Proc. Natl. Acad. Sci. USAVol. 86, pp. 104-108, January 1989Biochemistry

Prolactin and glucocorticoid hormones synergistically induceexpression of transfected rat 13-casein gene promoterconstructs in a mammary epithelial cell line

(growth hormone/insulin/inducible enhancer/terminal differentiation/milk protein gene regulation)

WOLFGANG DOPPLER*, BERND GRONER*, AND ROLAND K. BALL*Ludwig Institute for Cancer Research, Inselspital, 3010 Bern, Switzerland

Communicated by Elwood V. Jensen, October 3, 1988

ABSTRACT We have detected hormone response elementsin the promoter region of the rat f3-casein gene that confer thesynergistic action of prolactin and glucocorticoid hormonesupon transcription of chimeric gene constructs. A 2800-base-pair (bp) rat (3-casein gene fragment containing 2300 bpof 5' flanking sequence was placed in front of a chloramphen-icol acetyltransferase (CAT) reporter gene and stably trans-fected into the mouse mammary epithelial cell line HC11.Addition of prolactin or dexamethasone alone was sufficient fora modest induction of the fusion gene. The simultaneouspresence of both hormones produced a strongly synergisticeffect, which did not require the presence of insulin. Inductionof the fi-casein-CAT gene was only observed in stably trans-fected confluent cell cultures. Analysis of a 5' deletion series ofthe j3-casein-CAT gene construct revealed a stepwise loss ofhormone inducibility; 285 bp of 5' flanking sequence wassufficient to mediate the synergistic action of lactogenic hor-mones on expression. The response was reduced by half whencompared with the construct containing 2300 bp of the 5'flanking region. Synergistic inducibility further decreased indeletion mutants between -285 and -265 and was completelyabolished between -180 and -170. Thus, the 5' flankingregion between -285 and -170 contains cis-acting sequences,which are required for mediating the effect of prolactin anddexamethasone.

Recently, we described the isolation of the cloned mam-mary epithelial cell line HC11 (10). It was derived from theBALB/c mouse mammary epithelial cell line COMMA-1D(11) and is unique in maintaining the capability to produce themajor mouse milk protein f3-casein under the control oflactogenic hormones. HC11 cells do not require cultivationon exogenous extracellular matrix or cocultivation withfibroblasts or adipocytes for efficient induction of /-caseinprotein synthesis. Prolactin increases the transcription rate ofthe endogenous P-casein gene (10).We tested the potential of HC11 as a recipient cell line for

the analysis ofthe lactogenic hormone control of milk proteingene expression by gene transfer methods. The 5' flankingsequences of the rat ,B-casein gene were recombined with thechloramphenicol acetyltransferase (CAT) reporter gene andtransfected into HC11 cells. A strong induction of CATexpression by lactogenic hormones was observed in stablytransfected HC11 cells. The induction was dependent onprolactin, was enhanced by dexamethasone, and requiredsequences located upstream of nucleotide -170 of the ,B-casein gene. Thus, the milk protein ,3-casein is regulated atthe level of transcription and requires the synergistic actionof two different classes of hormones. This study provides abasis for a molecular description of the cis- and trans-actingelements used in control of the transcription of milk proteingenes by lactogenic hormones.

The lactogenic hormones prolactin, hydrocortisone, andinsulin have been shown to regulate milk protein expression(1). Induction of efficient milk protein synthesis by thesehormones is restricted to the terminally differentiated mam-mary epithelium. Complex cell-cell and cell-hormone inter-actions are required for terminal differentiation (1, 2).

Important insights into the mechanisms by which hor-mones and other agents control the expression of genes havebeen gained by the introduction of transfected genes andpromoter-gene constructs into cultured cells (3, 4). Suchstudies have led to the definition of a number of DNAsequences or response elements mediating the effect of theseinducers. Most of these response elements, such as those forinterferons, cAMP, serum growth factors, heavy metals, heatshock, or steroids (3, 5-7), reside in the 5' flanking region ofthe induced genes and have the properties of inducibleenhancers. The strategy to introduce milk protein geneconstructs into cultured mammary epithelial cells and mon-itor hormonal effects on their expression, to date, has beenunsuccessful. Cell lines isolated from the mammary glandeither lose their capability to produce milk proteins whenkept in culture or require growth on extracellular matrixcomponents (8, 9).

MATERIALS AND METHODS

Construction of CAT Plasmids. A 2.8-kilobase (kb) Eco-RI/EcoRI fragment of the genomic rat /8-casein gene waskindly provided by J. M. Rosen (12). It consists of 2.3 kb of5' flanking sequence, the first noncoding exon, and 0.45 kb ofthe first intron. The EcoRI sites were converted into BamHIsites by filling in the 3' recessed ends with the Klenowfragment of Escherichia coli DNA polymerase I and addingBamHI linkers (13). The fragment was cloned into the BamHIsite of pBLCAT3 (14) in the same orientation as CAT tocreate pfpc(-2300/+487)CAT (see Fig. 1). The numbers inparentheses indicate the 5' and 3' borders of 83-casein genesequences, according to the numbering given in ref. 12.The 5' deletions were introduced in the sequencing vector

pBluescript KS+ (Stratagene, LaJolla, CA). A 4.4-kb XbaI/Kpn I fragment containing the 2.8-kb rat ,8-casein fragment,CAT, and simian virus 40 sequences was excised frompI8c(-2300/+487)CAT and reinserted into the Xba I/Kpn Isites of the Bluescript polylinker to create pbs/3c(-2300/+487)CAT. Using the exonuclease III/mung bean nucleasetechnique (Stratagene; ref. 15) progressive 5' deletions werecreated starting from the HindIll site at -332 of the rat

Abbreviation: CAT, chloramphenicol acetyltransferase.*Present address: Friedrich Miescher Institut, P.O. Box 2543, 4002Basel, Switzerland.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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/3-casein. Bluescript sequences were protected by cleavingthe Sac I site. Deletion end points were determined bydouble-strand DNA sequencing (16).

Cell Culture and Hormones. Cells were grown in RPMI1640 medium with 10% heat-inactivated fetal calf serum, 5 Agof insulin per ml, 10 ng ofepidermal growth factor per ml, and50 Ag of gentamycin per ml (growth medium) (10). Themedium was changed every 2 days. Epidermal growth factorwas always omitted during lactogenic hormone induction.Ovine prolactin, bovine insulin, and dexamethasone wereobtained from Sigma; human growth hormone (iodinationgrade) was from UCB-Bioproducts (Braine-L'Alleud, Bel-gium).

Transfections. Cells were transfected by the calcium phos-phate precipitation technique (17). Then, 1-2 x 105 cells wereplated on a 9-cm culture dish. One day later, culture mediumwas changed to Dulbecco's modified Eagle's medium with10%o fetal calf serum and cells were cotransfected with 10 ,gof plasmid DNA and 1 Ag of pSV2-neo (18). Medium waschanged to growth medium (see above) 1 day later andreplaced the following day by the same medium containing200 ,ug of G418 per ml. Usually 100-1000 G418-resistantcolonies were obtained per 9-cm dish. The cells were pooledafter treatment with trypsin and expanded to 107 cells inG418-containing growth medium. The expanded cell poolwas used for the hormone induction experiments.RNase Protection Analysis. Total cytoplasmic RNA was

isolated (19) and subjected to RNase protection analysis (10).SP6-transcribed probes were generated from the Pvu II-linearized plasmids pSP65-/3c-HS and pSP65-pc/CAT-SP.These plasmids were created by inserting the 0.7-kbHindIII/Spe I and the 0.3-kb Spe I/Pvu II fragment frompI8c(-2300/+487)CAT into HindIII/Xba I or Xba I/Sma Idouble-digested pSP651, respectively (20).CAT Assays. Cells were harvested by treatment with

trypsin, washed twice with phosphate-buffered saline, andsuspended in 0.25 M Tris HCl (pH 7.8). Extracts wereprepared by four cycles of freeze-thawing. The lysed cellswere centrifuged at 10,000 x g for 5 min and the supernatantwas heated for 10 min at 60°C. Denatured protein waspelleted at 10,000 x g for 5 min and aliquots of the super-natant were taken for protein determinations (21) and assayof CAT enzyme activity, essentially as described (22).[14C]Chloramphenicol (20 ,uM) (Amersham) was used in theincubation mixture and the reaction products were resolvedby thin-layer chromatography. Conversion was determinedby cutting out the radioactive spots of nonacylated andacylated forms of chloramphenicol and measuring radioac-tivity by liquid scintillation counting.

RESULTSTranscription ofa Chimeric f-Casein-CAT Gene Is Induced

by Lactogenic Hormones in HCll Cells. To examine whetherDNA sequences responsible for the lactogenic hormonecontrol of P-casein expression are located in or around thepromoter region, we recombined a 2.8-kb restriction frag-ment from the 5' end of the rat ,B-casein gene with thebacterial CAT gene (Fig. lA; see Materials and Methods).This construct ppc(-2300/+487)CAT was cotransfectedwith the neomycin-resistance gene contained in pSV2-neointo the mammary epithelial cell line HC11.Two antisense RNA probes were made in vitro from

fragments cloned into the pSP65 vector. The probe HS (Fig.LA) spans the first exon of the f3-casein gene. If thepf3c(-2300/+487)CAT construct is initiated and spliced asthe rat 13-casein gene is (12), a fragment comprising the firstexon should be protected from RNase digestion (Fig. 1A).Two signals of43 and 46 nucleotides were found in protectionexperiments in which RNA from stably transfected HC11

A. rat B-casein (-2300/+487) . CAT -..

HindWu Spei Pvu T,

±zxIzI/2' /

"I-1 SP6 Probe HS

'--,__P___,SP6 ProbeSP

mRNA_

Protected Probe

B Probe HS

C 12 3 4

-67

,;,'46,,..,_ 43

43nt46nt

187nt

Probe SPCM N4

-309

-217-201

-180

- r-160

t 147

j*/-13FIG. 1. Structure and hormonal control of transcripts of

pfSc(-2300/+487)CAT stably transfected into HC11. (A) Probesused in RNase protection experiments. Initiation and splice donorsites were mapped by the SP6 probe HS. The HS probe of 916nucleotides (nt) contains 707 nt derived from the rat /3-casein gene(HindIII/Spe I). The probe SP contains the 298 nt of p,8c(-2300/+487)CAT between the Hindil site in the /-casein sequence and thePvu II site in the CAT sequence. (B) RNase protection analysis.HC11 cells were kept for 2 days in growth medium after reachingconfluence and then incubated with hormones for 2 days. Totalcytoplasmic RNA was isolated and 20 ,ug (probe SP, lane C) or 4 ,ug(remaining lanes) was used for RNase protection analysis. Protectedfragments ofprobe HS were analyzed on a 10% polyacrylamide/8 Murea gel and protected fragments of probe SP were analyzed on a 6%polyacrylamide/8 M urea gel. Fragments of pBR322 digested withHpa II and end-labeled with 32p were used as size markers (lane M).Size markers (in nt) are shown on the right. Arrows indicate theposition and size of protected fragments. RNA from transfectedHC11 cells treated with the following combinations of hormones wasanalyzed: lane N, no hormones; lane 1, insulin; lane 2, prolactin andinsulin; lane 3, dexamethasone and insulin; lane 4, prolactin, dex-amethasone, and insulin. In lane C, RNA from untransfected HC11cells, induced with prolactin, dexamethasone, and insulin wasanalyzed. Concentrations were as follows: prolactin, 5 ,ug/ml;dexamethasone, 1 ,AM; insulin, 5 ,ug/ml.

cells treated with lactogenic hormones was used (Fig. 1B).This corresponds to the previously described exon bound-aries of the ,B-casein gene (12). A second probe, SP (Fig. 1A),examined the splicing of the transcript from pf8c(-2300/+487)CAT in induced HC11 cells. This probe spans thejunction between the first intron of the ,B-casein gene and theCAT gene. A fragment of 187 nucleotides was protected,indicating the use of a cryptic splice acceptor site at position+476 of pfc(-2300/+487)CAT. The nucleotide sequence5'-TAAAAClTTllCTAG-3' precedes position +476. Thissequence is similar to the rodent splice acceptor consensussequence (23).

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A comparison of the intensity of the signals shown in Fig.1B indicates that the abundance of RNA transcripts fromp,8c(-2300/+487)CAT is hormonally controlled. Cellstreated with prolactin, dexamethasone, and insulin (Fig. 1B,lane 4) show increased levels of RNA detectable with bothprobes. Less p,3c(-2300/+487)CAT RNA was present incells treated with insulin (lane 1), prolactin and insulin (lane2), or dexamethasone and insulin (lane 3). A basal level oftranscription was detectable in cells cultured in the absenceof hormone (lane N). No signal was found in untransfectedcells (lanes C).

Synergistic Action of Lactogenic Hormones on p.ic(-2300/+487)CAT Expression. The introduction of pf3c(-2300/+487)CAT into HC11 cells results in accurate RNA initiationand the hormonal induction of RNA levels (Fig. 1B). Theactivity of the bacterial CAT gene linked to the /3-caseinpromoter sequence was used to quantitate the effects ofprolactin, dexamethasone, and insulin on the transcriptionalactivity of this promoter (Table 1). Confluent cultures ofppc(-2300/+487)CAT-transfected HC11 cells were inducedwith various combinations of hormones for 2 days and theCAT activity was determined in protein extracts. A basallevel ofCAT expression was detected in cells in the absenceof hormones, confirming the results in Fig. 1B. Insulin byitself had no effect on the basal activity, whereas dexameth-asone or prolactin caused a 4-fold induction. Insulin en-hanced the response to prolactin 2-fold but had no effect onthe dexamethasone inducibility. A strong synergism wasobserved when prolactin and dexamethasone were addedsimultaneously. This effect did not require the presence ofinsulin. The activity was increased 37-fold above the basalactivity (Table 1). Thus, signals generated by prolactin and byglucocorticoid hormones are required to cause a maximalinduction of the 13-casein gene promoter. A significant induc-tion of CAT activity was already seen 3 hr after addition ofdexamethasone together with prolactin. It continued toincrease over 6 days (data not shown). This is similar to thestimulation of the transcription of the endogenous /8-caseingene by prolactin (10).The CAT activity in p.Bc(-2300/+487)CAT-transfected

HC11 cells was measured as a function of the concentrationof prolactin, growth hormone, and dexamethasone in themedium (Fig. 2). In the presence of 10-6 M dexamethasone,a half-maximal response was obtained with 3 nM (68 ng/ml)ovine prolactin or 1 nM (22 ng/ml) human growth hormone.Both hormones have been shown to bind with similaraffinities to the prolactin receptor (24). Cells kept in 220 nM(5 ,ug/ml) ovine prolactin showed a half-maximal response to

Table 1. Effect of lactogenic hormones on expression ofp/3c(-2300/+487)CAT transfected into HC11

Hormone added CAT activity,*nmol per min Induction

Pri Dex Ins per mg of protein ratio, -fold- - - 1.02 0.57 1- - + 1.31 + 0.44 1.3- + - 4.16 ± 0.55 4.1- + + 3.56 ± 0.74 3.5+ - - 4.12 ± 0.95 4.1+ - + 8.39 ± 1.98 8.1+ + - 38.21 ± 3.04 37.5+ + + 32.11 ± 3.42 31.5HC11 cells derived from a pool of stably transfected cells were

kept for 2 days in growth medium after reaching confluence and thenincubated with hormones for 2 days. Concentrations are as follows:prolactin (Prl), 5 ,g/ml; dexamethasone (Dex), 1 uM; insulin (Ins),5 ,ug/mI.*Means ± SEM of three experiments with two pools of cells derivedfrom individual transfections.

100 *

T/~~~o111098 7 6(0~~~~~~~

E50-EX

E 25-

0 11 10 9 8 7 6

- log [hormone, M]

FIG. 2. Concentration requirements for ovine prolactin, humangrowth hormone, and dexamethasone for expression of p3c(-2300/+487)CAT in HC11 cells. Cells pretreated as described in Fig. 1Bwere incubated with hormones for 2 days. Dependence of CATexpression on ovine prolactin (o) or human growth hormone (e) wasdetermined in the presence of 1 gM dexamethasone and 5 ,ug ofinsulin per ml. Dependence of CAT expression on dexamethasone(n) was analyzed in the presence of 5 ,ug of ovine prolactin and 5 ,tgof insulin per ml. Bars indicate SEM of three experiments with twopools of cells from individual transfections.

dexamethasone at 8 nM, a concentration also determined forthe glucocorticoid receptor-mediated effects of dexameth-asone (25).The (3-Casein Gene Promoter Is Only Inducible in Confluent

Cell Cultures by Prolactin and Glucocorticoid Hormones. Theinduction of the casein-CAT construct by lactogenic hor-mones was found to be dependent on the density of the cellculture as shown in Fig. 3. Treatment of exponentiallygrowing transfectants with prolactin, dexamethasone, andinsulin for 2 days did not enhance expression ofCAT proteinwhen compared to cells treated with dexamethasone andinsulin for the same time period. Hormone inducibility wasonly detected in cells that were confluent for at least 1 dayduring the 2 days of hormone treatment. The response was

c166

._.0

8-

0 .4-

C 0-

T IT * -/---@0go 1¶Ij~~~~~~~~~~~~~~~~

/~~~/

0 1 2 3 4 8days after confluency

FIG. 3. Dependence of the lactogenic hormone response on cellculture conditions. Confluent pools of HC11 cells transfected withpBc(-2300/+487)CAT were split 1:4 and hormones were addedeither immediately or after different intervals of culture in growthmedium. Two days after addition of hormones, cells were harvestedand CAT activity was determined. The time indicates the number ofdays after the cell cultures have reached confluence. e, Prolactin,dexamethasone, and insulin; o, dexamethasone and insulin. Con-centrations are the same as in Fig. 1B. Bars indicate SEM ofexperiments from pools of cells derived from individual transfec-tions.

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further increased in cells that had reached confluence beforethey received hormones. Maximal CAT expression after a2-day stimulation with hormones was seen in cell culturesthat had reached confluence 2 days before hormone addition.After cultures of HC11 cells became confluent, the culturedensity increased -2-fold before a maximal density wasreached.

Analysis of Rat (8-Casein Regulatory Sequences Required toInduce CAT Expression by Prolactin and Dexamethasone. Theconferral of lactogenic hormone inducibility on the bacterialCAT gene was obtained by a 2.8-kb DNA fragment of the rat13-casein gene. To further delimit the sequence requirementsfor this response, the 2.8-kb ,B-casein fragment together withCAT and simian virus 40 sequences were inserted into thesequencing vector pBluescript KS+ to create pbs/3c(-2300/+487)CAT. The rat sequence was progressively deleted fromthe 5' end of this construct. Fig. 4 shows the ratios of CATactivities when constructs with decreasing lengths of 5'flanking sequence were induced with prolactin, dexametha-sone and insulin, or insulin alone in transfected HC11 cells.A maximal response (22-fold) was obtained with the con-struct containing 2.3 kb of 5' flanking sequence. Removal of=2 kb resulted in pbslc(-330/+487)CAT, retaining 330nucleotides 5' of the RNA initiation site. The inducibility ofthis construct was reduced to 10-fold. A further decrease ininducibility to -3-fold was observed in deletions retaining265-180 nucleotides of 5' flanking sequence, and the induc-ibility was entirely lost in deletion mutants containing 170nucleotides or less of 5' flanking sequence.The CAT activities of the parental construct and three of

the tested 5' deletion mutants in the presence of prolactin,dexamethasone, or both hormones are shown in Table 2. Thesynergistic response of pbs,8c(-2300/+487)CAT was lowerthan the 32-fold induction in p/c(-2300/+487)CAT (Table1), which differs only in the vector sequence. This mightreflect the effect of the vector on basal transcription levels.The inducibility by prolactin alone was reduced in pbsf3c-(-330/+487)CAT, while a 10.8-fold synergistic inducibilityremained. No significant inducibility by dexamethasonealone was observed with this construct. pbs(3c(-221/+487)-CAT showed a further decreased response to prolactin aloneand only a 2.1-fold synergistic induction. pbs,8c(-44/+487)-CAT was no longer inducible.These experiments indicate that multiple sequence ele-

ments are required for the lactogenic hormone response.

24-

20-

0°16-

h._0ff 12-c0* 8-

i 4-

I,,-2300

I

iji!lt | 1II---------t---

-300

Table 2. Induction of CAT expression by prolactin anddexamethasone in HC11 cells transfected with 5'deletions of pbspc(-2300/+487)CAT

Induction ratio (-fold) of CAT activity afterhormone addition

5' border Prolactin andof ,-casein Prolactin Dexamethasone dexamethasone

-2300 4.0 ± 0.7 2.6 ± 0.3 22.4 ± 3.9-330 2.9 ± 0.4 1.2 ± 0.2 10.8 ± 1.9-221 1.9 ± 0.2 0.72 ± 0.09 2.1 ± 0.4-44 0.87 ± 0.02 0.98 ± 0.03 0.95 ± 0.09

Pools of transfected HC11 cells were precultured and incubatedwith hormones as described in Table 1. Induced cells received insulinplus the indicated hormones. The induction ratio of CAT activity isrelative to the activity in the presence of insulin alone. Results areexpressed as means ± SEM from two to four individual transfectionexperiments. Mean CAT activities ofuninduced cells varied between0.83 and 1.21 nmol per min per mg of protein in these experiments.

Sequences between -180 and -265 confer a modest re-sponse. The response is sharply enhanced by sequencesbetween -265 and -285. Sequences between -330 and-2300 confer the inducibility by dexamethasone alone.

DISCUSSIONWe have shown that it is now possible to use gene transfermethods to study the lactogenic hormone control of milkprotein gene expression. This was dependent on the isolationof the hormone-responsive mouse mammary epithelial cellline HC11. The 5' flanking sequences mediate the synergisticinduction by dexamethasone and prolactin of the rat 8-caseinpromoter in HC11 cells, which have terminally differentiatedunder defined culture conditions.The dependence of the lactogenic hormone inducibility of

HC11 cells on culture conditions is reminiscent of the morecomplex requirements that primary mammary epithelial cellsdisplay for maintenance of their differentiated functions (8,9). These cells require a suitable substratum or the coculti-vation with adipocytes (2) to produce milk proteins inresponse to lactogenic hormones. In HC11 cells, the forma-tion of a confluent cell layer is the prerequisite to induceresponsiveness to lactogenic hormones. Confluent epithelialcells stop proliferating, deposit extracellular matrix, increasecell-cell contact, and polarize. All these events might con-

-250 -200 -150 -100position of 51 border of B-casein

-50 +1

FIG. 4. Synergistic inducibility of CAT expression by prolactin and dexamethasone in a 5' deletion series of pbsl3c(-2300/+487). PoolsofHC11 cells transfected with various 5' deletions ofthe rat 3-casein fragment (-2300/+487) in a Bluescript CAT expression vector were treatedwith hormone* as described in Fig. 1B. The induction ratios ofCAT activity in cells treated with prolactin, dexamethasone, and insulin comparedto the activities in cells that received insulin only are shown by bars (see Materials and Methods). CAT activities of uninduced cells variedbetween 0.46 and 2.36 nmol per min per mg of protein in the individual transfected cell pools.

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tribute to the establishment of the terminally differentiatedstate of HC11 cells.

Regulatory sequences that confer the synergistic action ofprolactin and glucocorticoid hormone upon the ,3-casein genepromoter are located at least within the first 2300 nucleotides5' of the RNA initiation site (Fig. 4). The region between-511 and +487 of the f3-casein gene promoter has recentlybeen shown to confer tissue-specific expression in transgenicmice (J. M. Rosen, personal communication). Thus, thesequences mediating tissue-specific expression and hormoneinduction are contained within the same region. The stepwisereduction of inducibility shown in Fig. 4 points to theexistence of discrete functional elements in this region.Various reports have documented the cooperative interac-tion between repeated enhancer elements or synergismbetween different neighboring enhancer and transcriptionfactor binding elements (3, 26, 27). We and others havescreened the functionally relevant DNA region for notewor-thy features. Hall et al. (28) and Yu-Lee et al. (29) haveidentified conserved sequences between -160 and -110 inseveral milk protein gene promoters. In the rat P-casein gene,this region harbors two inverted repeats: 5'-(-145)TTTCIT-GGGAAAGiAAAATAGAAAOAAACCATTTTCTA(-113)-3'. The spacing ofthe hexameric units ofthese motives is 10-11 nucleotides; thus, the repeated sequences are presented onthe same side of the DNA helix. Such structures could beimportant for the coordinate action of transcription factors.The sequence between -160 and -110 was not sufficient tomediate the lactogenic hormone response (Fig. 4) but mightbe necessary for the function of the hormone responseelements located 5' from that region. This is further sup-ported by our observation that the f3-casein sequencesbetween -167 and -2300 did not confer inducibility to thethymidine kinase promoter ofthe herpes simplex virus (W.D.and R.K.B., unpublished data).

Detailed information is available on the DNA sequencesthat interact with the glucocorticoid receptor complex (30). Aconsensus receptor binding sequence TGTTCT is found at-510 in the rat 13-casein gene. As shown in Table 2, deletionof the region containing this sequence abolished the responseto glucocorticoid alone. The region between -330 and +487does not contain a TGTTCT motif, but it is sufficient tomediate the effect of glucocorticoid in the synergistic re-sponse. Here, the effect of glucocorticoids could be indirectand dependent on the synthesis or repression of proteins. Westill cannot exclude a role for the small untranslated first exonof the rat /3-casein gene. However, 3' deletions up tonucleotide +46 of the 2.8-kb rat /3-casein fragment showedunimpaired hormone inducibility (data not shown), whichindicates that the fragment of the first intron is not involvedin mediating the effect of the hormones. In vitro mutagenesisand recombination of DNA elements, the use of specificinhibitors, and studies of DNA-protein interactions can beused to further elucidate the mechanism of lactogenic hor-mone regulation of milk protein gene expression.We are grateful to Dr. J. M. Rosen for critical reading of the

manuscript and providing us with the rat f3-casein fragment. Theauthors wish to thank A. Schlafli and H. Birk for excellent technicalassistance and C. Wiedmer and T. Diabat6 for typing the manuscript.

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