DNA AND CELL BIOLOGYVolume 18, Number 2, 1999Mary Ann Liebert, Inc.Pp. 147± 156

Multiple Promoter Elements Including a Novel Repressor SiteModulate Expression of the Chick Ovalbumin Gene

KARL R. SENSENBAUGH and MICHEL M. SANDERS

ABSTRACT

As is the case with many eukaryotic genes, regulation of the chick ovalbumin (Ov) gene involves both posi-tive and negative modulation. Recent studies indicate that positive regulation by steroids entails binding ofseveral proteins to a hormone-response unit called the steroid-dependent regulatory element (SDRE; 2 892 to2 780). In addition, gene activity is suppressed by factor(s) acting through the negative regulatory element(NRE; 2 308 to 2 88). Previous data suggested that the NRE is composed of multiple, independently actingnegative elements. The goal of the present studies was to define more precisely the locations of these negativeelements and to investigate their functional interactions. Transfection analyses of linker scanning mutants re-vealed a strong repressor site, designated the COUP-adjacent repressor (CAR) site, located between 2 119and 2 111. Gel mobility shift analyses with the CAR element suggested that it may play a role in the devel-opmental regulation of the Ov gene. A weaker repressor element was also identified at about 2 275. Surpris-ingly, two positive sites were found, one of which is the binding site for the estrogen-responsive transcriptionfactor d -EF1. These results demonstrate that the Ov NRE contains not only sites responsible for the repres-sion of the gene but also a positive element that is required for responsiveness to steroid hormones.

147

INTRODUCTION

DIFFERENTIAL GENE EXPRESSION provides the basis for the de-

velopment and function of differentiated tissues. The pro-

grammed activation and repression of specific genes in a coor-

dinated fashion requires the integration of many stimuli,

including hormonal, environmental, and nutritional cues. As a

result, eukaryotic genes must orchestrate converging positive

and negative signals to achieve an appropriate level of expres-

sion in the correct time frame.

The development and differentiation of the oviduct from a

10-mg tissue in the sexually immature chick to a 40-g organ in

the laying hen is directed primarily by estrogen (for review, see

Sanders and McKnight, 1986). Interestingly, estrogen coordi-

nates both the proliferation and differentiation of the tubular

gland cells in this organ through mechanisms that remain

largely unexplored. The net result is a tissue that can synthe-

size 3 3 1019 molecules of Ov per day, as well as large amounts

of the other egg white proteins (Garel et al., 1978).

Because of its exquisite sensitivity to estrogen at the tran-

scriptional level, the regulation of the Ov gene has been ex-

tensively characterized. The analyses indicate that it is a sec-

ondary response gene (Dean and Sanders, 1996), as the estro-

gen receptor does not appear to bind to the proximal 900 bp

that are required for responsiveness to steroid hormones

(Schweers et al., 1990), the induction by estrogen occurs only

after a 2 h lag (Palmiter et al., 1976), and protein synthesis in-

hibitors abolish induction (McKnight et al., 1980). Furthermore,

the synergistic actions of two steroid hormones, which are

thought to be estrogen and corticosterone in vivo (Sanders and

McKnight, 1985), are required. Deletion analyses identified two

major regulatory elements: a hormone-response unit located

from 2 892 to 2 780, called the steroid-dependent regulatory

element (SDRE), and a repressive element from 2 308 to 2 88

called the negative regulatory element (NRE; Sanders and

McKnight, 1988). Deletion of the SDRE eliminates respon-

siveness to steroid hormones, and transcriptional activity is at

basal levels. Deletion of both the SDRE and NRE restores max-

imal expression, but transcription is no longer dependent on

steroids (Sanders and McKnight, 1988). Thus, the NRE appears

to repress transcription of the Ov gene in the absence of steroid

hormones. Recent advances have identified the binding site for

a steroid-dependent, cycloheximide-sensitive protein called

Chirp-I (Dean et al., 1996). However, identification of numer-

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 435 Delaware St. SE, Minneapolis, Minnesota55455.

ous other protein-binding sites in the SDRE and the NRE in-

dicates that Chirp-I is just one of the many proteins involved

in modulating the expression of the Ov gene. In fact, the gene

appears to be actively repressed in the absence of steroid hor-

mones by four independently acting negative elements (Fig. 1B,

F1: 2 280 to 2 252, silencer: 2 237 to 2 228, F2: 2 175 to 2 132,

and F3: 2 132 to 2 87) (Ehlen Haecker et al., 1995).

The goal of the following studies was to map the four neg-

ative elements more precisely in the Ov NRE. In order to elim-

inate any distance-dependent effects, the studies were done such

that the correct spatial relations between the elements of the

NRE and the proximal promoter of the Ov gene were main-

tained. A strong repressor site was found in the NRE ( 2 119

and 2 111) that was designated the COUP-adjacent repressor

(CAR) site because of its proximity to the COUP-TF binding

site (2 85 to 2 73). However, the CAR site is not sufficient to

account for all repression seen with the NRE, and another neg-

ative site was identified from about 2 279 to 2 270. In support

of unpublished results (E.M. Chamberlain and M.M. Sanders,

manuscript submitted), an activator site ( 2 174 to 2 146) was

detected. This site binds the estrogen-responsive transcription

factor d -EF1 (E.M. Chamberlain and M.M. Sanders, manuscript

submitted). Another activator site is located between 2 204 and

2 175 but shows no resemblance to any known transcription

factor-binding site. These results indicate that the NRE is a com-

plex regulatory region, consisting of multiple positive and neg-

ative sites that all contribute to the regulation of the Ov gene.

MATERIALS AND METHODS

Construction of linker scanning mutants

Construction of linker scanning (LS) mutants V, W, X, O,

P, Q, R, N, Y, Z, AA, and S was done essentially as described

previously using a two-step polymerase chain reaction (PCR)

procedure (Ehlen Haecker et al., 1995). However, the PCR con-

ditions were slightly different: 93°C (45 sec), 45°C (2 min), and

72°C (2 min) for 30 cycles and 72°C (10 min) for 1 cycle. The

remaining LS mutations were made by a modification such that

the two PCR products were combined into a single product us-

ing primers homologous to the distal ends as described (E.M.

Chamberlain and M.M. Sanders, manuscript submitted). The

LS mutants covered the following bases in the NRE: V ( 2 308

to 2 299), W ( 2 298 to 2 289), X (2 288 to 2 279), O ( 2 279

to 2 270), P ( 2 268 to 2 259), Q ( 2 259 to 2 250), R (2 249 to

2 240), M2 ( 2 237 to 2 228), N (2 228 to 2 215), BB (2 213

to 2 204), CC (2 204 to 2 195), DD (2 184 to 2 185), EE ( 2 184

to 2 175), Y ( 2 174 to 2 161), Z (2 161 to 2 148), AA ( 2 148

to 2 136), U ( 2 134 to 2 120), T ( 2 119 to 2 106), S (2 104 to

2 90), ST1 ( 2 119 to 2 111), ST2 (2 109 to 2 100), and ST3

(2 99 to 2 90). The locations of the LS mutations in the NRE

are depicted in Figure 1C, and the actual sequences are given

in Figure 2. All mutations were confirmed by dideoxy se-

quencing.

Oviduct tubular gland cell culture and transfection

The isolation and transfection of primary oviduct cells has

been described in detail previously (Ehlen Haecker et al., 1995;

Sanders and McKnight, 1988). Briefly, chicks were given a pri-

mary treatment with estrogen in the form of two 10-mg di-

ethylstilbesterol (DES) pellets implanted subcutaneously in the

neck. After at least 2 weeks, the pellets were withdrawn from

the chicks for 2 days before sacrificing the animals. The mag-

num portion of the oviduct was isolated from multiple chicks

and pooled (about 0.4 g of oviduct/plasmid transfected). The

oviducts were then minced and the cells dissociated using

collagenase, protease, DNase I, and trypsin (Sanders and

McKnight, 1985). The DNA was transfected into the primary

oviduct cells by calcium phosphate coprecipitation using a fi-

nal DNA concentration of 15 m g/ml as described (Sanders and

McKnight, 1988). Each LS mutation was transfected in multi-

ple experiments using different DNA preparations. After 3 h of

transfection, the DNA was washed off, and the cells were cul-

tured for 22 to 24 h in medium with insulin (50 ng/ml) either

alone ( 2 S) or with 17 b -estradiol (1 3 10 2 7 M) plus corticos-

terone (1 3 10 2 6 M) ( 1 S). After the culture period, the cells

were harvested and assayed for the presence of chlorampheni-

col acetyltransferase (CAT) activity.

SENSENBAUGH AND SANDERS148

FIG. 1. Location of in vitro protein binding sites, functional sites, and LS mutations in the NRE. A. Schematic representationof the previously characterized in vitro protein-binding sites in the NRE. In vitro binding analysis was done by either DNase Ifootprinting (DF1±4 [Pastorcic et al., 1989]) or by gel mobility shift analysis (GSB1±4 [Ehlen Haecker et al., 1995]). B. Func-tional elements (F1, Silencer, F2, and F3) that were identified by transient transfection experiments using primary oviduct cells(Ehlen Haecker et al., 1995). C. The relative positions of the LS mutants in OvCAT-.900 ( 2 900 to 1 9) used for these studies.

CAT assays

The cells were scraped from dishes, pelleted, and resus-

pended in Reporter Lysis Buffer (Promega, Madison WI). The

cells were lysed, cell debris was spun down, and the supernatant

fluid containing cellular proteins was saved for further analy-

sis. These protein extracts were quantitated using the Bradford

Assay Kit from BioRad. The CAT assays were then set up with

a constant amount of protein (100±300 m g per tube), 4 mM

acetyl coenzyme A, and 3 m l of 14C-chloramphenicol (50±60

Ci/mmol) as previously described (Sanders and McKnight,

1988). Each figure represents the data pooled from multiple ex-

periments, typically four, that were done with at least duplicate

samples per hormone treatment in each experiment. The data

are presented as the mean 6 the standard deviation for the

pooled experiments and are normalized relative to the wildtype

(OvCAT-.900) construct. Data from all experiments with a

given plasmid were pooled and subjected to ANOVA.

Gel mobility shift assays

Oligonucleotides were synthesized by the University of Min-

nesota Microchemical Facility as single-stranded oligomers

with restriction enzyme-site overhangs. The oligomers used

were as follows:

CAR:

ctagaTATTGAAACTAAAATCTAACCCAATCCCATTt

CARmST1:

gatccTATTGAAACTAgctctagAgCCCAATCCCATTg

where capital letters represent Ov 5 9 flanking sequence (59 to

3 9 ), lowercase/plain text letters represent restriction enzyme

ends, and lowercase/boldface letters represent the mutant Ov

sequence, CARmST1. Both of the CAR oligomers span Ov

NRE sequences 2 130 to 2 100; and the CAR mutation,

CARmST1, spans Ov sequences 2 119 to 2 112. Complemen-

tary oligomers were annealed by incubating them in 50 mM

NaCl at 68°C for 15 min followed by cooling slowly to room

temperature. The annealed oligomers were labeled with the

Klenow fill-in reaction using [ a -32P]-dATP to a specific activ-

ity of at least 1 3 108 cpm/ m g. Nuclear protein extracts from

oviduct were obtained by previously described methods (Ehlen

Haecker et al., 1995). Binding reactions were set up as de-

REPRESSION OF OVALBUMIN GENE EXPRESSION 149

FIG. 2. Sequence of the wildtype NRE and of the LS mutations that were made in it. Wildtype nucleotides are indicated bycapital letters, whereas lowercase letters show mutated bases. Nucleotides that are represented by dashes are the same as wild-type. The COUP-TF binding site is underlined and in boldface (2 85 to 2 73).

scribed (Ehlen Haecker et al., 1995) and carried out in a wa-

terbath at 20°C for 30 min. The reactions were run in 0.53 TBE

on a 6% nondenaturing polyacrylamide gel at 4°C for 3.5 h at

160 V. When nonradioactive competitors were included in bind-

ing reactions, the probe and competitor DNA were mixed prior

to addition of the nuclear protein.

RESULTS

Linker scanning mutations in the NRE reveal bothpositive and negative regulatory elements

Early 5 9 -deletion experiments to define regulatory elements

in the Ov gene identified two large regulatory elements, the

SDRE and the NRE (Sanders and McKnight, 1988). All re-

sponsiveness to steroid hormones was lost with the 5 9 deletion

of a region including the SDRE (2 900 to 2 732), and tran-

scriptional activity remained at basal levels unless the NRE

(2 308 and 2 88) was also deleted. Additional experiments were

done to characterize the NRE, most of which placed fragments

of the NRE closer to the Ov promoter or adjacent to the het-

erologous thymidine kinase (TK) promoter (Ehlen Haecker etal., 1995). Those analyses revealed four negative elements that,

when used as isolated fragments, were capable of repressing

both the homologous and the heterologous promoters (Fig. 1B;

F1, silencer, F2, and F3). Those elements were also capable of

binding proteins, as demonstrated previously by gel mobility

shift assays (GMSAs; Ehlen Haecker et al., 1995) and DNase

I footprinting analysis (Pastorcic et al., 1989). The binding sites

identified in those GMSAs are depicted in Figure 1A as GSB1,

GSB2, GSB3, and GSB4; and those identified by the DNase I

footprinting are depicted as DF1, DF2, DF3, and DF4.

However, some concern exists because the functions of the

four elements were tested out of context and in the absence of

the SDRE. It is clear that many transcription factors have con-

textual requirements for their actions (Danilition et al., 1991;

Dawson et al., 1996; Perkins et al., 1993; Schanke and Van

Ness, 1994; Smith, 1995; Tansey et al., 1993; Wu and Berk,

1988), including repressors (Arnosti et al., 1996; Barolo and

Levine, 1997; Gray and Levine, 1996). Therefore, the goal of

these studies was to characterize the NRE in its normal context

to ascertain whether the functions ascribed to it earlier are in

fact correct and to extend those analyses by more precisely map-

ping the regulatory elements.

To keep Ov sequences in their wildtype context, LS muta-

tions were made throughout the NRE (Figs. 1C and 2) in Ov-

CAT-.900 (2 900 to 1 9), which contains all known regulatory

sequences (Dean et al., 1996; Ehlen Haecker et al., 1995; Nord-

strom et al., 1993; Sanders and McKnight, 1988; Schweers et

al., 1990; Schweers and Sanders, 1991) and is called wildtype.

The LS mutant constructs were transfected into primary oviduct

cells, which were subsequently cultured in the presence of in-

sulin (2 S) or of insulin, corticosterone, and estrogen ( 1 S). The

LS mutations in the 5 9 part of the NRE spanned in vitro bind-

ing sites GSB1, GSB2, DF1, DF2, and DF3 (Fig. 1A) and the

F1 and silencer functional regions (Fig. 1B). The results in Fig-

ure 3A show that none of these 59 LS mutations V±M2 had a

SENSENBAUGH AND SANDERS150

FIG. 3. Transfection of the NRE LS mutants into primaryoviduct cells. The locations of the LS mutations used in theseexperiments are shown relative to protein-binding sites and pre-viously identified functional sites in Figure 1, and the actual se-quences of the LS mutations are shown in Figure 2. A. Effectsof LS mutants in the 5 9 -end of the NRE (LS-V, -W, -X, -O, -P, -Q, -R, and -M2) on activity of the Ov promoter. B. Effectsof LS mutants in the center of the NRE (LS-N, -BB, -CC, -DD,and -EE) on the activity of the Ov promoter. C. Effects of LSmutants in the 3 9 end of the NRE (LS-Y, -Z, -AA, -U, -T, and-S) on the activity of the Ov promoter. Note that the scale isdifferent for this panel. D (inset). The activities of the LS mu-tations in the 3 9 end of the NRE on the same scale as used forpanels A and B. All LS mutants were transfected into primaryoviduct cells as described in Methods. The cells were culturedin serum-free medium containing either insulin alone (-S) or in-sulin, estrogen, and corticosterone ( 1 S). After 24 h, the cellswere harvested, and protein extracts were assayed for CAT ac-tivity. The results are reported as percent maximal conversionrelative to OvCAT-.900 (wildtype) cultured in the presence ofsteroids. Each mutant construct was tested with a minimum offour replicates per treatment. Most were done in multiple ex-periments with different plasmid preparations. Error is reportedas the standard deviation of replicates among experiments. Sta-tistical significance: * 5 P # 0.05, ** 5 P # 0.01, *** 5 P #0.0001.

significant effect on basal or steroid-induced reporter gene ex-

pression in the context of OvCAT-.900. Of particular note, mu-

tation of the silencer site (LS-M2) had no effect in the wild-

type context in these transient transfection assays.

In contrast, expression of all of the LS mutations in the cen-

tral section of the NRE (Fig. 3B) differed significantly from

wildtype. Basal expression of both LS-N and LS-BB was ele-

vated about twofold compared with the wildtype. Interestingly,

steroid-induced expression of LS-BB was also moderately, al-

though significantly, elevated. These results suggest that a neg-

ative element resides between 2 227 and 2 204 that preferen-

tially affects basal expression but that may impinge on

steroid-induced expression as well. However, mutations in the

region between 2 204 and 2 175 (LS mutants -CC, -DD, and

-EE) significantly attenuated the induction by steroids, indicat-

ing that this region is required for maximal expression (Fig. 3B;

1 S), although basal activity was unchanged (Fig. 3B: 2 S).

The LS mutations in the 3 9 end of the NRE (Fig. 3C) spanned

in vitro binding sites GSB3, GSB4, and DF4 and functional

sites F3 and F4 (Fig. 1A, B). Because of the large increase in

activity seen with LS-T and LS-S, the mutants LS-Y, -Z, -AA,

and -U are also graphed as an inset on the same scale used for

Figure 3A and 3B to show more clearly the effects of these mu-

tations (Fig. 3D). Both LS-Y and LS-Z exhibited significantly

reduced expression in the presence of steroids. This finding sug-

gests that the region from 2 174 to 2 148 contains a regulatory

element for one or more transcription factors involved in the

induction of the Ov gene by steroid hormones. Although some-

what variable, on average, LS-U exhibited wildtype levels of

expression in the presence of steroids but moderately elevated

expression in the absence of steroids. However, LS-T and

LS-S showed a dramatic increase in both the absence (5- and

11-fold, respectively) and presence (8- and 12.5-fold, respec-

tively) of steroids compared with the wildtype construct (Fig.

3C). This result suggests that a strong repressor element resides

between 2 119 and 2 90.

The dramatic results with LS-T and LS-S (note the change

in scale on the Y axis) prompted the division of this region into

three smaller LS mutations (Figs. 1 and 4A). The results of

transfection of these three mutations, designated LS-ST1 ( 2 119

to 2 111), LS-ST2 ( 2 109 to 2 100), and LS-ST3 (2 99 to 2 90),

are shown in Figure 4B. LS-ST1 produced a 4.5-fold increase

in activity in the absence of steroids and a 6.5-fold increase in

activity in the presence of steroids, which is similar to the in-

creases seen with the LS-T mutation. Surprisingly, neither LS-

ST2 nor LS-ST3 had an effect on transcriptional activity in ei-

ther the absence or the presence of steroids. Although it is not

clear why the LS-S mutation enhanced transcription, whereas

the LS-ST2 and LS-ST3 mutations that spanned the region did

not, search of a transcription factor-binding site database (Pre-

stridge, 1996) indicates that LS-S shares a high degree of ho-

mology with the site for the well-characterized activator, Sp1.

This is not true of the wildtype sequence or of the LS-ST2 or

LS-ST3 mutations. Therefore, it seems likely that LS-S creates

an activator site and that the nucleotides spanned by LS-S

(2 110 to 2 90) have no functional significance. In contrast, the

bases spanned by LS-ST1 ( 2 119 to 2 111) apparently bind a

strong repressor that affects both basal and steroid-hormone-in-

duced transcription. This repressor element has been designated

the CAR site because of its close proximity to the COUP-TF

binding site ( 2 85 to 2 73).

To summarize the transfection experiments with the LS mu-

tations, only the CAR site appeared to exert a sufficiently strong

effect to act as a repressor in the wildtype context. None of the

other four previously identified sites, F1, silencer, F2, or F3

(Ehlen Haecker et al., 1995), exerted an effect in this context.

This finding suggests either that they have no role, or that, as

postulated earlier (Ehlen Haecker et al., 1995), they are func-

tionally redundant such that all need to be mutated at once to

elevate basal transcription. Lastly, the LS mutations revealed

that the NRE contains two previously unsuspected positive sites

between 2 204 and 2 175 (LS-CC, -DD, -EE) and between

2 175 and 2 148 (LS-Y and -Z).

REPRESSION OF OVALBUMIN GENE EXPRESSION 151

FIG. 4. Mutational analysis of the repressor site in the 3 9 endof the NRE. A. Three LS mutations were made in OvCAT-.900that span LS-T and LS-S. B. The three LS mutations were trans-fected into primary oviduct cells, the cells subsequentlyprocessed, and the data analyzed as described in the legend forFigure 3. In the presence of steroid hormones, only the LS-T1mutant is significantly different from wildtype, with P ,0.0001. No significant differences were detected between wild-type and mutant constructs in the absence of steroids.

Binding of proteins to the CAR site varies with thestage of development

To investigate protein binding to the CAR site and to cor-

roborate the functional data, an oligomer containing the CAR

site ( 2 130 to 2 100) was subjected to GMSA analysis with

oviduct nuclear protein extracts from estrogen-withdrawn

chicks (Fig. 5). Although some were faint, complexes 1 through

3 could be seen reproducibly with the CAR oligomer (Fig. 5A;

lane 4). As indicated below, complexes 4 and 5 probably rep-

resent degraded or partially dissociated protein complexes.

None of the complexes was seen when the oligomer contained

the ST1 mutation (CARmST1) (Fig. 5A; lane 2). Instead, a band

of different mobility was observed with the mutant oligomer,

but that binding was not specific (data not shown). Further

analysis indicated that the binding to the wildtype CAR ele-

ment was specific because an excess of cold self-oligomer com-

peted for all five complexes (Fig. 5B; compare lanes 3±5 with

lane 2), but neither an excess of unlabeled CARmST1 (lanes

6±8) nor of a nonspecific oligomer (lanes 9±11) competed for

the any of the five complexes. Therefore, the repressive effects

of the CAR site can be attributed to protein(s) binding specif-

ically to it.

In an attempt to ascribe a biological function to the CAR

site, oviduct samples from different physiological states were

tested for altered binding activity. Gel mobility shift assays were

done with oviduct nuclear protein extracts from chickens at

three stages of development (Fig. 5C). Replicate nuclear pro-

tein extracts, each from different chickens, were used for each

condition. Although variability was apparent in the extent of

binding among preparations form the same treatment regime,

general patterns emerged. There was no difference in binding

with or without estrogen in sexually immature chicks of the

same stage of development, even though Ov was being pro-

duced in the estrogen-stimulated chicks but not in the estrogen-

withdrawn chicks (Fig. 5C; compare lanes 6±9 with lanes

10±12). Complexes 1 through 3 were present to various degrees

in all chick oviduct samples. Complexes 4 and 5 were present

only in samples prepared and stored for a long period of time

(Fig. 5C; lanes 7, 8, and 11) and may be attributable to degra-

dation of binding proteins or partial dissociation of protein com-

plexes. Thus, the binding of the CAR protein(s) does not ap-

pear to be dependent on estrogen. However, differences existed

between chick-derived samples and laying-hen samples.

Whereas complexes 1 through 3 were consistently present in

chick samples, only complex 1 appeared in laying-hen samples

(Fig. 5C; compare lanes 2±5 with lanes 6±9 and lanes 10±12).

The GMSA data indicate that as many as five protein±DNA

complexes were formed with the CAR site oligomer in sexually

immature chicks. Of these, complexes 1 through 3 probably rep-

resented some form of intact complex, as they were observed

consistently. None of the complexes bound to an oligomer with

the ST1 CAR site mutation, indicating that they were specific.

The data also suggest that the CAR site protein(s) plays a role in

the developmental expression of the Ov gene.

CAR site LS mutations in the NRE-only contextconfirm that additional repressor(s) elements arelocated in the NRE

The results with the LS mutants that are presented in Figure

3A are in conflict with our previous observations indicating that

two negative regulatory elements reside in the 5 9 end of the

FIG. 5. The CAR site binds multiple protein complexes. Gelmobility shift assays were carried out using standard conditions,as outlined in Methods. Five predominant complexes are markedby arrows. A. The CAR site oligomer (2 130 to 2 100) in lanes3 and 4 or the mutant CAR site oligomer, CARmST1, lanes 1 and2, were radioactively labeled. Lanes 1 and 3 show the probesalone, whereas lanes 2 and 4 show the probes incubated withoviduct nuclear proteins isolated from chicks that had been with-drawn from estrogen for 2 days. B. The CAR site oligomer wasthe radioactive probe. Lane 1, probe alone; lanes 2±11, nuclearproteins from chicks that had been withdrawn from estrogen for2 days. Competitors were included in 1003 (lanes 3, 6, and 9),3003 (lanes 4, 7, and 10), or 5003 (lanes 5, 8, and 11) molar ex-cess. The competitors were nonradioactive self-oligomer (lanes3±5), the CARmST1 oligomer (lanes 6±8), or a nonspecificoligomer homologous to the polycloning region of pTZ18R (lanes9±11). C. The CAR site oligomer was used as the radioactiveprobe. Lane 1, probe alone; lanes 2±5, four independently pre-pared nuclear protein samples from laying-hen oviduct; lanes 6±9,four independently prepared samples from estrogen-stimulatedchick oviduct; and lanes 10±12, three independently preparedsamples from chicks withdrawn from estrogen for 2 days.

NRE: one from about 2 280 to 2 252 (LS-O, -P, and -Q) and

the silencer from 2 237 to 2 228 (LS-M2) (Ehlen Haecker et

al., 1995). These differences are not particularly surprising, as

those earlier experiments suggested that all four negative ele-

ments in the NRE are functionally independent and that knock-

ing out one is insufficient to restore transcriptional activity.

However, that interpretation was based on the observation that

none of the deletion constructs restored expression to the same

level as OvCAT-.087C (Fig. 6A), a construct lacking the NRE

and a region that we now know includes the CAR site. The

CAR site had not been discovered, and all of those deletion

constructs retained an intact CAR element. Therefore, the pos-

sibility exists that the lack of full activity of those constructs

was attributable to repression by the CAR site, not to residual

negative elements. Therefore, two additional experiments (Figs.

6B and 7) were done in attempts to reconcile the lack of effect

of LS mutants in the 5 9 end of the NRE with the earlier results.

The goals of the first experiment were to test whether the

CAR site is in fact responsible for all of the repressive activity

of the NRE and, if not, whether the silencer makes a signifi-

cant contribution. Two constructs used in previous studies

define the characteristic activity of the NRE (Sanders and

McKnight, 1988). OvCAT-.308N (308N in Fig. 6A; 2 308 to

1 9) contains the NRE but not the SDRE, while OvCAT-.087C

(087C in Fig. 6A; 2 87 to 1 9) contains neither. The activity of

OvCAT-.308N was very low and was at the same level as wild-

type OvCAT-.900 in the absence of steroid hormones (Fig. 6B),

while OvCAT-.087 activity was as high as that of OvCAT-.900

in the presence of steroid hormones (Sanders and McKnight,

1988). To determine whether the repressive activity of other re-

gions of the NRE was masked by the CAR site, the CARmST1

mutation was made in the OvCAT-.308N context (Fig. 6A;

ST1.308N). The mutation was then tested in transfection ex-

periments, with OvCAT-.308N and -.087C as controls. If the

CAR site were the only repressor site in the NRE, then Ov-

CAT-ST1.308N should have the same activity as OvCAT-

.087C. Although the ST1 mutation did show approximately a

twofold increase in activity in these experiments compared with

OvCAT-.308N, it did not match the activity of OvCAT-.087C

(Fig. 6B; compare 308N, ST1.308N, and 087C).

While these experiments confirm the importance of the CAR

site, they support previous results that indicate that other ele-

ments in the NRE contribute to its overall repressive activity.

As the silencer (2 237 to 2 228) has been well characterized

and appears to be present in a number of genes (Ehlen Haecker

et al., 1995), an experiment was done to test whether it might

be another essential repressor in the Ov NRE. To this end, ad-

ditional constructs were made in the OvCAT-.308N context.

One contained a single LS mutation in the silencer, and the

other contained LS mutations in both the ST1 and silencer sites

(Fig. 6A; M2.308N and ST1M2.308N, respectively). The si-

lencer mutation did not show an increase in activity over Ov-

CAT-.308N (Fig. 6B; compare 308N and M2.308N), and the

ST1/silencer double mutation showed the same twofold in-

crease as the single ST1 mutation (Fig. 6B; compare 308N,

ST1.308N, and ST1M2.308N). None of these constructs

showed the same activity as OvCAT-.087C (Fig. 6B), con-

firming that, indeed, other repressor sites reside in the NRE but

raising the possibility that the silencer plays no role in the nor-

mal context.

The results of the CAR and silencer double mutations in the

NRE-only context indicate that another repressor site(s) exists

in the NRE that cannot be detected with the LS mutants in the

context of the wildtype Ov gene because of their redundant na-

ture. Because previous studies indicated that a repressive ele-

ment resides in the region between 2 280 and 2 252 (Ehlen

Haecker et al., 1995), all of the other potential repressor ele-

ments (Fig. 1; Silencer, F2, and F3) were removed except for

the putative one in the 59 end (Fig. 1; F1). The expectation was

that this construct (OvCAT-.239D 3 9 ) should exhibit reduced ac-

tivity compared with OvCAT-.087C but that a mutation in the

putative negative site should raise expression if it was in fact a

repressive element. OvCAT.239 D 3 9 (Fig. 7A; 239) is a 39 dele-

tion of the NRE that exhibited increased activity compared with

its parent construct, OvCAT-.308F (Fig. 7A; 308F and Fig. 7B;

compare activities of 308F and 239), presumably because of

the removal of the CAR site and, perhaps, other repressor sites.

In order to determine whether the F1 element is responsible for

REPRESSION OF OVALBUMIN GENE EXPRESSION 153

FIG. 6. Results of transfections with the ST1 or silencer LSmutations or both in the NRE-only context. A. Schematic rep-resentation of mutants ST1, M2, and the ST1/M2 double LSmutant in the context of the NRE alone. B. Transfection dataof mutants ST1, M2, and ST1/M2 in the context of the NREalone. The mutations were made and transfected into primaryoviduct cells as described in Methods. The cells were culturedin medium without steroid hormones, as OvCAT-.308N is notresponsive to steroid hormones (Ehlen Haecker et al., 1995).Protein extracts from the cells were assayed for CAT activity,and the results are reported as percent conversion normalizedto that of OvCAT-.308N. Error is reported as the standard de-viation of replicates in all experiments.

repressing OvCAT-.239, two mutations were made that corre-

spond to the LS-O and LS-P mutations in the wildtype context

(Fig. 2). These mutations were picked because in vitro binding

data suggest these sites might be important (Ehlen Haecker et

al., 1995; Pastorcic et al., 1989). In these transfection experi-

ments, mutant OvCAT-239LSO showed about a twofold in-

crease in activity in the presence of steroids (Fig. 7B) compared

with intact OvCAT-.239. OvCAT-.239LSP had no effect, sug-

gesting that the bases spanned by LS-O ( 2 279 to 2 270) in-

clude the repressor site. These results therefore confirm that the

F1 site is, in fact, a weak negative element whose activity is

masked in these transient transfection assays by other negative

elements. The challenge will be to determine its normal phys-

iological role in the regulation of the Ov gene.

DISCUSSION

Although each tubular gland cell in laying hens contains ap-

proximately 50,000 copies of Ov mRNA, only a few copies ex-

ist per cell in the absence of steroid hormones. Furthermore,

expression of the Ov gene is strictly limited to avian oviduct.

However, the mechanisms that restrict expression of this gene

to the requisite spatial and temporal framework remain obscure.

Presumably, the NRE plays a central role. The goal of these

studies was therefore to characterize the NRE more thoroughly

in hopes of elucidating the mechanisms responsible for the ex-

quisitely regulated expression of the Ov gene. To this end, LS

mutations were made throughout the NRE in the wildtype con-

text (OvCAT-.900) so that appropriate spacing of DNA ele-

ments and protein±protein interactions could be maintained.

The expectation was that several negative elements would be

identified that would repress expression in the absence of

steroid hormones, which proved to be the case. However, two

positive elements were defined as well. The activities of LS-Y

and -Z (2 174 to 2 146) were consistently reduced in response

to steroids, although basal activity was unaffected (see Fig. 3C).

This result implies that any transcriptional activator(s) acting

through this site(s) is involved in the induction of Ov gene tran-

scription by steroids. In vitro binding studies, both by DNase I

footprinting (Pastorcic et al., 1989) and by GMSAs (Ehlen

Haecker et al., 1995), support the contention that a factor(s) is

binding to and acting through this functionally important site.

Interestingly, an estrogen-inducible transcription factor has

been identified in the chick oviduct (E.M. Chamberlain and

M.M. Sanders, manuscript submitted). The factor, d -EF1, binds

to the core sequence C¤TNT¤CACCTGT¤A. An 8/9-bp homologous

sequence is located in the LS-Z mutant region from 2 146 to

2 154 (CTTACCTcT). Gel mobility shift studies with a large

oligomer covering this region and oviduct nuclear proteins show

slight differences in binding with and without estrogen (Ehlen

Haecker et al., 1995). Independent binding studies with a

smaller oligomer containing the putative d -EF1-binding site in-

dicate that indeed, d -EF1 is the factor binding and that binding

is increased by estrogen (E.M. Chamberlain and M.M. Sanders,

manuscript submitted). Thus, the Ov gene is the first example

of a gene regulated by two estrogen-inducible primary-response

genes, d -EF1 and Chirp-I (Dean et al., 1996).

The other region that contains a positive site(s) is the area

between 2 204 and 2 175 (LS-CC, -DD, and -EE). Like the

d -EF1 site, this site appears to affect only the steroid-induced

activity, not basal activity. This site is less well characterized

than other sites in the NRE, but it does not appear to have any

repressive effect on either the Ov or TK promoters (Ehlen

Haecker et al., 1995). Furthermore, DNase I footprinting failed

to show any proteins binding to this region (Pastorcic et al.,

1989). However, the transfection and DNA-binding experi-

ments were all done in the absence of the SDRE and estrogen,

so it is likely that the results do not accurately reflect what is

happening at this element in the intact gene in response to es-

trogen treatment. A database search for transcription factor-

binding sites (Prestridge, 1996) revealed no striking homology

with any previously identified binding sites, so it may be nec-

essary to clone this protein before a definitive functional role

can be ascribed to it.

The mutagenesis of the NRE in the wildtype context revealed

SENSENBAUGH AND SANDERS154

FIG. 7. Identification of another negative element in the NRE.A. Schematic representation of LS-O and LS-P in the contextof OvCAT-.239 D 3 9 (Ehlen Haecker et al., 1995). B. Trans-fection of OvCAT-.239LS-O and LS-P into primary oviductcells. The cells were transfected as described in Methods andthen cultured in medium containing insulin alone ( 2 S) or in-sulin plus estrogen and corticosterone ( 1 S). Protein extractsfrom the cells were assayed for CAT activity, and the resultsare reported as percent conversion normalized to that of Ov-CAT-.900 (wildtype) cultured with insulin alone. Error is re-ported as the standard deviation of replicates in all experiments.

a novel negative element, the CAR site (2 119 to 2 111). Mu-

tating this site in both the wildtype and NRE-only contexts in-

creased transcription directed by the Ov promoter. Furthermore,

both basal and steroid-induced transcription was elevated in the

wildtype context. Thus, the CAR-binding protein is most likely

a direct repressor of transcription. Direct repressors are domi-

nant repressors, requiring specific activators to overcome their

expression (Cowell, 1994; Johnson, 1995). Direct repressors

may act to prevent leaky expression of genes by general acti-

vators that are present even in the absence of the true inducing

stimuli.

Gel mobility shift binding data indicated that a protein(s)

binds to the CAR site. Although an exact function cannot yet

be ascribed to the CAR-binding protein(s), the binding data sug-

gest that it may have a developmental role in the expression of

the Ov gene. There are three shifted complexes in fresh, im-

mature chick-derived nuclear protein extracts, but only one in

oviduct extracts from laying hens. The one band from laying

hens aligns with the slowest migrating complex found in the

immature chick. We postulate that this larger complex repre-

sents the inactive complex, as the Ov gene in laying hens is

transcriptionally very active. Additional experimentation will

be required to elucidate how the CAR repressor protein is in-

activated by steroid hormones, but the binding experiments with

laying hen extracts suggest that another protein(s) interacts with

the repressor complex, thereby inactivating it. Unfortunately,

the CAR site sequence shares no homology with other sites in

the transcription factor-binding site databases, so identification

of the pertinent transcription factor(s) must await cloning.

As the LS data suggest that the CAR site is the only strong

negative element in the NRE, which is in contrast to our pre-

vious results, the CAR site was mutated in the context of the

NRE alone and its activity compared with that of constructs

containing either the intact NRE (OvCAT-.308N) or no NRE

(OvCAT-.087C). The activity of the CAR-site mutant NRE was

higher than that of the entire intact NRE but did not reach the

level achieved when no NRE was present (see Fig. 6). The most

straightforward explanation, as proposed previously (Ehlen

Haecker et al., 1995), is that other repressor sites exist. Muta-

genesis of the silencer in this same context did not additionally

alter the promoter activity. Therefore, either the silencer is not

active in the Ov gene or, as we believe, its activity is being

masked by the presence of still another negative element. The

O site ( 2 279 to 2 270) is a candidate for this other negative el-

ement, as its mutation in the OvCAT-.239LSO construct con-

sistently produced an increase in activity (see Fig. 7). However,

caution must be used in interpreting these results because the

O site is moved adjacent to the minimal promoter in those con-

structs, which dramatically changes its context.

These studies demonstrate that the Ov NRE is considerably

more complex than originally thought. Earlier data indicated

that it contains four independent negative elements (Ehlen

Haecker et al., 1995) and a positive element that binds the es-

trogen-inducible factor d -EF1 (E.M. Chamberlain and M.M.

Sanders, manuscript submitted). The LS mutational analysis

herein limited the most distal negative element to the region

between 2 279 and 2 270 and revealed two previously unsus-

pected regulatory elements, one positive (2 204 to 2 175) and

one negative (2 119 to 2 111). The functional roles of these el-

ements await additional characterization. However, the proxi-

mal negative element, designated the CAR element, appears to

be a novel DNA sequence that binds a repressor protein or pro-

tein complex involved in the developmental regulation of the

Ov gene. Clearly, determining the mechanisms by which the

NRE orchestrates the appropriate level of Ov gene transcrip-

tion will be a challenge.

ACKNOWLEDGMENTS

This work was supported by NIH grants R01DK40082 and

R01DK49698 to MMS. K.R.S. was supported in part by NIH

grant T32GM07323. The statistical analysis by Dr. Diane Dean

is gratefully acknowledged.

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Address reprint requests to:

Dr. Michel M. Sanders

Department of Biochemistry, Molecular Biology,

and Biophysics

University of Minnesota4± 225 Millard Hall

435 Delaware St. SE

Minneapolis, MN 55455

Received for publication June 22, 1998; accepted in revised

form October 13, 1998.

SENSENBAUGH AND SANDERS156