -331 /-I 35 and -135/-26) had, in conjunction with the initiator, promoter activity which was, in most of the cases, not dependent upon the orientation of the fragment. In BRL3A cells, each one of these three domains had a level of promoter activity which was between l l-42% of the promoter activity of the -455/+30 proximal 5’flanking region. Thus, the constitutive promoter activity of the IGF-I-R gene cannot be explained by a minimal promoter element model, but, rather, by a model in

which contiguous &-elements act together to activate transcription. A somewhat similar model was recently proposed for the POMC gene, a gene which is also highly regulated during development (33). In this case, three domains of the promoter were defined which have distinct and complementary activities. Within these domains, which require each other for full activity, at least nine regulatory elements were defined which bind different nuclear proteins and which act synergistically to control POMC gene expression. In CHO cells, on the other hand, the promoter activity of a construct containing the -494/-331 fragment upstream of the initiator was approximately 91% of the

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MOL 1552

ENDO.

1992

Vo16No.10

CHO

extract

_

Competitor Molar

0.3

(ug)

1

_

3

3

3

3

3

-

IGFR/AP2

AR2

Spl

SPl

SPl

3200

9

320

3200

3200

excess

3

PROBES 5'-GGCTCCCCACGCCCGC-3' 3'-CCGAGGGGTGCGGGCG-5'

IGFR AP2 oligo

AP2 oligo

5'-GATCGAACTGACCGCCCGCGGCCCGT-3' 3'-CTAGCTTGACTGGCGGGCGCCGGGCA-5'

SPl oligo

5'-GATCGATCGGGGCGGGGCGATC-3' 3'-CTAGCTAGCCCCGCCCCGCTAG-5'

Fig. 7. Mapping of Protein Binding Sites in the -331/-l 35 Region by Gel-Retardation Assays A, This schematic diagram shows the presence of two consensus Spl binding sites (open bars) and a putative binding site for transcription factor AP-2 (28; dotted bar). B, Gel-retardation assays were performed as indicated in Results and in the legend to Fig. 6. Two oligomers directed against the AP-2 site were employed: IGFR AP2 oligo, specifically designed against the AP-2 consensus sequence found in the IGF-I-R promoter, and AP2 oligo (Stratagene), which is directed against a different motif of the AP-2 site (30).

exhibited by the -455/+30 fragment, whereas the -331/-135 and -135/-26 domains exhibit approximately 70% of the activity of the -455/+30 fragment. Thus, each one of these fragments can partially substitute for the -455/+30 region. Therefore, promoter activity in this cell line should be considered on the basis of complex interactions between positive and negative regulatory factors. Alternatively, the similarity in the luciferase values observed with all of the subfragments in both cell lines (Fig. 4) can be interpreted to suggest that no major differences exist between the different domains, and activity

that the only difference lies in the higher level activity of the complete construct in BRL3A cells. This could reflect the presence of a rat-specific Vans-acting factor in BRL3A cells which preferentially activates transcription from the complete construct. Sequences in the proximal 5’-untranslated region are apparently devoid of any promoter activity, as suggested by the observation that a construct with a minimal 5’untranslated region [p(-455/+3O)LUC] had the same activity as p(-416/+232)LUC. It is possible, however, that the long 5’-untranslated region of the

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IGF-I-R

Nuclear

Gene

Promoter

1553

I P

Extract

2 t3 Neonate

Brain

2

Cold Competitor (fold molar excess)

Free

Probe

-w

Fig. 8. Gel-Retardation Analysis of the -416/-331 Fragment of the IGF-I-R Gene Promoter One microgram of crude nuclear extract from rat neonate brain or adult liver was incubated with 10,000 dpm 3*P-labeled -416/-331 fragment, in the absence or presence of 25 to 250-fold molar excess of the unlabeled DNA fragment, and the reaction products were resolved on a 4% polyacrylamide gel. The arrows at the left indicate the shifted bands corresponding to the DNA-nuclear protein complexes.

IGF-I-R mRNA is involved in translational regulation via mechanisms which remain to be elucidated. The models proposed above are consistent with the results of gel-retardation assays which showed that all of the fragments analyzed, with the exception of the initiator region, bind, to some extent, to proteins present in nuclear extracts from CHO cells and from neonate brain, an organ in which the levels of IGF-I receptor mRNA and protein are especially high. On the other hand, nuclear proteins from adult liver bind to the regulatory region of the IGF-I-R gene only at very high concentrations, consistent with low levels of IGF-I-R mRNA in this organ (4). Using Drosophila cells, which under normal conditions do not express Spl mRNA and protein (29), we have been able to show that this transcription factor activates transcription from the IGF-I-R promoter. Furthermore, the fact that an out-of-frame Spl expression vector was unable to stimulate promoter activity strongly suggests that this effect was due to the Spl protein. The results of gel-retardation assays with specific oligonucleotides corresponding to consensus Spl , AP-2, and ETF binding sites, in conjunction with DNase I footprinting assays in which purified Spl and AP-2 proteins were employed, suggest that, at least in brain,

Spl is probably one of the main regulators of transcription of the IGF-I-R gene. ETF and AP-2, on the other hand, are not obviously involved in DNA binding, although an oligomer corresponding to a consensus AP2 sequence was somewhat active as a competitor in gel-retardation assays. The importance of Spl in the regulation of IGF-I-R gene expression can be further inferred from comparison of the rat and human proximal 5’-flanking sequences (Fig. 11). Six Spl consensus binding sites were found in the human promoter, with three of them being conserved in the rat (34). Similarly, the initiator element was fully conserved in the human gene, in which species transcription initiation also starts at a single site. Interestingly, the AP-2 consensus site in the rat promoter, which was apparently not very active in binding assays, is conserved in the human receptor gene sequence (34). Spl activates a specific group of promoters transcribed by RNA polymerase II in vertebrates. It binds GC-rich sequences by means of three zinc fingers and activates transcription via glutamine-rich domains (25, 35). Although present in most tissues, a recent developmental study in mouse showed that certain tissues have especially high levels of Spl mRNA and protein at defined ontogenic stages (36). For instance, the levels of Spl transcripts in 64-day-old liver were approximately 11 times lower than those in 5day-old cerebellum. This finding may explain the fact that neonate brain extracts were able to interact with promoter fragments in gel-retardation assays and DNase I footprinting, whereas adult liver extracts were ineffective. Moreover, different Spl sites in the 5’-flanking region apparently differ in their binding affinity since brain extracts were able to generate footprints in some of them (such as the site at -399/-392) but not in others. It has been recently demonstrated that a cluster of four GC boxes located -593 to -618 bp upstream of the ATG translation initiation codon are required for efficient expression of the human insulin receptor gene (37). In the IGF-I-R gene promoter, on the other hand, a cluster of GC boxes in positions -378 to -355 upstream of the transcription initiation site (Fig. 6A) are not apparently crucial for efficient promoter activity (Fig. 4). Interestingly, transfection of BRLBA and CHO cells with constructs containing either one or two copies of the -494/-331 fragment upstream of the initiator resulted in similar levels of promoter activity. These results are consistent with an interplay of positive and negative regulatory elements in the control of IGF-I-R gene transcription, in that additional copies of this region would not necessarily result in an increased promoter activity, since stimulatory and inhibitory elements would neutralize each other. In summary, we have shown that the IGF-I-R promoter exhibits high basal activity but with specific features which are cell type-specific. These results may explain the widespread distribution of IGF-I-R mRNA and protein in most body tissues under basal conditions as well as its regulation in certain physiological (i.e. development and differentiation) (38, 39) and patholog-

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MOL 1554

ENDO.

1992

Vo16No.10

NEONATE - .

COMPETITOR MOLAR

EXCESS

-

-

ETF oliao x140

x1400

.

BRAIN *

ETF/SPl xl4

Fig. 9. Gel-Retardation Analysis of the -416/-331 Region One microgram of crude nuclear extract from neonate brain was incubated presence of 14- to 1400-fold molar excess of ETF oligomer, ETF/Spl oligonucleotides are shown in Fig. 6B.

ical (i.e. diabetes and tumors) states (5-7). It appears that transcription factor Spl is probably the main regulator of gene expression, and that cis-elements spread over several hundred bases upstream of the transcription start site contribute either additively (as in BRLBA cells) or through more complex interaction mechanisms to the promoter activity of this gene. The mechanisms of this activation, as well as the possible contribution of additional regulatory factors, are currently under investigation.

MATERIALS AND METHODS Isolation

and Characterization

of IGF-I-R

Genomic

Clones

An approximately 3-kilobase Rsal genomic fragment containing 2.35 kilobases of 5’-flanking sequences and 640 bp 5’untranslated region was isolated from a previously described IGF-I-R rat aenomic clone (XIGF-I-R-3) bv hvbridization with a ‘*P-labeled synthetic oligonucleotide ‘probe directed against sequences -323 to -295 with respect to the transcription start site (17). The purified Rsal fragment was subcloned into a pGEM-4Z vector (Promega) and the double-stranded plasmid DNA sequenced as described (40). The previously described Alul (-416 to +232) and Smal fragments (-331 to +520), encoding the proximal promoter region, are contained within

NUCLEAR

x140

oliqo x1400

EXTRACT +

.SPl xl4

ok+ x140

with 32P-labeled -416/-331 oligomer, or Spl oligomer.

DNA in the absence or The sequences of the

this Rsal genomic clone (17) (Fig. 1). Sequencing of this fragment showed total identity in the region overlapping the previously published cDNA and genomic sequences (Fig 1) (4, 17) with the exception of two additional G residues located at positions -72 and -99, which were omitted from the previously published sequence (17). Plasmid

Constructions

for Luciferase

Assays

The promoter activity of the 5’-flanking and 5’-untranslated regions was studied by transient transfection assays in which putative promoter fragments were fused to a promoterless firefly luciferase reporter gene (pOLUC) (41). Rsal and Alul fragments were isolated from vector DNA by agarose gel electrophoresis and inserted into the HindIll site of pOLUC, which had been converted to a blunt end by filling in with Klenow fragment. The orientation of the inserts within the luciferase vector was determined by sequencing with an SP6 promoter primer. A 485-bp Hphl genomic fragment consisting mostly of 5’flanking sequences (-455 to +30) was obtained by digestion of the Rsal plasmid with Hphl, followed by treatment with Klenow and ligation into the blunt-ended HindIll site of pOLUC. A 52-bp double-stranded synthetic oligonucleotide encoding the transcription start region (-27 to +21; 5’-CCCCGAGAGCGCGCGCGTAGAGCCCCCAGTGTGTGGCGGCGGCGGCGC-3’) flanked by BarnHI sites was inserted into the BarnHI site of pOLUC (Fig. 3). Clones were generated which contained the initiator in both sense and antisense orientations [p(-27/+21)LUC and p(+21/ -27)LUC, respectively]. In addition, three different fragments

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IGF-I-R

Gene

1555

Promoter

analysis of IGF-I binding sites in these cell lines was performed using competition-inhibition curves generated with [“51]IGF-I and increasing amounts of cold IGF-I. The number of IGF-I binding sites in this clone of CHO cells under basal conditions was approximately 7800 binding sites per cell, whereas in BRLBA it was approximately 760 sites per cell. Supercoiled plasmid DNAs were transfected by electroporation as described (42). Briefly, confluent cells (30-60 x 1 O6 cells/l50-mm dishes) were trypsinized, washed once with serum-containing media and once with ice-cold electroporation buffer (20 mM HEPES, pH 7.05, 137 mM NaCI, 5 mM KCI, 0.7 mM Na2HP04, and 6 mM dextrose) and finally resuspended in 1 ml electroporation buffer. Upon addition of 20 pg luciferase plasmid and 20 pg P-galactosidase plasmid, cells were transferred to disposable electroporation chambers and electroporated at 300 V and 800 microFaradays at 0 C using a BRL Cell-Porator electroporation apparatus (BRL, Gaithersburg, MD). Transformed cells were seeded in loo-mm Petri dishes using serum-containing media, and the percentage of viable cells was determined by trypan blue exclusion. Media was replaced 24 h after transfection and, after an additional 24 h, the cells were washed three times in ice-cold PBS and lysed in 1 ml 1% Triton X-100, 25 mM glycylglycine, pH 7.8, 15 mM MaSOd. 4 mM EGTA. and 1 mM dithiothreitol (DTT). To measure luciferase activity, 0.2 ml of a 0.2 mM o-luciferin solution was added to the extract in the presence of 2 mM ATP and 2 mrv DTT, and the emission of light was monitored using an automated luminometer (Berthold Clini-Lumat, London Diagnostics, Eden Prairie, MN). The amount of light emitted (at 390-620 nm) was proportional to the amount of cell extract added (data not shown).

-359

-392

-399

Identification

Fig. 10. DNase I Footprinting Analysis of the -416/-359 Region of the IGF-I-R 5’-Flanking Region A 32P-labeled probe extending from -416 to -135 was incubated with increasing amounts of purified Spl protein, or with crude nuclear extracts from adult liver or neonate brain, and subsequently digested with DNase I as indicated in Materials and Methods. Protected sequences are indicated by a bar. The open box extending from -399 to -392 corresponds to the Spl site shown at the 5’-end of the schematic in Fig. 6A. The footprint obtained in this region with 10 ng Spl was 10-l 2 bp larger than the consensus Spl binding site. The open box extending from -378 to -359 corresponds to the two Spl sites which overlap with the ETF site (Fig. 6A). The arrow at the bottom of the gel denotes a hypersensitive site at -411, which was seen upon incubation with high concentrations of liver nuclear extract.

of the proximal 5’-flanking region (-494 to -331, Pvull/Aval; -331 to -135, AvallSau3A; and -135 to -26, Sau3AjAval) were subcloned in both orientations immediately upstream of the initiator element in p(-27/+21)LUC. In one case, (-494 to -331) clones were generated which contained two copies of the fragment in a tandem array in both orientations. As a positive control, a high-expression plasmid (pSV2LUC) containing the SV40 enhancer/promoter was used (41). As a control for transfection efficiency, cells were cotransfected with a p-galactosidase expression vector (pCMV@; Clontech, Palo Alto, CA) in which the @-galactosidase gene is under the control of a cytomegalovirus enhancer/promoter. Transient

Transfection

CHO and BRL3A mixture containing

Assays

and Luciferase

Assays

cells were grown in Ham’s F-12 nutrient 10% or 5% fetal bovine serum. Scatchard

of Transcription

Initiation

Sites

The transcription initiation site of IGF-I-R promoter-luciferase fusion mRNAs was determined by primer extension as described (17). Briefly, 12 pg poly(A)+ mRNA prepared from CHO cells which were transiently transfected with the p(-416/ +232)LUC or p(-2350/+64O)LUC constructs (Fig. 3) were annealed to a “P-labeled 22-base oligonucleotide complementary to bases +28 to +49 of a previously described IGF-I-R cDNA (clone 1; Ref. 4). After incubation at 42 C for 18 h, cold nucleotides and avian myeloblastosis virus reverse transcriptase were added and the reaction continued for 1 h at 42 C. The extended product was extracted with phenol-chloroform, precipitated, and analyzed on an 8% polyacrylamide/8 M urea denaturing gel. A DNA ladder was generated using the same DNA template and oligonucleotide primer and run in adjacent lanes. Coexpression

Studies

SL2 cells were grown in M3 media (Quality Biological, Inc., Gaithersbura. MD) containina 10% heat-inactivated fetal bovine serum,> mM glutamine,&d 20 Kg/ml gentamicin sulfate. Cells were kept at 23 C in tightly closed flasks and split every 3-4 days using approximately one-third of the conditioned media. Twenty-four hours before transfection, cells were replated onto loo-mm petri dishes (15 x 1 O6 cells/l 0 ml media). Cells were transfected using a calcium phosphate transfection kit (Specialty Media, Inc., Lavallette, NJ), as previously described (29). Each plate received 5 pg reporter plasmid p(-416/+232)LUC and variable amounts of Spl expression plasmid pPadh.Spl -in (0, 2, and 15 pg), as well as pG4Z DNA to bring the total amount of transfected DNA to 20 rg per plate. A group of plates were also cotransfected with the expression plasmid pPadh.Spl-out, a construct in which the presence of an extra nucleotide shifts the Spl coding sequence out of frame (29). After transfection, cells were left undisturbed for 48 h at which time cells were collected by pipetting, washed once with ice-cold PBS, and the cell pellet resuspended in 0.5 ml 0.25 M Tris/HCI, pH 8.0, containing 1 mM DTT. Cells were lysed by freezing on dry ice and thawing,

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MOL 1556

ENDO.

Vo16No.10

1992

Fig. 11. Potentially Functional Conserved Sites in the Rat IGF-I-R Gene Promoter Arrows denote potentially active Spl sites as determined by gel-retardation assays or DNase I footprinting. Spl sites conserved in the human IGF-I-R promoter are indicated by an asterisk. Spl site at -399/-392 was also footprinted with neonate brain nuclear extracts. Gel-retardation and DNase I footprinting assays provided only weak evidence for an active AP-2 site, although the fact that it is perfectly conserved in the human gene sequence, together with the observation that IGF-I-R mRNA levels are modulated by CAMP-mediated mechanisms (14) suggest that AP-2 may be functional.

followed by sonication on an Ultrasonic Processor (Heat Systems-Ultrasonics, Inc., Farmingdale, NY) set at the microtip limit, for 1 min. The lysate was clarified by centrifugation (10 min at 14,000 rpm), after which aliquots were obtained for luciferase activity assays and protein determination (BioRad, Richmond, CA). Gel-Retardation

Assays

Nuclear extracts from CHO cells, neonate rat brain, and adult rat liver were prepared as described (43). Four different restriction fragments in the proximal 5’-flanking region were employed in gel-retardation assays: a 85-bp Alul-Aval fragment including nucleotides -416 to -331, a 196-bp Aval-Sau3A fragment (-331 to -135) a 109-bp Sau3A-Aval fragment (-135 to -26) and a 214-bp Haelll fragment (-29 to +185) (Fig. 3). Fragments were labeled with 32P-deoxynucleotide triphosphates using the Klenow fragment of DNA polymerase I, followed by purification over ELU-TIP columns (Schleicher and Schuell, Keene, NH). In addition, a 52-bp double-stranded synthetic oligonucleotide encoding the initiator element (-27 to +21) was labeled following the same protocol. Binding assays were done by preincubating 0.3-7.2 pg crude nuclear extract and 100-500 na oolv(dl-dC) (Pharmacia. Piscataway, NJ), with or without theindicated unlabeled DNA as a competitor, in 20 ~1 12 mM HEPES, pH 7.9, 12 mM KCI, 0.6 mM MgCI,, 1.2 mM DTT, 75 mM NaCI, and 10.2% glycerol for 30 min at room temperature; 10,000 dpm (-40 pg) of the labeled fragment were then added, and the reaction was incubated for an additional 30 min. The reaction products were analyzed by electrophoresis on a 4% polyacrylamide gel followed by autoradiography at -70 C. The sequences of the oligonucleotides used in gel-retardation assays are shown in Figs. 6B and 7B. DNase

I Footprinting

For DNase I footprinting, the following DNA fragments were employed: an Alul-Aval fragment including nucleotides -416 to -331, an AlulSau3A fragment extending from -416 to -135 and a Sau3A-Aval fragment extending from -135 to +115 (Fig. 3). Double-stranded DNA probes were cloned into the multiple cloning site of pGEM vectors; supercoiled DNAs were linearized with either EcoRl or Hindlll, phenol/chloroformextracted, and ethanol-precipitated. The DNA pellets were resuspended in 40 ~1 water and end-labeled with avian myeloblastosis virus reverse transcriptase and radioactive nucleotides. Subsequently, the reaction mixtures were diluted to a total volume of 200 ~1 with an appropriate buffer, and the inserts were excised with a restriction enzyme at the unlabeled end of the probe. To this mixture, 50 ~1 formamide loading buffer were added, and the labeled probes were gel-purified

on a 6% nondenaturing polyacrylamide gel. The wet gel was exposed briefly (3-4 min) to Kodak XAR2 film (Eastman Kodak Co., Rochester, NY) to locate the probe fragment, and the gel bands containing the probes were excised, macerated, and incubated for 5-l 5 h at 37 C with 600 ~1 Gilbert’s buffer (500 mM NH, acetate, 10 mM Mg acetate, 1 mM EDTA and 0.1% sodium dodecyl sulfate). After a phenol/chloroform extraction and ethanol precipitation, the probe pellets were reconstituted in distilled water at 1 O,OOO-20,000 dpm/pl and stored at 4 C. Footprinting reactions were performed using a Hotfoot footprinting kit (Stratagene, San Dieqo, CA). Briefly, reactions were carried out in a total vol of 56~1 and digested with 0.0080.032 U DNase I for 2 min. Purified SD~ orotein. kindlv orovided by R. Tjian (University of California, ‘Berkeley, ‘CA) and recombinant AP-2 protein (Promega) or crude nuclear extracts from rat neonate brain or adult liver were used in the assays, The reaction mixtures were phenol/chloroform-extracted and ethanol-precipitated before application to a 12% polyacrylamide/urea gel. Maxam-Gilbert G and C sequencing reactions were run as markers.

Acknowledgments We thank Drs. A. R. Brasier and J. F. Habener for the pOLUC and pSV2LUC plasmids, Drs. S. Jackson and R. Tjian for the Spl protein and expression vectors, and Drs. C. Wu and J. Wisniewski for the SL2 cells. We also thank Drs. C. McKeon, D. Accili, and M. Reitman for critical reading of the manuscript and Ms. Violet Katz for expert processing of the manuscript.

Received April 10, 1992. Revision received June 5, 1992. Accepted July 9, 1992. Address requests for reprints to: Dr. Haim Werner, Section on Molecular and Cellular Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Building 10, Room 88243, Bethesda, Maryland 20892. This work was supported in part by a grant from the American Diabetes Association, Washington, DC Area Affiliate (to C.T.R.).

REFERENCES 1. Rechler MM, Nissley SP 1985 The nature and regulation of the receptors for insulin-like growth factors. Annu Rev Physiol 471425-442 2. Werner H, Woloschak M, Stannard B, Shen-Orr Z, Roberts Jr CT, LeRoith D 1991 The insulin-like growth factor

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IGF-I-R

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I receptor: molecular biology, heterogeneity and regulation. In: LeRoith D (ed) Insulin-Like Growth Factors: Molecular and Cellular Aspects. CRC Press, Boca Raton, p 17-47 Ullrich A, Gray A, Tam AW, Yang-Feng T, Tsubokawa M, Collins C, Henzel W, LeBon T, Kathuria S, Chen E, Jacobs S, Francke U, Ramachandran J, Fujita-Yamaguchi Y 1986 Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 5:2503-2512 Werner H, Woloschak M, Adamo M, Shen-Orr Z, Roberts Jr CT, LeRoith D 1989 Developmental regulation of the rat insulin-like growth factor I receptor gene. Proc Natl Acad Sci USA 86:7451-7455 Lowe Jr WL, Adamo M, Werner H, Roberts Jr CT, LeRoith D 1989 Regulation by fasting of rat insulin-like growth factor I and its receptor: effects on gene expression and binding. J Clin Invest 84:619-626 Werner H, Shen-Orr Z, Stannard B, Burguera B, Roberts Jr CT, LeRoith D 1990 Experimental diabetes increases insulin-like growth factor I and II receptor concentration and gene expression in kidney. Diabetes 39:1490-l 497 Cullen KJ, Yee D, Sly WL, Perdue J, Hampton B, Lippman ME, Rosen M 1990 Insulin-like growth factor receptor expression and function in human breast cancer. Cancer Res 50148-53 Glick RP, Gettleman R, Pate1 K, Lakshman R, Tsibris JCM 1989 Insulin and insulin-like growth factor I in brain tumors: binding and in vitro effects. Neurosurgery 24:791797 Minuto F, DelMonte P, Barreca A, Alama A, Cariola G, Giordano G 1988 Evidence for autocrine mitogenic stimulation by somatomedin-C/insulin-like growth factor I on an established human lung cancer cell line. Cancer Res 48:3716-3719 Stracke ML, Engel JD, Wilson LW, Rechler MM, Liotta LA, Schiffman E 1989 The type I insulin-like growth factor receptor is a motility receptor in human melanoma cells. J Biol Chem 264:21544-21549 Kaleko M, Rutter WJ, Miller AD 1990 Overexpression of the human insulin-like growth factor I receptor promotes ligand-dependent neoplastic transformation. Mol Cell Biol 10:464-473 Stewart AJ, Johnson MD, May FEB, Westley BR 1990 Role of insulin-like growth factors and the type I insulinlike growth factor receptor in the estrogen-stimulated proliferation of human breast cancer cells. J Biol Chem 265:21172-21178 Papa V, Hartmann KKP, Rosenthal SM, Maddux BA, Siiteri PK, Goldfine ID 1991 Progestins induce downregulation of insulin-like growth factor I (IGF-I) receptors in human breast cancer cells: potential autocrine role of IGF-II. Mol Endocrinol 5:709-717 Ota A, Shen-Orr Z, Roberts Jr CT, LeRoith D 1989 TPAinduced neurite formation in a neuroblastoma cell line (SHSY5Y) is associated with increased IGF-I receptor mRNA and binding. Mol Brain Res 6:69-76 Hernandez ER, Hurwitz A, Botero L, Ricciarelli E, Werner H, Roberts Jr CT, LeRoith D, Adashi EY 1991 Insulin-like growth factor receptor gene expression in the rat ovary: divergent regulation of distinct receptor species. Mol Endocrinol 5:1799-l 805 lsaksson OGP, Lindahl A, lsgaard J, Nilsson A, Tornell J, Carlsson B 1991 Dual regulation of cartilage growth. In: Spencer EM (ed), Modern Concepts of Insulin-Like Growth Factors. Elsevier, New York, pp 121-l 27 Werner H, Stannard B, Bach MA, LeRoith D, Roberts Jr CT 1990 Cloning and characterization of the proximal promoter region of the rat insulin-like growth factor I (IGFI) receptor gene. Biochem Biophys Res Commun 169:1021-1027 Araki E, Shimada F, Uzawa H, Mori M, Ebina Y 1987

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27.

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29.

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33.

34.

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36.

37.

38.

39.

Characterization of the promoter region of the human insulin receptor gene. J Biol Chem 262:16186-l 6191 McKeon C, Moncada V, Pham T, Salvatore P, Kadowaki T, Accili D, Taylor SI 1990 Structural and functional analysis of the insulin receptor promoter. Mol Endocrinol 4:647-656 Seino S, Seino M, Nishi S, Bell GI 1989 Structure of the human insulin receptor qene and characterization of its promoter. Proc Natj Acad Sci USA 86:114-l 18 Tewari DS. Cook DM. Taub R 1989 Characterization of the promoter region and 3’ end of the human insulin receptor gene. J Biol Chem 264:16238-l 6245 Biggin MD, Tjian R 1988 Transcription factors that activate the Ultrabithorax promoter in developmentally staged extracts. Cell 53:699-711 Perkins KK, Dailey GM, Tjian R 1988 In vitro analysis of the Antennapedia P2 promoter: identification of a new Drosophila transcription factor. Genes Dev 2:1615-l 626 Smale ST, Baltimore D 1989 The “initiator” as a transcription control element. Cell 57:103-l 13 Briggs MR, Kadonaga JT, Bell SP, Tjian R 1986 Purification and biochemical characterization of the promoterspecific transcription factor Spl Science 234:47-52 Kageyama R, Merlin0 GT, Pastan I 1989 Nuclear factor ETF specifically stimulates transcription from promoters without a TATA box. J Biol Chem 264:15508-l 5514 Kageyama R, Pastan I 1989 Molecular cloning and characterization of a human DNA binding factor that represses transcription. Cell 59:815-825 Roesler WJ, Vandenbark GR, Hanson RW 1988 Cyclic AMP and the induction of eukaryotic gene transcription. J Biol Chem 263:9063-9066 Courey AJ, Tjian R 1988 Analysis of Spl in viva reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell 55:887-898 Williams T, Tjian R 1991 Analysis of the DNA-binding and activation properties of the human transcription factor AP2. Genes Dev 5:670-682 Petty KJ, Morioka H, Mitsuhashi T, Nikodem V 1989 Thyroid hormone regulation of transcription factors involved in malic enzyme gene expression. J Biol Chem 264:11483-l 1490 Smale ST, Schmidt MC, Berk AJ, Baltimore D 1990 Transcriptional activation by Spl as directed through TATA or initiator: specific requirements for mammalian transcriotion factor II D. Proc Natl Acad Sci USA 87:45094513 ’ Therrien M, Drouin J 1991 Pituitary pro-opiomelanocortin gene expression requires synergistic interactions of several regulatory elements. Mol Cell Biol 11:3492-3503 Cooke DW, Banker-t LA, Roberts Jr CT, LeRoith D, Casella SJ 1991 Analysis of the human type I insulin-like growth factor receptor promoter region. Biochem Biophys Res Commun 177:1113-l 120 Courey AJ, Holtzman DA, Jackson SP, Tjian R 1989 Synergistic activation by the glutamine-rich domains in human transcription factor Spl Cell 59:827-836 Saffer JD, Jackson SP, Annarella MB 1991 Developmental expression of Spl in the mouse. Mol Cell Biol 11:21892199 Araki E, Murakami T, Shirotani T, Kanai F, Shinohara T, Shimada F, Mori M, Shichiri M, Ebina Y 1991 A cluster of four Spl binding sites required for efficient expression of the human insulin receptor gene. J Biol Chem 266:39443948 Bondy CA, Werner H, Roberts Jr CT, LeRoith D 1990 Cellular pattern of insulin-like growth factor-l (IGF-I) and type I IGF receptor gene expression in early organogenesis: comparison with IGF-II gene expression. Mol Endocrinol4:1386-1398 Bondy CA, Werner H, Roberts Jr CT, LeRoith D 1991 Cellular pattern of type-l insulin-like growth factor receptor gene expression during maturation of the rat brain: com-

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MOL 1558

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ENDO.

parison with insulin-like growth factors-l and -II. Neuroscience 46:909-923 Hattori M, Sakaki Y 1986 Dideoxy sequencing method using denatured plasmid templates. Anal Biochem 152:232-238 Brasier AR, Tate JE, Habener JF 1989 Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechniques 7:1116-l 122

40. 41.

Eleventh

international Recent

Symposium

42.

43.

Chu G, Hayakawa H, Berg P 1987 Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res 15131 l-l 326 Hennighausen L, Lubon H 1987 Interaction of protein with DNA in vitro. In: Berger SL, Kimmel AR (eds) Methods in Enzymology, Guide to Molecular Cloning Techniques. Academic Press, Orlando, vol 152:721-735

of the Journal

Molecular Biology Advances in Steroid Biochemistry May 30-June 2, 1993, Seefeld, Scientific

Organizing

of Steroid Biochemistry and Molecular Tyrol, Austria

and

Biology

Committee

H. Adlercreutz, Helsinki, Finland E. Gurpide, New York, E. V. Jensen, Hamburg, Germany L. Martini, Milan, Italy A. Munck, Hanover, U.S.A. B. W. O’Malley, Houston, U.S.A. J.-P. Raynaud, Paris, France J. R. Pasqualini, Paris, France The 11 th International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, will be held in Seefeld, Tyrol, Austria, from May 30 to June 2, 1993. The following topics will be considered: 1. 2. 3. 4. 5.

Steroid receptor structure and function on gene expression; Steroids, antisteroids, growth factors, and cancer; Molecular biology of enzymatic processes in steroid transformation; Steroids in the neuroendocrine system (including reproduction); and Adrenal steroids: new developments related to hyptertension.

Lectures (approximately 30-35) will be by invitation of the Scientific Organizing Committee, and in addition there will be a poster section. All poster presentations will be subject to selection by the Scientific Organizing Committee, and abstracts (maximum 200 words) must be addressed to Dr. J. R. Pasqualini by Tuesday January 12, 1993 (postmark) at the latest (original + 10 copies). The total number of participants will be limited to 150. For further details please contact: General Scientific Secretariat Dr J. R. Pasqualini C.N.R.S. Steroid Hormone Research Unit Foundation for Hormone Research 26 Boulevard Brune, 75014 Paris, France Tel.: (33) (1) 45.39.91.09 Fax: (33) (1) 45.42.61.21.

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