May 12, 1992 - 1980), extracellular matrix (ECM) (Bissell and Hall,. 1987), and growth factors (Taketani and Oka, 1983). Milk proteins appear during pregnancy ...
Molecular Biology of the Cell Vol. 3, 699-709, June 1992
A Novel Transcriptional Enhancer is Involved in the Prolactin- and Extracellular Matrix-Dependent Regulation of fl-Casein Gene Expression Christian Schmidhauser,* Gerald F. Casperson,t Connie A. Myers,* Kimberly T. Sanzo,t Suzanne Bolten,t and Mina J. Bissell* *Cell and Molecular Biology Division, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720; and tMonsanto Corporate Research AA3C, Chesterfield Village Parkway, Chesterfield, Missouri 63198 Submitted February 20, 1992; Accepted May 12, 1992
Lactogenic hormones and extracellular matrix (ECM) act synergistically to regulate f-casein expression in culture. We have developed a functional subpopulation of the mouse mammary epithelial cell strain COMMA-1D (designated CID 9), which expresses high level of 3-casein, forms alveolar-like structures when plated onto the EHS tumor-derived matrix, and secretes f-casein unidirectionally into a lumen. We have further shown that ECMand prolactin-dependent regulations of f-casein occur mainly at the transcriptional level and that 5' sequences play an important role in these regulations. To address the question of the nature of the DNA sequence requirements for such regulation, we analyzed the bovine ,B-casein gene promoter in these cells. We now have located a 160-bp transcriptional enhancer (BCE1) within the 5' flanking region of the 3-casein gene. Using functional assays, we show that BCE1 contains responsive elements for prolactin- and ECM-dependent regulation. BCE1 placed upstream of a truncated and inactive f-casein promoter (the shortest extending from -89 to +42 bp with regard to the transcription start site) reconstitutes a promoter even more potent than the intact promoter, which contains BCE1 in its normal context more than 1.5 kb upstream. This small fusion promoter also reconstitutes the normal pattern of regulation, including a requirement for both prolactin and ECM and a synergistic action of prolactin and hydrocortisone. By replacing the milk promoter with a heterologous viral promoter, we show that BCE1 participates in the prolactin- and ECMmediated regulation. INTRODUCTION The mechanisms governing the development and differentiation of the mammary gland are complex and are guided by several hormones (Topper and Freeman, 1980), extracellular matrix (ECM) (Bissell and Hall, 1987), and growth factors (Taketani and Oka, 1983). Milk proteins appear during pregnancy and are highly abundant markers for the differentiated state. A number of tissue culture models have been developed to analyze the regulation of the milk proteins. These includes organ cultures (Guyette et al., 1979), cells cultured on plastic substratum, attached type I collagen and floating collagen (Emerman et al., 1977), cells on an acellular matrix derived from the mammary gland (Wicha et al., 1982), cells in or on top of a reconstituted basement membrane C) 1992 by The American Society for Cell Biology
(EHS) (Blum et al., 1987; Li et al., 1987; Barcellos-Hoff et al., 1989), and single cells embedded in gelatinous substrata (Streuli et al., 199 lb). These models allow manipulation of regulatory events in the absence of the more complex environment of the gland. The expression of specific genes can be perturbed selectively by altering microenvironmental variables such as ECM and hormones. The importance of the interaction of cells with ECM for gene expression has been demonstrated for several systems. Cultured mouse mammary epithelial cells, for example, differentiate only when in contact with ECM, either provided exogenously (Li et al., 1987) or in systems, which allow the cells to lay down their own basement membrane (Streuli and Bissell, 1990). The ap699
C. Schmidhauser et al.
pearance of a basement membrane coincides with the expression of milk proteins during alveolar development in the pregnant animal. During involution, exogenous tissue inhibitor of metalloproteinases (TIMP) delays the regression of alveoli and prolongs the lactating status of the mammary epithelia (Talhouk et al., 1992). Two essential environmental cues positively regulate the transcription of fl-casein in culture: ECM and prolactin. Other hormones such as hydrocortisone synergistically increase the expression of f-casein and allow maximal transcription in HC 11 cells (Ball et al., 1988; Doppler et al., 1989, 1990). The results from the CID 9 cells show transcriptional activity in the absence of hydrocortisone (Schmidhauser et al., 1990). There is increasing evidence that ECM regulates specific genes through ECM-dependent expression of transcription factors, which allow tissue specific transcription of the target gene. An ECM-responsive element located in the enhancer of the albumin gene binds liver specific factors, which are more abundant when the cells are maintained in the presence of an ECM (Di Persio et al., 1991; Liu et
al., 1991).
We have shown previously that important regulatory elements are located more than 800 bp upstream in the bovine f,-casein promoter (Schmidhauser et al., 1990). In this study we address the question of DNA sequence requirements for the ECM- and prolactin-dependent regulation of ,3-casein transcription. We show that a unique 160-bp enhancer element lies 1.5 kb upstream of the bovine fl-casein transcription initiation site. We provide evidence that a small proximal portion of the bovine f3-casein promoter in conjunction with this transcriptional enhancer reconstitutes the ECM- and prolactin-dependent regulation in a cell type-specific manner. We further show that the enhancer can function from a heterologous promoter and that it remains ECMand prolactin-responsive.
MATERIALS AND METHODS Cell culture and differentiation CID 9 cells (Schmidhauser et al., 1990) and their transfected derivatives proliferate in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (1:1; GIBCO, Grand Island, NY) containing 5% heat-inactivated fetal calf serum (FCS), insulin (5 ,g/ml; Sigma, St. Louis, MO), and gentamicin (50 ,ug/ml; Sigma). EHS matrix was prepared from EHS tumors (Kleinman et al., 1986) passaged in C57BL mice. Two hundred microliters EHS was applied per 35-mm prechilled culture dish and gelled at 37°C for 30 min. For hormone-dependent differentiation, cells were plated in the presence of 2% FCS (to support attachment to plastic dishes) and insulin at a concentration of 6.2 X 104 cells/cm: to the relevant substratum. Hydrocortisone (1 yg/ml; Sigma) and/or prolactin (3 Ag/ml ovine prolactin; National Institutes of Health, Bethesda, MD) was added as indicated. Twelve hours after plating, the cells were washed twice and cultivated for additional 5 d in serum-free medium containing the relevant hormone combination. The medium was changed every other day. were allowed to
Plasmids Casein constructs. The construction of b(cas-1790+42/CAT (chlor-
amphenicol acetyl transferase) and b,Bcas-791+42/CAT are described 700
elsewhere (Schmidhauser et al., 1990). Deletions from the 5' end of the b,lcas promoter in each of the plasmids were made by exonuclease III digestion with the Erase-a-base kit (ProMega, Madison, WI) and done according to the protocol of the manufacturer. The exact 5' border was determined by sequencing. ER-1 and ER-2 were obtained by deletion of b,3cas DNA upstream of the EcoRI site, located at position -121 and -89, respectively. BCE1 was synthesized by polymerase chain reaction (PCR, Cetus, Norwalk, CT) with bflcas-1790+42/CAT as a template and its correct synthesis confirmed by sequencing. MMTV constructs. A mouse mammary tumor virus (MMTV) promoter fragment (extending from -114 to +82bp relative to the transcription start site) was synthesized by PCR. The PCR primers served to add a Sal I site to the 5' end and a BamHI site to the 3' end of the fragment. The fragment was cloned into the Sal I/BamHI digested pMON3605, the SV40 polyadenylation signal/transcription terminator in pUC18 (Highkin et al., 1991), such that a unique Sal I site lay upstream of the promoter with a unique BamHI site between promoter and terminator. CAT, excised from pCM-4 (Pharmacia, Piscataway, NJ) as a BamHI fragment, was cloned into the BamHI site. BCE1, synthesized as a Sal I fragment by PCR, was inserted into the Sal I site upstream of the MMTV promoter. This fragment of the MMTV promoter has been reported to display only basal transcription with no glucocorticoid inducibility when transfected into L cells (Majors and Varmus, 1983). SV40 constructs. The Sal I BCE1 fragment described above was cloned into the Sal I site of p1229 (the 205 bp SphI to HindIII fragment of the SV40 early promoter fused to CAT and the SV40 terminator in pML2 (a generous gift of J. Majors, Washington University, MO). The SV40 early promoter fragment in p1229 contains the transcription start site and 70 bp of the 5' untranslated sequences, and the three 21-bp repeats (SP1 binding sites) but not the SV40 enhancers. -
Transfection and CAT assays To avoid passage-dependent variation of function, all transfections utilized CID 9 cells in passage 12. One day before the transfection the cells were plated at a density of 2.4 X 104/cm2. A calcium phosphate precipitation (Gorman, 1986) was carried out in 100-mm dishes with 30 gg of plasmid DNA and 3 jig of SV2 neo as a selectable marker. After 4 h incubation the cells were glycerol shocked (25% glycerol, in 25 mM HEPES, 140 mM NaCL) for 90 s, washed 3X with medium, and incubated in proliferation medium (5% FCS plus insulin). Selection was started with G418 (Gibco; 400 ,g/ml) 36 h after transfection. Colonies (200-500) were pooled and expanded for stocks and differentiation assays. The transfections were done in duplicates (except for the data presented in Figure 2). The total magnitude of CAT expression between the two transfections sets varied, up to 19% in some experiments, whereas the regulation patterns remained constant. For CAT assays, the selected cells were differentiated for 6 d on the appropriate substrate in the presence of the hormones indicated. Cells were harvested from either EHS or plastic using 1 ml of dispase (Collaborative Research, Bedford, MA) per 35 mm dish for 40 min at 37°C. The cell suspension was washed 3 times with tris-buffered saline [TBS, 25 mM tris(hydroxymethyl)aminomethane (Tris) 8, 40 mM KCl, 3.5 mM NaCl], lysed with 0.5% NP40, further solubilized by two freezing-thawing cycles, and heated to 60°C for 10 min to destroy endogenous CAT-like activities. To normalize the lysates, cell proteins were quantified with a micro Bradford assay (Bio-Rad, Richmond, CA). To confirm that the extract had been normalized correctly and that the EHS excess had been removed successfully, 3 gg of the cell proteins were separated on a reducing SDS polyacrylamide gel and stained with silver. To measure CAT enzyme activity, 10 zg of cell lysate was used either in a thin-layer chromatography (TLC) assay (Sambook et al., 1989) or in a two-phase diffusion assay as described by Neumann et al. (1987). For TLC assays extracts were incubated with 10 MACi 14Cchloramphenicol (Sigma, 55 mCi/mmol diluted to 0.01 uCi/Il) and acetyl coenzyme A (70 ,ug) for 6 h at 37°C, extracted with ethyl acetate and separated by TLC. The levels of acetylated chloramphenicol were quantified by scrapping the radioactive spots from the TLC plates and Molecular Biology of the Cell
ECM- and Prolactin-Dependent Enhancer for 3-Casein
by counting them in a scintillation counter. CAT activity was calculated as substrate conversion per minute per micrograms of lysate.
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Northern blot analysis Total RNA was isolated as described by Chomczynski and Sacchi, 1987. [32P]-Labeled random primed probe (Feinberg and Vogelstein, 1984) was prepared with gel-purified insert sequence from a plasmid containing 540 bp of 0-casein cDNA kindly provided by J. M. Rosen (Baylor College, Houston, TX).
RESULTS Localization of a transcriptional enhancer in the 5' flanking region of the fi-casein gene CID 9 cells express milk proteins under the tight control of ECM and the lactogenic hormones, insulin (i), hydrocortisone (h), and prolactin (p). We have shown that a major regulatory element for the ,B-casein gene was located more than 0.8 kb upstream of the transcription start site of the bovine f-casein (b3cas) gene (Schmidhauser et al., 1990). CID 9 cells stably transfected with b3cas-1790+42/CAT (the bovine 3-casein promoter extending from -1790 to +42, relative to the transcription start site), linked to the chloramphenicol acetyl transferase (CAT) gene produced 17-fold more CAT than cells transfected with b3cas-791+42/CAT. In addition, we observed a striking ECM dependency for the longer promoter, whereas the shorter one, although showing the same level of expression on plastic, responded poorly to ECM. To localize precisely the regulatory element(s) within this region we made eight promoter deletions with 5' ends between -1790 and -791 (Figure 1A). These constructs were introduced into CID 9 cells and stable transfectants were selected with G418. The cells were then allowed to differentiate by plating onto either EHS or plastic culture dishes in the presence of the lactogenic hormones (ihp). Deletion of the region extending from -1677 to -1517 bp caused a more than 10-fold decrease in promoter activity (Figure 2). No other deletion resulted in a significant change in CAT expression. This finding suggested that an important regulatory element resides within the region between -1677 and -1517. We refer to this sequence as BCE1 (3-casein element 1). The sequence of BCE1 is shown in Figure 3. To reconstitute the promotor activity of bfcas- 1790/ CAT, we placed BCE1 upstream of the bfcas-791+42/ CAT construct (Figure 1B). BCE1 stimulated CAT expression 3.6-fold (Figure 4, lane 1 vs. 5) in the plus or normal orientation and 8.5-fold (Figure 4, lane 1 vs. 9) in the inverted orientation compared with cells transfected with b,Bcas-791+42/CAT alone. Unless otherwise noted, comparisons of promoter strength refer to cells cultured on EHS in the presence of insulin, hydrocortisone, and prolactin. In addition, ECM induction of CAT expression was increased by the addition of BCE1 to bocas-791+42/CAT. Whereas bfcas-791+42/ CAT alone showed a 4.6-fold induction by ECM (lane Vol. 3, June 1992
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Figure 1. Constructs with 5' flanking region of bovine /3-casein fusion genes, MMTV promoter BCE1 fusions, and SV40 early promoter BCE1 fusion. (A) Exonuclease III deletions of bflcas-1790+42/CAT and bfcas-791+42/CAT resulted in the constructs b,Bcas-1706+42/CAT to b,Bcas-942+42/CAT and bfcas-696+42/CAT to bocas-312+42/ CAT, respectively. Construct ER-1 and ER-2 were obtained by a partial EcoRI digestion and religation to delete sequences upstream of position -121 and -89. Each of the construct contains a unique 5' terminus and is fused to CAT at position +42 (relative to the transcription start site) in the first noncoding exon of the bovine #-casein gene (black box). (B) BCE1 (0) linked in both directions to the 5' end of bfcas791+42/CAT, ER-1 (-121 bp), ER-2 (-89 bp), to position -114 bp of the MMTV promoter, and to 140 bp of the SV40 early promoter. The arrow indicates the transcription start site. E, EcoRI; X, XbaI; H, -
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GACAGTC ATTAGGAAAT TCTCTGTTTA TTGCACAATA TGTAAAGCAT -1680 -1630 CTTCCTGAGA AAAGGGAAAT GTTGAATGGG AAGGACATGC TTTCTTTTGT -1580 ATTCCTTTTC TCAGAAATCA CACTTTTTTG CCTGTGGCCT TGGCAACCAA -1530 AAGCTAACAC ATA
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Figure 3. DNA Sequence of BCE1. The sequence starts at position -1676 and ends at -1517 with regard to the transcription start site.
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Figure 2. Effect of 5' exonuclease III deletions on CAT expression. CID 9 cells were stably transfected with the constructs with their 5' border indicated at the x-axis of the graph covering the region between bflcas-1790+42/CAT and b,Bcas-791+42/CAT (see Figure 1) and plated in defined medium for 6 d on EHS in the presence of insulin, hydrocortisone, and prolactin. Continuous deletions from the 5' end of the bovine /3-casein promoter region with exonuclease III resulted in a sharp drop of activity between deletion with the 5' border at position -1677 and -1517 bp. This region was spanned by 160 bp (BCE1). Relative CAT activity was calculated from the slope of different time points in a two-phase diffusion system as described in MATERIALS AND METHODS. The data are obtained from a single transfection set.
1 vs. 2), the BCEi-containing promoter exhibited a 12to 16-fold increase (lane 5 vs. 6; 9 vs. 10). A similar upregulation of the ECM effect could be observed under conditions where hydrocortisone was omitted (Figure 4, E ip vs. P ip). None of these constructs, either with or without BCEi, were active in the absence of prolactin. Thus BCE1 not only enhanced the total promoter activity, it also increased the ECM-dependent induction of the fl-casein promoter. This effect of ECM was hy-
drocortisone independent. To locate important regulatory elements in the proximal promoter region downstream of -791, we made an additional set of exonuclease III deletions from the 5' end of b3cas-791+42/CAT (Figure 1A). These constructs were transfected stably into CID 9 cells, and CAT activity was measured from cells plated on different substrata in the presence of lactogenic hormones. Deletion of the region between -791 and -673 resulted in an eightfold decrease in CAT expression (Figure 5). Surprisingly, deletion of the next region (to -588) caused a 28-fold increase in promoter activity. This observation suggests the possibility of a negative regula702
tory sequence between -588 and -673. Further deletions from -588 led to progressive decrease in promoter activity until all activity was lost with the deletion of the region between -312 and -121. Neither of the two shorter promoters, bfcas-121+42 (designated ER-1) nor bfcas-89+42 (ER-2), displayed activity under any conditions (Figure 5). We also tested BCE1 in conjunction with the inactive b,Bcas promoter fragments ER-1 and ER-2 (Figure 1B). BCE1 linked upstream of either ER-1 or ER-2 reconstituted promoters with considerable activity. Indeed, BCEl/ER-1/CAT expressed 1.5- to 2.5-fold more CAT activity than b3cas-1790+42/CAT, which contains BCE1 in its normal context (Figure 5). These studies showed that BCE1 restored the transcriptional activity to the inactive ER-1 (bfcas-121+42/ CAT) and ER-2 (bfcas-89+42/CAT) promoter. We therefore investigated the hormonal and ECM-depen6-
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Figure 4. CAT activity from one representative set of transfected CID 9 cells demonstrating the enhancer activity of BCE1. BCE1 was linked in both orientations (+/-) to bf,cas-791+42/CAT, and the stably transfected cells were plated under various hormone and substrata conditions. The activities were obtained from a two-phase diffusion CAT assay and express the relative substrate conversion per micrograms per minute calculated from the slope of different time points. Two independent sets of transfections resulted in comparable CAT expression (up to 14% difference between sets 1 and 2) and identical regulation patterns. The background value obtained from nontransfected cells was subtracted. Substrata used are indicated in capital letters EHS (E) and tissue culture plastic (P). Hormones are indicated in lower case letters (i, insulin; h, hydrocortisone; p, prolactin). Molecular Biology of the Cell
ECM- and Prolactin-Dependent Enhancer for ,B-Casein
(P ihp) expressed 5- to 23-fold less CAT. ECM stimulation also occurred in the absence of hydrocortisone: CAT expression was 33- to 45-fold reduced when the cells were plated onto plastic compared with EHS (Figure 6, B and C, E ip vs. P ip). As observed previously, there was no CAT expression in the absence of prolactin (Figure 6, B and C, E ih, P ih, E i, P i). For the BCE1/ ER-2 fusion, the absolute amount of promoter activity, but not the qualitative regulation, was dependent on the orientation of BCE1 (Figure 7). In contrast to the results with bf3cas-791+42/CAT and ER-1, the enhancer activity of BCE1 in the shorter construct (ER-2) became orientation independent. The negatively oriented enhancer (Figure 7C) displayed similar activity and regulation pattern as observed for BCE1/ER-1 in either orientation. When BCE1 was added to ER-2 in the positive orientation (Figure 7B), the total activity was greatly reduced (6-fold); however, it was still regulated by prolactin and ECM. The regulation of the ER-1/BCE1 and ER-2/BCE1 fusions by hormones and ECM reflected the expression pattern of the endogenous mouse f-casein gene (Figure 8) and also the CAT expression pattern of 3bcas1790+42/CAT (Figure 6D and Schmidhauser et al., 1990). Thus the bfcas-89+42 of the bovine f3-casein promoter and the 160-bp enhancer BCE1 are sufficient to display the complete spectrum of regulation by hormones and ECM.
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Replacement of ,3-casein promoter with a truncated form of the MMTV promoter
dent regulation of ER-1 and ER-2 fused to BCE1. CAT expression from both BCE1/ER-1 fusions were strongly dependent on the presence of ECM (Figure 6, B and C, E ihp vs. P ihp). Cells plated on tissue culture plastic
To investigate whether or not BCE1 can exert an influence on a heterologous promoter in the absence of its 0.0Q M
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Figure 6. Effect of BCE1, substratum and hormones on CAT expression by the -120+42bf3cas promoter (ER-1). CID 9 cells were stably transfected with ER-1 (A), ER-1 plus the BCEi enhancer in positive orientation (B), ER-1 plus BCE1 enhancer in the negative orientation (C). G 418 resistent CID 9 cells were plated on EHS (E) or tissue culture plastic (P) and maintained for 6 d in culture. The hormonal conditions are indicated in lower case letters as follows: insulin (i), hydrocortisone (h), prolactin (p). Ten micrograms total lysate of cells was used for each thin layer CAT assay. b,Bcas1790+42/CAT was used as a positive control because it contains BCE1 in its normal context (D). The activity in the bar graph represents counts per minute converted per microgram of lysate per minute. Two individual transfection sets resulted in comparable regulation patterns and a variation of up to 19% in the total magnitude of CAT expression. Vol. 3, June 1992
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own promoter, we replaced the f-casein promoter with two viral promoters. We first fused the SV40 early promoter to BCE1 (Figure 1B). None of these constructs (with or without BCE1) showed any significant expression in CID 9 cells plated on EHS under any hormonal conditions. Cells on plastic, on the other hand, expressed a weak uniform signal that was not regulated by hormones nor affected by BCE1 (see DISCUSSION). Thus the SV40 early promoter, although useful for many nondifferentiated cells in the presence of serum, was not suitable for use in differentiated mammary epithelial
cells. We then placed BCE1 upstream of a truncated form of the mouse mammary tumor virus long terminal repeat (MMTV LTR). This MMTV promoter extended from -114 to +82bp relative to the transcription start site (Figure 1B). Although this segment of the MMTV promoter contains two glucocorticoid-responsive elements (GRE), it has been reported to be nonresponsive to glucocorticoids and to exhibit only basal activity in L cells (Majors and Varmus, 1983). A comparison of the regulation of this promoter with and without BCE1 is shown in Figure 9. CAT expression from the MMTV promoter alone was relatively weak (Figure 9A) but was slightly stimulated by hydrocortisone (1.9-fold stimulation) and by ECM (3-fold). The -114+82 MMTV promoter lacked any response to prolactin. The effect of BCE1, on the other hand, was striking (Figure 9, B and C). BCE1 stimulated transcription from the MMTV pro704
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moter to very high levels (11.5-fold stimulation in the positive orientation, 7.9-fold increase when inverted, Figure 9). Indeed, the BCE1/MMTV promoter fusion was 2- to 3-fold more active than the BCE1/ER-1 promoters (see Figure 7). BCE1 altered the pattern of regulation of the MMTV promoter as well as the level of its activity. The BCE1/ MMTV fusion promoter responded to prolactin (4.5fold stimulation in cells cultured on EHS, Figure 9) whereas the MMTV promoter alone did not. ECM-dependent induction of promoter activity was also greater for the BCE1-containing promoter. ECM stimulated CAT expression by the BCE1/MMTV fusion promoter
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Figure 8. Endogenous mouse ,B-casein mRNA levels in CID 9 cells plated under various substrata and in different hormonal conditions. CID 9 cells were maintained for 6 d in defined medium containing the hormone conditions as indicated in lower case letters: insulin (i), hydrocortisone (h), and prolactin (p). Total RNA (2 sg) from cells cultured on EHS (E) or tissue culture plastic (P) were separated on a northern blot and probed with a cDNA specific for mouse ,B-casein.
Molecular Biology of the Cell
ECM- and Prolactin-Dependent Enhancer for f,-Casein W::L X
Figure 9. Effect of BCE1 on the CAT expression with a viral promoter. CID 9 cells were stably transfected with a truncated form of the 15 MMTV promoter (-114 bp) linked to CAT (A), and with the same construct but BCE1 linked either in the positive orientation (B), or in the 10 negative orientation (C) to the 5' end of this promoter. After G418 selection the cells were cultured for 6 d in defined medium on either EHS (E) or tissue culture plastic (P) in the presence of insulin (i)', hydrocortisone (h), prolactin (p), or a combination of these. Two individual 0 transfection sets varied up to 19% in total activity whereas the regulation patterns remained constant. Ten micrograms of protein lysate were used for each of the CAT assays. Activity: cpm . jg-l . min-.
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was 12-fold compared with only 3-fold for MMTV alone (E ihp vs. P ihp, Figure 9). Interestingly, the ECM- and prolactin-dependent stimulation apparently mediated by BCE1 were mutually independent. Prolactin stimulated CAT expression from the BCE1/MMTV fusion promoter 2.3-fold on plastic; ECM stimulated the same promoter 5.9-fold in the absence of prolactin.
The activity of BCE1 and ER-1 is cell type specific To see whether the truncated form of the bovine icasein promoter (ER-1) when attached to BCE1 is cell type specific, we transfected ER-1 with and without BCE1 (in both orientations) into Madin Derby Canine Kidney epithelial cells (MDCK). CAT expression was measured in the presence of ECM and lactogenic hormones. Neither ER-1 nor ER-1 plus BCE1 were expressed in these cells. Thus bflcas-121+42/CAT fused to BCE1 regulates CAT in a cell-type specific manner. DISCUSSION Previously we demonstrated that the bovine f-casein gene is regulated at the transcriptional level by ECM and lactogenic hormones in mouse mammary cells (Schmidhauser et al., 1990). We now have localized a transcriptional enhancer (BCE1) within the 5' flanking region and have shown that this enhancer plays a key role in the ECM- and prolactin-dependent regulation of the f3-casein gene. BCE1 is a novel enhancer, in that Vol. 3, June 1992
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it does not share any significant sequence homology with any known, well-characterized enhancer elements. BCE1 does share sequence homology with similarly placed regions of the rat f3-casein promoter (unpublished data) and the bovine a-lactalbumin promoter (G. Bleck and R. Bremel, University of Wisconsin, personal communication). The fact that genes other than caseins have sequences similar to BCE1 in their 5' flanking region indicates that elements related to BCE1 may play an important role in the regulation of these genes as well. It also raises an evolutionary issue because a-lactalbumin is not thought to be related to the casein gene family. Between nucleotides -1575 and -1565, BCE1 also contains some homology (6 of 9 bp match) for the consensus binding site for the putative, mammary-specific DNA-binding protein MGF (Schmitt-Ney et al., 1991) found in the proximal region of the rat fl-casein promoter. The significance of this homology can not be determined at the present time because the crucial base contacts of MGF (or indeed its definitive role in major milk protein gene expression) have not been determined. We discuss later the importance of the proximal MGF binding sites that are conserved in bovine and other species. To analyze the enhancer activity further, we linked BCE1 to two different forms (-121+42 and -89+42) of truncated ,B-casein promoter, which by themselves have no activity. Addition of BCE1 to the inactive promoters was sufficient to reconstitute transcriptional ac705
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tivity. Furthermore, regulation of promoter activity by both ECM and hormones was essentially the same as with the wild-type promoter. We therefore conclude that regulatory elements for both ECM and prolactin must be either located within BCE1 or in the remaining truncated form of the promoter (between -89 and +42). If these elements are located in the promoter, they only could become active in the presence of the enhancer. To distinguish between these two possibilities, we replaced the casein promoter with a truncated viral promoter (MMTV). The viral promoter alone was not prolactin responsive and displayed only a very faint substratum dependency. The addition of BCE1 conferred a strong prolactin- and ECM-responsiveness, indicating that BCE1 contains regulatory elements for both prolactin and ECM. This does not, however, rule out the possibility that additional ECM- and prolactin-responsive elements reside within the ER-2 promoter. Various prolactin receptors in liver (Boutin et al., 1988; Davis and Linzer, 1989) and mammary cells (Edery et al., 1989; Lesueur et al., 1991) have been isolated and characterized and functionally expressed in cells in culture. However, little is known about the transduction of the hormonal signal from the receptor to the nucleus. BCE1 will be of interest in elucidating aspects of the mechanisms of prolactin action. We have not yet succeeded in separating the prolactin and the ECM response in terms of sequence requirements. However, we have shown functionally that 1) the ECM response is glucocorticoid independent because the ECM effect was also observed under conditions where hydrocortisone was omitted and 2) the BCE1-MMTV constructs demonstrated an appreciable substratum dependency in the absence of prolactin. Because prolactin is absolutely essential for 13-casein expression, we needed a prolactin-independent promoter to demonstrate this effect. We therefore suggest that these two events are functionally separable and may be mediated by distinct sequence elements within BCE1, although the final proof will have to await additional experiments. The results from the shortest construct (ER-2/BCE1) indicates that at least one of the critical elements is located in the 3' region of BCE1: the activity of BCE1 linked in the positive orientation to the shortest (bf3cas89+42) of the casein promoter was very weak. The inverted orientation of BCE1 resulted in an activity comparable with the bfcas-120+42/CAT promoter. Nuclear protein(s) binding to the 3' end of the enhancer might sterically interfere or be interfered with by the proteins on the promoter. When BCE1 is inverted and its 3' end is now distal, this critical region might be sufficiently far away so as not to hinder its interaction with the promoter. Although ECM regulates many genes, the mechanisms are poorly understood and various different types 706
of regulation are observed. Whereas f3-casein transcription is induced by an ECM signal, TGF#31 transcription is downregulated by the same external cue (Streuli et al., 1991a). Transfected and transgenic whey acidic protein (WAP) genes appear to be expressed in the absence of ECM, whereas endogenous WAP is tightly regulated indicating that the endogenous gene is maintained in a repressed stage by sequence elements missing in the introduced constructs (Dale et al., 1992). There is accumulating data that growth and differentiation factors are attached to the ECM and can thereby be presented to the cells (Bradley and Brown, 1990; Klagsbrun, 1990; Rathjen et al., 1990). It can be argued that these factors, rather than the ECM, regulate 3-casein gene expression. Three lines of evidence argue against a direct involvement of such factors in the regulation of the 1-casein gene in our system: 1) factorpurified EHS (Taub et al., 1990) did not affect either the level of endogenous 1-casein expression or its regulation in mouse mammary epithelial cells (Streuli et al., 199 lb); 2) a blocking antibody against 31 subunit of integrins blocked the induction of the 13-casein gene (Streuli et al., 1991b), indicating that signal transduction is through integrins and not through receptors for growth or other factors; and 3) purified laminin is sufficient to induce the expression of f3-casein in CID 9 cells, as we have recently shown (Streuli, Schmidhauser, Yurchenco, and Bissell, unpublished data). The efforts to replace the casein promoter with the SV40 early promoter basically failed because there was only a weak hormone-independent activity on plastic; no matter which construct was tested, no activity was observed in cells cultured on EHS. It is important to note that the SV40 promoter was active on plastic, but BCE1 neither regulated nor increased the expression under these conditions. One possible explanation for this finding is that the cells in contact with ECM downregulate or do not express Spi, a nuclear binding protein necessary for the SV40 promoter activity. Studies in mice (Saffer et al., 1991) have shown that Spl is downregulated in fully differentiated cells. Well-developed tissues with specialized functions normally exhibit little Spl in the animal (Saffer et al., 1991). Thus differentiated cells cultured on EHS will not drive the SV40 promoter, whereas those on plastic, which are less differentiated, might allow some expression of SP1. Prolactin and hydrocortisone are known to act synergistically in activating the transcription of the rat 13casein gene (Doppler et al., 1989). Hydrocortisone is believed to interact indirectly through an unknown pathway and not through a glucocorticoid-responsive element (GRE) located within the promoter (Doppler et al., 1990). Analysis of the rat f,-casein promoter in HCl 1 cells (Ball et al., 1988), a clonal cell line also derived from COMMA-lD cells (Danielson et al., 1984), resulted in a model for transcriptional repression. Nuclear factors binding to two palindromic sequences (between -100 Molecular Biology of the Cell
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and -150) were suggested to function as transcriptional inhibitors. Binding of these factors decreased with increasing differentiation at high density (Schmitt-Ney et al., 1991). Mutations in these DNA binding sites caused an increase of basal promoter activity in the absence of hormones. Our data do not support such mechanisms for the bflcas promoter despite the high degree of sequence homology between rat and bovine for both suggested binding sites. Both binding sites are deleted in the b,Bcas-89+42 promoter of the bovine 3-casein gene (only the distal site is present in the bflcas-121+42 promoter). Neither of these promoters fused to BCE1 were active in the absence of the lactogenic hormones. So far it is not clear whether this discrepancy is due to the nature of the different cell systems employed (HC11 cells are responsive to prolactin only at high density), or because of small differences in the promoter sequence between rat and bovine, or because the rat constructs did not include BCE1. Recently a mammary-specific DNA binding protein (MGF) was described (Schmitt-Ney et al., 1991; Watson et al., 1991). The MFG binding site is highly conserved in the casein gene family of different species including bovine. It was suggested that MGF contributes to the tissue-specific expression of fl-casein. Mutagenesis of the MGF binding site of the rat f-casein promoter resulted in a complete abrogation of the promoter activity. ER-1 (bflcas-121+42/CAT), which contains the consensus for MGF, was not active in CID 9 cells. Only the addition of BCE1 to this construct reconstituted activity and the regulation pattern known for the endogenouse 3-casein (Figure 6). Deletion of two of three contact points of the MGF consensus in the bovine promoter (b3cas-89+42/CAT) did not affect the prolactin- and ECM-dependent regulation when BCE1 was linked to this promoter. Indeed activity remained very high in CID 9 cells. These findings raise the interesting question of whether BCE1 and the MGF binding site have some similar functional properties. Another feature of the 5' flanking region is a sequence that negatively regulates transcription. This is located between -588 and -673 relative to the transcription start site. Not only is there an increase in the total activity after the deletion of this region (Figure 5) but also the constructs where BCE1 is located upsteam of this sequence (b3cas-1791+42 and BCE1 fused to bfcas791+42) consistently expressed 2- to 4-fold lower activity than promoters containing BCE1 but not the region between -588 and -673. Transcriptional repression as a mechanism for regulation is known for several other genes (Kageyama and Pastan, 1989; Bahniamad et al., 1990). However, further studies are needed to produce conclusive data about the nature of this putative inhibitory sequence in the bovine f-casein promoter. We previously showed that the bovine f,-casein promoter functioned in a cell type-specific manner because Vol. 3, June 1992
no CAT expression was observed in CHO cells (Schmidhauser et al., 1990). Transfections of HC1 1 cells, NIH 3T3 cells, and NOG-8 cells with WAP/CAT, and rat ,B-casein/CAT constructs resulted in the transcription of these constructs only in the HC1 1 cells (Doppler et al., 1991). Using MDCK, a kidney epithelial cell line, we show here that constructs as short as the BCE 1/ER1 fusion in the presence of lactogenic hormones and ECM are not expressed in MDCK cells whereas the same constructs under identical conditions have high activity in CID 9 cells. It is possible that the consensus for MGF still located in this construct contributed to the cell typespecific expression. On the other hand, because prolactin is essential for the expression of 3-casein and it is unlikely that CHO and MDCK cells have prolactin receptors, lack of activity is not surprising. To see whether additional mammary specific factors are necessary, the expression needs to be tested in the presence of a functional prolactin receptor. CHO cells containing a ,3-lactoglobulin/CAT fusion expressed CAT activity only when cotransfected with a constitutively expressed cDNA for a prolactin receptor (Lesueur et al., 1991). In hepatocytes, where the transcription and expression of the albumin gene is also controlled by ECM (Rojkind et al., 1980; Bissell et al., 1987; Friedman et al., 1989), a substratum-responsive region could be localized far upstream in an enhancer of the albumin gene (Di Persio et al., 1991; Liu et al., 1991). Furthermore, the differential expression of a nuclear protein binding to this region was found to be dependent on the substratum on which the cells were plated. It is therefore likely that DNA regions with functions analogous to BCE1 are present in other tissue-specific genes in epithelial cells. We observe three shifted bands from nuclear proteins binding to BCE1 in a bandshift experiment. Additionally, preliminary results indicate two footprints within BCE1 located at either end. However, none of these binding activities appear to be changed with nuclear extracts from cells cultured in the absence of either ECM or prolactin. Therefore additional studies about the state of phosphorylation of these proteins and sitespecific mutagenesis within BCE1 need to be done to prove their functional significance. BCE1 should provide an important tool for analysis of the additional steps involved in ECM and prolactin signal transduction.
ACKNOWLEDGMENTS We are grateful to C. Streuli, R. Talhouk, A. Holwett, and T. Warren for critical reading of the manuscript and helpful discussions during the work. We also thank C. Fairman and K. Sanzo for excellent technical assistance. This work was supported by the Health Effects Research Division, U.S. Department of Energy (Contract DE-AC0376sfO0098) a gift for research from Monsanto Company (M.J.B., C.A.M., and C.S.) and by Monsanto Corporate Research (G.F.C., S.Z.B., and K.T.S.). C.S. is a fellow of the Schweizerische Nationalfonds.
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Taketani, Y., and Oka, T. (1983). Tumor promoterl2-O-tetradecanoylphorbol 13-acetate, like epidermal growth factor, stimulates cell proliferation and inhibits differentiation of mouse mammary epithelial cells in culture. Proc. Natl. Acad. Sci. USA 80, 1646-1649. Talhouk, R.S., Bissell, M.J., and Werb, Z. Co-ordinated expression of ECM-degrading proteinases and their inhibitors regulate mammary epithelial function during involution. J. Cell Biol. (in press) Taub, M., Wang, Y., Szczesny, T.M., and Kleinman, H.K. (1990). Epidermal growth factor or transforming growth factor a is required for kidney tubulogenesis in matrigel cultures in serum free medium. Proc. Natl. Acad. Sci. USA 87, 4002-4006. Topper, Y.J., and Freeman, C.S. (1980). Multiple hormone interactions in the developmental biology of the mammary gland. Physiol. Rev. 60, 1049-1105. Watson, C.J., Gordon, K.E., Robertson, M., and Clark, A.J. (1991). Interaction of DNA-binding proteins with a milk protein gene promoter in vitro: identification of a mammary gland-specific factor. Nucleic Acids Res. 19, 6603-6610. Wicha, M.S., Lowrie, G., Kohn, E., Bagavandoss, P., and Mahn, T. (1982). Extracellular matrix promotes mammary epithelial growth and differentiation in vitro. Proc. Natl. Acad. Sci. USA 79, 3213-3217.
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