Ceil, Vol. 53, 371-382, May 6, 1988, Copyright 0 1988 by Cell Press

Differential Gene Activation by Glucocorticoids and Progestins through the Hormone Regulatory Element of Mouse Mammary Tumor Virus Georges Chalepakis, Jutta Arnemann, Emily Slater, Hans-Joachim Briiller, Bernhard Gross, and Miguel Beato lnstitut fiir Molekularbiologie und Tumorforschung Philipps-UniversitBt Emil-Mannkopff-Strasse 1 D-3550 Marburg, Federal Republic of Germany

Summary

The hormone regulatory element (HRE) of mouse mammary tumor virus can mediate activation of an ad- jacent promoter by glucocorticoids and progestins. A detailed comparison of the DNA binding of receptors for both hormones using DNAase I footprinting and methylation protection detects clear differences in their interactions with the HRE region between posi- tions -130 and -100. Binding studies and gene trans- fer experiments with a variety of mutants covering the entire HRE demonstrate differences in the relevance of the individual sequence motifs for induction by each hormone. The influence of changes in the angu- lar orientation of receptor binding sites is also differ- ent for glucocorticoid and progesterone induction. In transfection experiments with mutated HREs, we find a functional cooperation between the receptor bind- ing sites that does not correlate with variations in the in vitro affinity of the receptors for the corresponding DNA fragment.

Introduction

Gene regulation by steroid hormones is mediated by an interaction of the hormone receptors with specific DNA se- quences located in the vicinity of the regulated promoter (Yamamoto, 1985; Beato et al., 1987). Such regulatory ele- ments were first identified in the long terminal repeat (LTR) region of the mouse mammary tumor virus (MMTV), where they were found to be involved in binding of the glu- cocorticoid receptor (GR) and in glucocorticoid induction (Chandler et al., 1983; Payvar et al., 1983; Scheidereit et al., 1983). Later we found that the same regulatory ele- ments bind the progesterone receptor (PR) and mediate progesterone induction of adjacent promoters (von der Ahe et al., 1985; Cato et al., 1986). Thus the correspond- ing DNA sequences have been called hormone regulatory or responsive elements (HREs).

In the chicken lysozyme gene, which also responds to glucocorticoids and progesterone, binding of the recep- tors for these two hormones to an overlapping region of the promoter has also been observed (Renkawitz et al., 1984; von der Ahe et al., 1985). However, a detailed analy sis of this region by methylation protection and DNAase I footprinting showed that each hormone receptor con- tacts different bases within the regulatory element, and suggested that the molecular recognition mechanism may

be different for each receptor (von der Ahe et al., 1986). It was not possible to assess the functional significance of these differences because the lysozyme system is not suitable for a mutational analysis in vivo. Since this type

of analysis can be performed with the HRE of MMTV, we decided to analyze in detail the interaction of the GR and the PR with the LTR region of MMTV, anld to study the in- fluence of mutations affecting individual sequence motifs or nucleotides on the response to each hormone.

Optimal progesterone inductions of MMTV-carrying plas- mids in gene transfer experiments were obtained with a clone of the human mammary tumor cell line, T47D, that has low levels of GR. Since we wanted to study glucocorti- coid and progesterone induction in the same cell line, the response to dexamethasone was analyzed by cotransfect- ing rat GR cDNA (Miesfeld et al., 1986) in an expression vector.

Here we show that the intimate molecular interactions of the GR and PR with the HRE of MMTV differ, and that certain mutations at relevant nucleotides or insertions be- tween the binding sites selectively affect inducibility by one or the other hormone.

Results

Binding of PR to the MMTV LTR In protection studies with exonuclease Ill it was shown previously that the rabbit uterine PR binds to the HRE of MMTV between -190 and -75, the same region pro- tected by the GR (von der Ahe et al., 1985). Further purifi- cation of the PR has allowed us to investigate its interac- tion with the LTR region by means of DNAase I footprinting and methylation protection experiments The results of these analyses are summarized in Figure 1, and examples of the experimental results are shown in Figure 2. The limits of the DNAase l-protected region between -162 and -192 are identical on both DNA strands to those pre- viously found for the rat liver GR (Scheidereit et al., 1983). Also, the same guanine residues are protected by the PR against methylation by dimethyl sulfate, as observed in this region with the GR (Scheidereit and Beato, 1984). The only difference is the guanine at -176 in the antisense strand, which is hypermethylated in the presence of GR (Scheidereit and Beato, 1984) but not influenced upon binding of PR (Figure 28).

In the promoter-proximal region the footprints obtained with each receptor are clearly different. Whereas the GR generates three separate DNAase I footprints, corre- sponding to each of the TGTTCT motifs (Figure 1; Scheid- ereit et al., 1983), PR binding leads to almost continuous protection extending from -132 to -71, interrupted only at -84/-85 in the upper or sense strand (1Figures 2A and 2B, lanes 2). Thus the footprint generated by the PR is longer than the one obtained with the GR, and covers 9110 additional base pairs between -1221-123 and -132 (Fig- ure 1). In this region there are two guanlne residues, at -127 and -128 in the upper strand, that are protected

Cell 372

714” 3 ’ c: I z 3 ’ -pm; g-K? IIIIIIIIIII,,,,,,I IIIIIIIIIIII, IIISI,,/IIIII

CTGRGTTGGTTTGGTRTCRRR~GRTCTGRG~TCTTRGT~RTTTTC~TR~TTT~RTCTRTCCR

GACTCRRCCARRCCATRGTTTRCRRGRCTRGRCTCGRGRRTCRCRRGRTRRRRGGRTRCRRGRRRRCCTTR~RT~ +

IIII,,I,III,,,, IIIII!IIIIIII111,II II.IIIIIII 4 9 *o Q c, . a . 0 c!

; 60 ;48 ;x! 1, CRP

AGTCTTRTGTARRTGCTTATGTRRRCCR~RR~R~~~RRGRGTGCTGRTTTTJTGRGTRRRCTTGCRR~RGTCCTRRCR

TCRGRATRCRTTTRCGRRTRCRTTTGGTRTTTTCTCRCGRCTRRRRRRCTCRTTTGRRCGTTGTCRGGRTTGT

A

4 5 1 2345 2

Figure 1. Nucleotide Sequence of the Pro- moter Region of the MMTV LTR

Regions protected by the GR against digestion by DNAase I (Scheidereit et al., 1983) are indi- cated by the dotted lines above (sense strand) and below (antisense strand) the sequence. Regions protected by the PR are indicated by continuous lines. The open vertical arrows point to guanine residues protected against methylation by dimethyl sulfate in the presence of GR; guanines hypermethylated after incuba- tion with GR are indicated by the black vertical arrows (Scheidereit and Beato, 1984). Arrow- heads are used to indicate similar changes in- duced by binding of the PR. Numbers refer to the distance from the transcription start point (labeled CAP).

Figure 2. DNAase I Footprint and Methylation Protection Experiments with the PR and the Wild-Type MMTV LTR

(A) Sense strand. The plasmid pLTR-wt was digested with Hindlll, labeled at the 5’end, and redigested with Xhol. The isolated 364 bp frag- ment (4 ng) was incubated with (lanes 1 and 5) or without (lanes 2 and 4) PR (130 ng), and sub- mitted to digestion with DNAase I (lanes 1 and 2) or treated with dimethyl sulfate (lanes 4 and 5) as described in Experimental Procedures. Shown is an autoradiogram of a 6.5% poly acrylamide-8 M urea sequencing gel (Maxam and Gilbert, 1980). Lane 3 shows a guanine- specific sequence reaction (Maxam and Gil- bert, 1980). (B) Antisense strand. The plasmid pMMTV-wt was digested with Pvull, labeled at the 5’ end, and redigested with Hindlll. The resulting 310 bp restriction fragment was isolated, incubated with (lanes 1 and 6) or without (lanes 2 and 5) PR, and subjected to DNAase I digestion (lanes 1 and 2) or dimethyl sulfate treatment (lanes 5 and 6). Lanes 3 and 4 show purine and guanine sequencing reactions, respectively. Numbers refer to distance from the transcrip- tion start point. DNAase I footprints are in- dicated by vertical lines, and DNAase l-hy- persensitive sites are marked by horizontal arrows. Open triangles denote protected gua- nines, and closed triangles point to hyper- methylated guanines.

against methylation by the PR (Figure 2A, lanes 4 and 5) tected or hypermethylated in the presence of GR respond

but not by the GR (Scheidereit and Beato, 1984). In addi- in a similar way to binding of the PR (Figures 1 and 2).

tion, the G at -103 in the lower or antisense strand is only In the region between the receptor binding sites we find

protected by the PR (Figure 2B, lanes 5 and 6). All the two guanine residues (positions -146 and -153 in the up-

other guanine residues in this region that are either pro- per strand) that are hypermethylated in the presence of

Differential Regulation of MMTV by Two Steroids 373

SEloI Hind111

Es + \I- LS-203 * w --a is-175 * Y" LS-119 * WE LS-98 *

.m LS-83

/ Ret k *

NC-illter Elut1on ) * AWI

,

*- electro-

- phoresls

Figure 3. Influence of Qligonucleotide Mutants on Binding of the GR and PR

(A) The positions of the different oligonucleotide mutations in the MMTV LTR are shown. The sequences of the individual mutants are shown below the nucleotide sequence of the wild-type sense strand (Figure 1). The mutants are named “Ls” followed by a number denoting the position of the first exchanged nucleotide (5’). Also shown are mutations that introduce new Hindlll and Sal1 sites at positions -190 and -155. (B) Strategy for the binding experiments. An equimolar mixture of the indicated mutants was digested with Hindlll, labeled at the 5’ end, and redigested with Bglll. The resulting mixture of 360 bp fragments was isolated, incubated with different amounts of GR or PR, and filtered through nitrocellulose (Geisse et al., 1982). The protein-bound DNA was eluted, ethanol-precipitated, and digested with Aval before electrophoresis through a polyacrylamide gel (C). (C) Autoradiogram of the 8% polyacrylamide gel. Lanes 1 and 5, control input DNA. Lanes 2-4, DNA retained on the filters in the presence of 10 ng of PR along with 40 ng (lane 2), 80 ng (lane 3) or 120 ng (lane 4) of poly(dl-dC).poly(dl-dC). Lanes 6-8, DNA retained in the presence of 15 ng of GR along with 40 ng (lane 6), 80 ng (lane 7), or120 ng (lane 8) of poly(dl-dC).poly(dl-dC). The LS mutants from which the individual bands are derived are indicated on the right. (D) Quantitative evaluation of the autoradiogram. The autoradiogram shown in (C) was evaluated by scanning with an integrating microdensitometer. Relative areas of the individual bands, referred to as the input bands, were averaged from lanes 2-4 for the PR and from lanes 6-8 for the GR, and are expressed as fractions of the values observed with the LS-203 mutant,

the PR (Figure 2A, lanes 4 and 5), as observed after bind- ing of the GR (Scheidereit and Beato, 1984). However, methylation in this region is altered at four additional posi- tions upon binding of the GR, but none of these is affected by binding of the PR (Figure 1). In addition, binding of the PR induces DNAase l-hypersensitive sites at positions -84 and -94 of the adjacent thymidine kinase (TK) pro-

moter (Figure 2A, lane 1). In conclusion, the intimate molecular interactions of the

GR and the PR with the MMTV HRE have many common

features, but can be clearly distinguished by DNAase I footprinting and methylation protection.

Influence of Oligonucleotide Mutations on Receptor Binding To further understand the relevance of individual se- quence motifs for the binding of the two horrnone recep- tors, we made use of a series of oligonucleotide mutations within and around the HRE. Six of these mutants in- troduce a new Smal restriction site (Figure 3.A). The four

Cell 374

mutants that eliminate each of the TGTTCT motifs were mixed with the LS-203 mutant, which does not influence receptor binding in vitro (data not shown); after restriction digestion and end labeling, the mixture of five fragments Of equal length was incubated with each receptor. The receptor-bound fragments were isolated by filtration through nitrocellulose, digested with Aval, and then ana- lyzed by gel electrophoresis (Figures 36 and 3C). This ap- proach was possible because after restriction with Aval, each mutant yields a labeled fragment of different size, thus allowing a comparison of the relative affinity of the receptor for each mutant DNA. A quantitative evaluation of the autoradiogram (Figure 3D) shows clear differences in the DNA affinity of individual mutants for binding of the GR versus the PR. Whereas mutation of the motif at -83 seems to have little influence on binding of the PR, it markedly inhibits binding of the GR. A similar but less dra- matic difference is observed with the LS-98 mutant, On the other hand, mutation of the TGTTCT at -175 reduces binding of the PR more significantly than that of the GR, and the LS-119 mutant exhibits an intermediate behavior.

These binding experiments suggest that there is no strong cooperativity between the individual sequence mo- tifs in terms of receptor binding to a DNA fragment, since mutation of any single site has only small effects (max- imally 2-fold) on the affinity of the receptors for the whole fragment.

To analyze the functional significance of these differ- ences in receptor binding, we cloned the different LS mu- tations in front of the herpes simplex virus TK promoter (-105 to +57) in the pTK-CAT.3 plasmid, and performed transient gene transfer experiments in T47D human mam- mary cells (Cato et al., 1986). Since the cell line we used has low levels of endogenous GR, in experiments de- signed to study glucocorticoid induction we cotransfected the rat GR cDNA within a construct driven by the Rous sarcoma virus (RSV) promoter (Miesfeld et al., 1986). We had previously found that the RSV promoter is efficiently expressed in this cell line, and is not significantly in- fluenced by treatment of the cells with steroid hormones (data not shown). In fact, cotransfection with pRSV-/acZ and measurement of f3-galactosidase activity was used to normalize the values of CAT assays. The results of these experiments are summarized in Table 1.

None of the LS mutants tested significantly affected the basal CAT activity in the absence of hormone, which was very low in all cases (Table 1). Those mutants that elimi- nate the hexanucleotide motif at position -175, -119, or -98 respond very poorly to either glucocorticoids or progestins. pLS-119 is unresponsive to progestins but still weakly inducible by dexamethasone. The mutant LS-83, which exchanges the TGTTCT most proximal to the pro- moter, shows a different behavior; it reduces glucocorti- coid induction but has no effect on the response to R5020. The reverse is true for the LS-108 mutant, which ex- changes 6 nucleotides including a G residue (-103 in the lower strand) that is contacted only by the PR (see Figure 26). This mutant still responds well to dexamethasone, but induction by R5020 is reduced by 78% (Table 1).

In DNAase I footprint experiments with LS-108 (Figure

Table 1. Influence of LS Mutations on Hormonal Induction

pLTR-wt pLS-203 PLS-175 pLS-147 PLS-119 PLS-I 08 PLS-98 pLS-83

CAT Activity (% Conversion)

Control R5020 Dex

0.17 18.4 17.2 0.27 13.8 19.5 0.35 3.6 2.3 0.34 28.4 20.1 0.20 0.21 1.04 0.26 5.7 14.3 0.17 0.59 1.2 0.18 17.9 12.0

Supercoiled DNA (2 pg) from the indicated mutants was transfected into T47D cells together with 2 ug of pRSV-/acZ and, for dexametha- sone inductton, 2 ug of pRSV.GR. Previous experiments showed that transfection of pRSV.GR had no influence on the basal CAT activity of the cells not treated with hormones. Therefore, a single control value was used for each construction, in which no pRSV.GR was added. Twenty-four hours after transfection the medium was exchanged, and either ethanol alone (control), lo-* M R5020 (R5020), or lo-’ M dexa- methasone (Dex) was added. After 48 hr at 37%, the cells were har- vested and the CAT and b-galactosidase activities were determined rn the extracts (see Experimental Procedures). The numbers represent the average of two experjments in which 10 kg of cellular protein was used for the CAT assay. The effect of each mutation relative to the wild type was reproducible (less than 20% variation between the two experiments).

4A), a clear weakening of the protection by the PR recep- tor in the region between -107 and -122 is observed. In- stead of a continuous footprint between -131 and -71, as seen in the wild type, only a few bands are protected. In fact, the footprints obtained with PR and GR in this region of the mutated HRE are very similar. In addition, the promoter-distal footprint is also less evident in the mutant.

The LS mutations at -203 and -147, located outside of the receptor binding sites, do not influence receptor bind- ing in vitro and have less dramatic effects on the response to both hormones (Table 1). In fact, the absolute CAT activ- ity observed with LS-147 was always higher than with the wild-type LTR, but because the basal activity was also high the relative hormonal response was lower.

We have previously shown that the TK promoter is ac- curately transcribed in T47D cells following hormonal in- duction of constructions containing the MMTV HRE (Cato et al., 1986). To make sure that this is also the case for cells cotransfected with the GR cDNA, we analyzed the RNA from these cells by the quantitative RNAase protec- tion procedure (Figure 5). It is clear that dexamethasone treatment of these cells leads to an accumulation of tran- script correctly initiated at the TK promoter (Figure 5, lane 4).

In conclusion, although mutation of individual hexa- nucleotides only reduces binding affinity by less than 3-fold, many of these LS mutants inhibit hormonal response in gene transfer experiments by over lo-fold. Thus there is a functional cooperation between the receptor binding sites that is not reflected by parallel changes in the in vitro affinities of the hormone receptors for the mutated HRE. In addition, some mutants, like LS-108 and LS-83, exhibit a partial dissociation of the response to progestins and glucocorticoids.

Differential Regulation of MMTV by Two Steroids 375

WT

- ‘,” -7l ----- --

“,

: . w

2

“21 -- - - - - - - - -*

.LC c e3C=c *--

-x81-.

A

LS -108

Figure 4. DNAase I Footprint Analysis of the Binding of PR and GR to MMTV LTR Frag- ments Derived from pLS-108 or pMMTV-21

(A) The plasmid pLS-108, was digested with Hmdlll, 5’labeled to analyze the sense strand, and redigested with Bglll. L.ane 4, G+A se- quencing reaction; lane 2, control reaction, in- cubated with buffer; lane 1, protection ob- served with 170 ng PI?; lane 3, protection observed with 700 ng GR Symbols are as in Figure 2. Protection of the sense strand of the wild-type MMTV in the presence of PR, shown at left (see Figure 2A for details), is given for comparison. (8) The plasmld pMMTV-21 was digested with BamHI, 5’labeled, and redigested with Pvull to analvze the antisense strand. Lane 1, auanine- specific sequencing reaction; lane 2, G+A se- quencing reaction; lane 3, control reaction, in- cubated with buffer; lane 4, protection seen with 130 ng PR.

Localized Deletions and Inversions To understand the cooperativity between the two blocks of binding sites, at -190 to -160 and at -130 to -70, we analyzed the functional activity of each separate group of binding sites and introduced insertions of different lengths between them.

A comparison of the constructions pTK-CAT-5A and pMMTV-A-SA shows that, under the conditions of our as- say, sequences upstream of -370 are not essential for op- timal inducibility by either glucocorticoids or progestins (Figure 6). Indeed, all the sequences upstream of -200 can be removed without impairing hormonal response (Figure 6, pMMTV-21H). Thus only the previously de- scribed region containing the receptor binding sites is needed for induction by both glucocorticoids and proges- tins in transient gene transfer experiments (Scheidereit et al., 1983).

The promoter-distal receptor binding site by itself was ineffective in the functional assay (Figure 6, pMMTV-23, pMMTV-10, or pMMTV-l1), although we know that it binds both GR and PR very tightly (Scheidereit and Beato, 1984;

unpublished results). The group of receptor binding sites between -130 and -70 confers weakglucocorticoid (18% of wild type) and progestin (26% of wild type) inducibilities to the TK promoter (Figure 6, pMMTV-22). Exonuclease Ill protection experiments with such deletion mutants show that the GR binds efficiently to the promoter-proximal sites even when they are separated from the distal element (Scheidereit and Beato, 1984; unpublished Iresults). Thus the lack of glucocorticoid inducibility is not clue to a low affinity for the GR.

Figure 6 also shows that inverting the whole region be- tween -189 and -157 does not interfere with hormonal response (pBMU-37). On the contrary, this construction was even more inducible than the wild-type configuration (compare to pMMTV-21). DNAase I footprint experiments show a normal pattern of protection by both GR and PR in this inverted region (data not shown). Thus the relative orientation of the receptor binding sites to each other seems to be irrelevant for hormonal induction.

The conclusion of this type of experiment is that there is a strong functional cooperation between the two blocks

Cell 376

MP - D R c

1 23456

Figure 5. Analysis of CAT RNA after Transfection

Both dexamethasone and R5020 increase the concentration of TK-CAT mRNA following transfection of T47D ceils with plasmid pTK-CAT-5A. Lane 1, pBR322 restricted with Hpall and end-labeled (M). Lane 2, the 347 nucleotide probe used for hybridizations (P). Lane 3, RNA ex- tracted from transfected cells incubated without hormones (-). Lane 4, RNA from cells cotransfected with RSV GR, treated with 2 x toe7 M dexamethasone (D). Lane 5, RNA from transfected cells treated with 2 x 10-s M R5020 (R). Lane 6, RNA from untransfected cells (C). Cor- rectly initiated TK-CAT mRNA protects 210 nucleotides of the probe (upper arrow). The 298 nucleotide band originates from readthrough transcripts upstream of the TK promoter. RSVCAT mRNA gives rise to a 150 nucleotide band.

of binding sites, since each separated region is ineffective and both have to be included in the constructions to obtain optimal induction. However, this functional cooperation is orientation-independent and does not appear to correlate with equivalent changes in affinity of the hormone recep- tors for the corresponding linear DNA fragments in vitro.

insertions between the Binding Sites To further analyze the nature of the functional interaction between the two blocks of receptor binding sites, we took advantage of the LS-147 mutant, which only slightly in- fluences hormone inducibility (Table 1). In this position oli- gonucleotides of defined length and sequence were intro- duced and the influence of these manipulations on the inducibility by glucocorticoids and progestins was deter- mined in gene transfer assays (Table 2). The introduction of 5 bp in this region resulted in opposite changes of in- ducibility by each hormone: whereas expression in the presence of R5020 was inhibited by 30010, the effect of dexamethasone was twice that observed with the parental construction. In interpreting these data we refer to the ab- solute CAT activity as percent conversion of chloramphen- icol. Since the basal activity of these constructions in the absence of hormones was also increased, the extent of in- duction is lower. Taking this into account, a result opposite to that described with the 5 bp insertion was obtained with the 10 bp insertion mutant: this mutant showed no change in the presence of glucocorticoid but was more active in

the presence of R5020 than the parental construction. In- creasing the length of the insertions progressively de- creased inducibility by dexamethasone, whereas induc- tion by progestins was lower than wild type with the 18 bp insertion, but optimal with the 20 bp insertion. indeed, even the construction with a 30 bp insertion showed wild- type inducibility by progestins. An insertion of 60 bp dra- matically reduced inducibility by both hormones. In filter binding experiments we showed that this insertion mutant had affinities for the GR and the PR only 18% lower than those observed with the wild-type HRE (Figure 7B). Thus these experiments again suggest that there is a strong functional cooperation between the two blocks of receptor binding sites, but that this is not due to differences in the in vitro affinity of the corresponding DNA for the hormone receptors.

Point Mutations As a preliminary step in the generation of a series of point mutations within the promoter-distal receptor binding site, we introduced two new restriction sites flanking this re- gion. At -190 we replaced a C with a T and thus created a new Hindlll site, and at -155 we exchanged the se- quence TGA with GTC and created a new Sal1 site (Figure 3A). A similar set of mutations has been constructed by Hutchison et al. (1986). As expected, this mutation did not influence the binding of the GR (data not shown). The mu- tant constructions also yielded DNAase I footprints with the PR identical to those obtained with the wild-type HRE (compare Figures 46 and 28). In agreement with these results, the inducibility of this mutant by glucocorticoids was only somewhat lower than that of the wild-type HRE (Figure 6). Unexpectedly, response to progestins was markedly reduced (30% of wild type), and this effect could be attributed to the mutations at -155 since eliminating these mutations restores wild-type inducibility (compare constructions pMMTV-20 and pMMTV-21 with pMMTV- 20H and pMMTV-21H in Figure 6). Thus this mutant pro- vides an additional example for a differential contribution of individual DNA sequences to response by either glu- cocorticoids or progestins, which again cannot be ex- plained in terms of changes in receptor binding affinity to DNA.

Discussion

Interaction of the Hormone Receptors and DNA In Vitro Our previous studies on the binding of GR and PR to the MMTV LTR showed that both proteins cover the same re- gion of the LTR as determined by exonuclease III protec- tion (von der Ahe et al., 1985). Our present results confirm the identity of the outer limits of the nuclease-protected re- gion, but reveal differences in the length of the individual DNAase I footprints and in the purine residues contacted by each receptor. The methylation protection studies show that the two guanine residues (one in the sense strand and another in the antisense strand) within the hexa- nucleotide TGTTCT that are known to be protected by the GR (Scheidereit and Beato, 1984) are also in intimate con-

Differentiai Regulation of MMTV by Two Steroids 377

pTK-CAT-5A 0.33 39.4

pXXTV-iSA 0.53 39.6

plmTv-20B 0.56 40.7

p--20 0.35 12.0

pKnTV-21H 0.36 40.5

pmxrv-21 0.35 14.7

pBRl-37 0.28 21.5

pmrrv-22 0.36 10.2

pHXTV-23 0.41 0.62

pNMTv-10 0.72 0.61

pxxrv-11 0.42 0.58

CAT ACTIVITY C’L con.7ersion>

uJ*RoI x5020 DEX

_--------------------------------

44.5

42.8

43.8

31.7

44.5

28.2

39.5

8.1

0.36

ll.d.

n. d.

A

Figure 6. Influence of Different Deletions in the MMTV LTR on Hormone lnducibility in T47D Cells

The indicated deletions and point mutations within the MMTV LTR were constructed as de- scribed in Experimental Procedures. The con- struction pMMTV-20 contains two new sites for Hindlll and Sal1 introduced by Ml3 mutagene- sis at positions -190 and -155, respectively (see Figure 3A). All mutated LTR fragments were cloned in front of the TK promoter in pTK- CAT3 (Cato et al., 1986). Vertical lines in pTK- CAT-5A indicate only the positions of relevant restriction sites. The same lines in the MMTV constructs indicate the corresponding mu- tants. The black boxes represent the TGTTCT motifs in the MMTV LTR region. Values for CAT activity represent the average of two experi- ments and are expressed as percent chloram- phenicol conversion In an assay containing 20 Kg of protein. The absolute values varied in’ each experiment, but the efiect of mutations relative to the wild type were highly reproduc- ible. CAT activity was assayed as described in Experimental Procedures and Table 2.

tact with the PR. In addition, the PR contacts three G residues that are not protected by the GR, and some of the hypermethylated sites are also different. That these differ- ences and similarities are real and do not reflect the in- fluence of variations in the purification protocols or cross- contamination of the receptor preparation is suggested by the following considerations. The strategies followed for purification of each receptor were essentially identical, and were based on differential binding of the receptors to DNA-cellulose (see Experimental Procedures). A PR puri- fied in a very different way (by steroid affinity chromatog- raphy) from chick oviduct (von der Ahe et al., 1986) yielded results similar to those reported here (data not shown).

Thus, as in the case of the chicken lysozyme promoter (von der Ahe et al., 1986), the interactions of the GR and the PR with the HRE of MMTV exhibit both common and different features. Within the promoter-distal binding sites only very subtle differences are found, whereas in the more promoter-proximal region the PR generates a longer footprint and contacts three additional guanine residues.

Table 2. Influence of Insertion Mutations on Hormone lnducibility

CAT Activity (% Conversion)

Mutant Control R5020 Dex

pLS-147 0.31 42.5 26.7 pBMl-5 1.11 30.1 48.2 pBMI-10 2.84 54.5 25.2 pBMI-18 1.25 35.1 18.9 pBMI-20 0.54 56.3 18.1 pBMI-30 0.86 41.3 14.4 pBMI-60 1 .o 4.7 1.7

- DNA from the indicated constructions (2 pg) was transfected together with pASV-/acZ and with (Dex) or without (Control, R5020) 1 vg of pRSV.GR. The rest of the experiment was performed as described in the legend to Table 1 and in Experimental Procedures, except that 20 pg of cellular protein was used for the CAT assay.

1 LS-147

PTCGAT

1 Synthetic Polylinker

/\ / Jb& E Hae I

\ CGGRTRTCTAGRCAGTCGACTT[~CCL~[~CAT

Eco RU Ea I

6 GR PR

12345 6 7

Figure 7. Insertion Mutations in the MMTV LTR between the Two Blocks of Receptor Binding Sites

(A) Strategy followed to construct the insertions. The piasmid LS-147 was digested with Clal, and a synthetic polylinker with the restriction sites for the enzymes Xbal, Hael, EcoRV, Sall, and Clal was inserted into this position so that only one Clal site was regenerated. The ob- tained plasmid has a 30 bp insertion (pBMI-30). Dualication of the poly- linker leads to a 60 bp insertion (pBMI-60). Elimination of short se- quences inside the polylinker by restriction digestion gives rise to the plasmids pBMI-5, pBMI-IO, pBMI-18, and pBMI-20. (B) Binding of PR and GR to a mixture of a wild-type MMTV LTR frag- ment, a MMTV LTR fragment with the 60 bp insertion, and a non- specific vector fragment. The specific fragments were obtained by digestion of either pLTR-wt or pBMI-60 with Hindllf, 5’ labeling, and redigestion with Bglll. The nonspecific vector fragment was derived from pLTR-wt after redigestion with Aatll. The three fragments were mixed, incubated with or without the receptor, and filtered on nitrocellu- lose; the retained DNA was analyzed on a 5% polyacrylamide-8 M urea sequencing gel. Lane 4, control, without receptor; lanes 1-3, 20 ng GR; lanes 5-7, 15 ng PR; lanes 1 and 5, 40 ng poly(dl-dC),poly(dl- dC); lanes 2 and 6, 120 ng poly(dl-dC).poly(dI-dC); lane:s 3 and 7, 180 ng poly(dl-dC).poly(dl-dC).

Cell 370

Figure 8. Computer Graphic Representation of the Promoter-Proximal Set of Receptor Binding Sites

(A) Lateral view. The upper or sense strand is shown in blue, the lower or antisense strand in yellow. The first base pair on the left is -133 and the last on the right is -77. The projectron is such that the base pairs are seen from the side and the bases cannot be identified. The van der Waals spheres of the N-7 positions of those G residues protected by the GR and the PR are shown in green; those protected only by the PR are shown in red. For a better orientation some of these positions are labeled. (B) Axial projection of the same region of the LTR shown in (A). For simplicity, all atoms with the exception of the helrx backbone and the protected N-7 positions of guanines have been eliminated. The color code for the van der Waals spheres is as in (A).

However, contrary to what we found in the lysozyme gene, computer graphic analysis does not suggest that each receptor contacts the DNA double helix in this region at a different angle (Figure 8). With the exception of the gua- nine residue at -113 that is contacted by both receptors, all the other protected N-7 positions of guanine are local- ized within a narrow sector of the helical circle (Figure 8B).

The results of binding experiments with linear DNA frag- ments of the different mutant constructions do not suggest a strong cooperativity between the four individual binding sites previously reported (Scheidereit et al., 1983). Muta- tion of any one of the four TGTTCT motifs eliminates bind- ing of the receptor to the mutated site, but does not reduce the affinity of the receptor for the whole HRE by more than 3-fold. Similar results were derived from binding studies with localized deletions and insertions, none of which had a dramatic effect on the affinity of the receptors for the HRE-containing fragments.

Effect of HRE Mutations on the Hormonal Response Contrary to the in vitro binding results, gene transfer ex- periments demonstrate a strong functional interaction be- tween the different receptor binding sites. Mutations that only affect individual binding sites have a dramatic in- fluence on the hormonal response of the corresponding constructions. These results confirm the relevance of the four conserved motifs TGTTCT for glucocorticoid induc- tion (Scheidereit and Beato, 1984), and show that at least three of these motifs are also essential for progesterone induction. The dramatic reduction of glucocorticoid induc-

ibility observed with the LS mutants -119 and -98 is in apparent contradiction to previously published results (Hynes et al., 1983; Lee et al., 1984; Buetti and Kiihnel, 1986; Kijhnel et al., 1986). However, in all of these studies fibroblasts were used for the transfection experiments. We have previously shown that deletion of the promoter- proximal receptor binding sites completely inhibits gluco- corticoid response in mammary cells, but only reduces in- ducibility of the same construction in fibroblasts by about 3-fold (Cato et al., 1986). Thus the differences observed could be due to cell-specific requirements, an aspect to keep in mind when comparing results from different groups. In addition, the MMTV strain used for the studies by Lee et al. (1984) differs at essential positions from that used in our experiments. In particular, the T at position -119 of our MMTV strain (Figure 1) is a G in the strain

used by Lee et al. (1984) (see Figure 4 in their paper). We would expect this difference to lead to marked reduction in receptor binding at this site, a fact that could explain the relatively low hormonal induction seen by these authors (ca. IO-fold).

The results obtained with the LS-83 mutant are peculiar in that this mutation inhibits response to glucocorticoids much more than to progestins. In this case a correlation with in vitro binding data is observed, since this mutant has lower relative affinity for GR than for PR. It is tempting to speculate on the possible relevance in this context of the binding site for nuclear factor I (NFI) overlapping the most promoter-proximal receptor binding site (Nowock et al., 1985; Cordingley et al., 1987). It has been suggested

Differential Regulation of MMTV by Two Steroids 379

that binding of NFI to this site is needed for glucocorticoid induction of the MMTV promoter (Miksicek et al., 1987). It will be interesting to know whether progesterone re- sponse is equally dependent on binding of NFI. These considerations have no relevance for our results, however, since the intact binding site for NFI is absent from all our constructions. As previously shown, the half of the NFI binding site present in our constructs is not sufficient to generate a DNAase I footprint nor a shift of the corre- sponding DNA fragment in the gel retardation assay (Mik- sicek et al. 1987).

A similar dissociation between the influence of a muta- tion on induction by glucocorticoids and progesterone is observed with the construction pMMTV-20. The ex- changes relevant for inhibition of progesterone response (TGA to GTC at -157) are localized outside of the DNAase I footprints and do not influence receptor binding to the

HRE. Interestingly, this mutation changes the pattern of cutting by topoisomerases I and II within the HRE (U. Briiggemeier, G. C., and M. B., unpublished), thus rais- ing the possibility that DNA secondary structure is dif- ferentially involved in the induction by each hormone. In this context it is interesting to note that binding of the PR generates two DNAase l-binding hypersensitive sites within the adjacent TK promoter (see Figure 2).

Oligonucleotide directed mutations located outside of the main receptor binding sites have less dramatic effects on hormonal response. LS-147, which substitutes the se- quence TGGTTT, does not reduce hormonal inducibility. Similar results were obtained by Buetti and Ktihnel (1986) using a series of “linker scanner” mutants in this region. These results clearly exclude a functional contribution of a potential or cryptic receptor binding site in this region, which has been reported to be protected against DNAase / by purified GR (Payvar et al., 1983). We have not found a receptor footprint in this region, but we detected changes in the guanine methylation pattern after binding of GR (Scheidereit and Beato, 1984). Whatever the reason for the altered reactivity with dimethyl sulfate, this se- quence, which shows homology to the so-called SV40 en- hancer core motif (Weiher et al., 1983), can be mutated

without inhibiting hormonal response. In fact, inversion of the -189 to -157 region has no influence on receptor binding or on hormonal induction. Any structural interac- tion between receptor molecules bound to this region and other factors bound to the -150 to -140 region should be perturbed by this inversion. From these results we con- clude that there is no indication that regulatory elements are located between the previously described receptor binding sites (Scheidereit et al., 1983).

Influence of Insertions between the Receptor Binding Regions Taking advantage of LS-147, which generates a new re- striction site, we introduced oligonucleotides of different lengths between the two receptor binding regions and analyzed their influence on the response to glucocorti- coids and progesterone. The response to progesterone followed the pattern expected if the DNA-bound receptor

proteins need to interact with each other on one side of the double helix. Insertions of 10 and 20 bp-integral multi- ples of the heiical pitch that do not alter the relative angu- lar orientation of the bound receptor molecules-did not inhibit but rather enhanced progesterone response. Even insertion of 30 bp did not interfere with induction by progestins.

Response to glucocorticoids follows a completely differ- ent pattern. Introduction of 5 bp (one-half helical turn) en- hances dexamethasone response to the level observed

with progestins. This finding suggests that the natural configuration of receptor binding sites in the MMTV LTR is optimally designed to respond to progestins rather than to glucocorticoids. Insertions of longer oligonucleotides resulted in a progressive loss of glucocorticoid inducibil- ity. Constructions containing insertions of 20 and 30 bp re- spond poorly to dexamethasone but very efficiently to

progestins. A much longer insertion, of 60 bp, dramati- cally reduces inducibility by both hormones.

The behavior of the insertion mutants in the functional test cannot be explained in terms of the relative affinity for the receptors in vitro. A comparison of the wild-type re- sponse element and the 60 bp insertion mutant detects only a very slight reduction in affinity for either GR or PR. We interpret these results as additional evidence for a functional cooperation of DNA-bound receptor molecules that is facilitated by a particular array of regulatory se- quences.

Whether the differences between the progesterone and glucocorticoid responses reflect the different sizes of the N-terminal regions of the corresponding receptors re- mains to be established. These results, whatever the ex- planation, clearly demonstrate that the functional interac- tion between DNA-bound molecules of the PR differs from that occurring between GR molecules. Since the MMTV HRE also appears to mediate the response to androgens (Darbre et al., 1986; Cato et al., 1987), it will be interesting to test how this set of insertion mutants influences the an- drogen inducibility of an adjacent promoter.

Conclusions

Three general conclusions can be drawn from these studies. First, a functional interaction between receptor molecules bound to the different receptor bindling sites is required for optimal glucocorticoid and progesterone in- duction mediated through the HRE of MMTV. Second, the details of the interaction of the hormone receptors with DNA, as well as the cooperation between DNA-bound receptor molecules, differ for glucocorticoid and proges- terone. And third, there is no indication of the involvement in the hormonal response of other factors interacting with sequences located between the two sets of receptor bind- ing sites.

These studies were directed toward understanding the physical and functional relationships between different receptor molecules bound to the MMTV HRE. The ques- tion still remains about how the binding of the hormone receptors to the HRE is converted into a functional re-

Cell 380

sponse in terms of promoter utilization. Further experi- ments designed to analyze the relationship between re- ceptor molecules and other promoter elements and factors involved in transcription of the relevant promoters are needed to answer this question.

Experimental Procedures

Purification of Hormone Receptors GR was purified from liver cytosol of adrenalectomized rats according to previously published procedures (Geisse et al., 1982). Since the ma- terial eluting from DNA-cellulose contains contaminating calf thymus DNA, the preparation was passed through a 1 ml column of DEAE- Sephacel (Pharmacia) equilibrated with 20 mM Tris-HCI (pH 7.5), 0.3 M NaCI, 1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol. The flow- through was supplemented with bovine serum albumin (0.1 mglml final concentration), aliquoted, frozen in liquid NP, and stored at -80%. The purity of the preparation was 50%-70%.

PR was purified by a very similar procedure from uterine cytosol of rabbits treated with estrogen for 1 week. The extract was supple- mented with protease inhibitors (von der Ahe et al., 1985). The final purity of the preparations, assuming a molecular mass of 110,000 dal- tons for PR, varied from 10% and 20%. The final steps in the prepara- tion were as described for the GR.

Plasmid Constructions The plasmids pTK-CAT.3 and pTK-CAT-SA have been previously de- scribed (Cat0 et al., 1986).

The oligonucleotide mutants in the MMTV LTR, here called the LS mutants, were constructed by P. Skroch and H. Ponta (Cato et al., 1988). After digestion with Hinfl and generation of blunt ends with the Klenow fragment of DNA polymerase, the -236 to -70 region of the LTR was subcloned in the filled-in Sall site of pTK-CAT.3 to produce the plasmids pLS-203, pLS-175, pLS-147, pLS-119, pLS-108, pLS-98, and pLS-83. Subcloning of the wild-type fragment leads to the plasmid pLTR-wt. The construction pLS-175 also contains two substitutions (Gs for As at positions -167 and -164), and the sequence from -158 to -144 has been deleted. However, these changes do not significantly influence the inducibility of the promoter-proximal binding sites (com- pare p.MMTV.22 and pLS-175).

For the generation of deletions and point mutations the Hindlll- BamHl fragment of the MMTV LTR from positions -430 to -70 was isolated from pTK-CAT-5A and subcloned in M13mp9 between the Hindlll and the BamHl sites. To generate a new Hindlll site upstream of the receptor binding region, the T at -190 was replaced by a C using a mutated 23 bp long oligonucleotide corresponding to LTR sequences from -202 to -180 (5’-GTAACCATAAGCTTATTTAAACC-3’).

The Sal1 site at position -155 was generated by changing the se- quence TGA (-156 to -154) to GTC, using a 32 bp mutagenic primer corresponding to the LTR sequences -171 through -140 (S-GGAA- ACCACTTGTCGACCATCCTTGTTTTAAG-3’).

Oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer 380A using the phosphite triester method in a solid-phase synthesis with P-cyanoethyl-phosphoamidites (Sinha et al., 1984). The tritylated oligonucleotides were passed through C18-SEP-PAK dispos- able columns and purified by high-pressure liquid chromatography on a Cl8 column. Alternatively, the detritylated oligonucleotides were passed through Sephadex G-50 columns and purified through poly- acrylamide-8 M urea gels (Maxam and Gilbert, 1980).

The oligonucleotide primers were annealed to the single-stranded templates, extended by the Klenow fragment of DNA polymerase, and ligated with T4 DNA ligase (Zoller and Smith, 1982). Competent JMlOl cells were transformed, phage plaques were screened using the radio- active oligonucleotide as hybridization probe, and the isolated DNA was chemically sequenced (Maxam and Gilbert, 1980).

The Rsal-BamHI fragment of the MMTV LTR (-362 to -70) contain- ing the appropriate mutations was subcloned in pTK-CAT.3 (Cat0 et al., 1986) between the filled-in Hindlll and EamHl sites in front of the TK promoter, yielding the plasmids pMMTV-20 and pMMTW20H (Figure 6). The Hindlll-BamHI fragment (-190 to -70) from these construc- tions fused to the TK promoter produces the plasmids pMMTV-21 and pMMTV-21H, respectively. The plasmid pMMTV-5A contains the wild-

type MMTV LTR sequences from -362 to -70. pBMU-37 is derived from pMMTV-21 after digestion with Hindlll and Sall, followed by the Introduction of the synthetic oligonucleotide 5’AGCTTCATCCTTG. TTTTAAGAACAGTTTGTAACCATG-3’, which is the HRE from -189 to -157 in the inverted orientation. The following deletion mutants are de- rived from pMMTV-20: pMMTV-22 contains the Sall-BamHI fragment (-155 to -7O), pMMTV-10 contains the Rsal-Sau3A fragment (-362 to -113), and pMMTV-II contains the HindWSau3A fragment, each cloned in front of the TK promoter. The plasmid pMMTV-23 is produced by digesting pMMTV-20 with Sal1 and BamHI, treating with Klenow fragment to introduce blunt ends, and then religating wtth T4 DNA ligase.

The 30 bp insertion between the two sets of receptor binding sites was created by Clal digestion of the oligonucleotide mutant pLS-147, followed by insertion of a 30 bp synthetic polylinker with Clal overlap- ping ends (5’~CGGATATCTAGACAGTCGACTTGCCGGCAT-3’) to yield pBMI-30 (Figure 7A). Subsequent digestion of this plasmid with differ- ent enzymes and religation yielded the various insertion mutants. pMMTV-wt was derived from ~13-13 (Hynes et al., 1983). The BamHI- Hinfl fragment containing the LTR sequences from -236 to -70 was treated with the Klenow fragment of DNA polymerase to generate blunt ends and then subcloned into the Hincll sites of pUC9, with the pro- moter-proximal region close to the EcoR! site of the polylinker.

DNA Binding Assays Nitrocellulose filter binding experiments with end-labeled restriction fragments were performed as previously reported (Geisse et al., 1982; Scheidereit et al., 1983). The concentrations of MgC12 and NaCl in the incubation mixture were as indicated in the appropriate figure legends. The incubations were performed at 25% for 40 min in a final volume of 50-100 aI. After filtration and washing, the bound DNA was eluted from the filters by incubation at room temperature for 60 min with 200 ~1 of 0.14% sodium dodecylsulfate (SDS) in Tris-EDTA buffer (10 mM Tris-HCI [pH 7.51, 1 mM EDTA) containing 30 Kg/ml carrier tRNA. SDS was precipitated with 0.1 M KCI on ice, and the DNA was extracted with phenol-chloroform (1:l) and precipitated with ethanol prior to second- ary restriction and/or gel electrophoretic analysis.

DNAase I footprints were performed as previously described (Scheid- ereif et al., 1983). End-labeled DNA fragments (2-4 ng) were incubated with different amounts of partially purified receptors (from O-130 ng for PR and O-700 ng for GR) in a final volume of 200 PI containing 10% glycerol (vollvol), 1 mM EDTA, 1 mM B-mercaptoethanol, 1 mM dithio- threitol, 1 mM MgCI,, 80-100 mM NaCI, and 0.1 mglml bovine serum albumin in IO mM Tris-HCI (pH 8.0). After a 40 min incubation at 25”C, the temperature was lowered to 20°C and incubation continued for 4 min before the addition of 26.5 1.11 of prewarmed DNAase I mixture: 10 U of DNAase I (Boehringer), 48 mM MgCIz, and 0.5-1.0 ug of poly(dA- dT).poly(dA-dT). Digestion with DNAase I was finished after 30 set by addition of 6 mM EDTA and extraction with phenol-chloroform (l:l). The DNA was ethanol-precipitated and analyzed in 6.5% polyacryl- amide-8 M urea gels (Maxam and Gilbert, 1980).

Methylation protection experiments were performed essentially as described previously (Scheidereit and Beato, 1984). Methylation was carried out by addition of 1 PI of 98.9% dimethyl sulfate to a 200 +I foot- print assay (see above) without poly(dA-dT).poly(dA-dT). After 30 set at 20°C, the reaction was stopped by addition of 50 VI of 1.5 M sodium acetate (pH 7.0) containing 1 M B-mercaptoethanol and 5 Kg of tRNA. After ethanol precipitation and extraction with phenol-chloroform the samples were analyzed in sequencing gels.

Transfections and CAT Assay T47D cells were grown in RPM1 1640 medium supplemented with !O% fetal calf serum and bovine insulin (0.6 pglml). DNA transfections were carried out by the DEAE-dextran method as previously described (Cat0 et al., 1986).

Generally, 2 pg of supercoiled DNA of the construction to be tested and, when needed, 2 pg of pRSV.GR (Miesfeld et al., 1986) were trans- fected per plate (10” cells). The total amount of DNA was maintained constant at 10 pg by the addition of pUC8. If used, hormones were added 20 hr after transfection. The final concentration was 10e7 M for dexamethasone and 10-s M for R5020. After 48 hr the cells were har- vested and then lysed by repeated freezing and thawing in 0.25 M su- crose, 0.25 M Tris-HCI (pH 7.8); cell extracts were prepared by centri-

Differential Regulatron of MMTV by Two Steroids 361

fugation at 10,000 x g for 5 min. The protein concentration was determined according to Bradford (1976) and the CAT assay was per- formed using the same amount of protein for each reaction (lo-20 pg), as described by Gorman et al. (1982b). Quantitation was accomplished by scratching the individual spots of the thin-layer chromatogram and counting the radioactivity by liquid scintillation. Whenever the percent conversion to acetylated chloramphenicol exceeded 80%, the CAT as- say was repeated using lower amounts of extracted protein.

For most experiments an internal marker gene, pRSV-/acZ, known to be expressed independently of hormonal treatment was cotrans- fected to correct for experimental variation. The p-galactosidase activ- ity in the extracts was determined photometrically (Hall et al., 1983), and used to normalize the CAT activity measurement. These values never varied by more than a factor of 2.

Analysis of the Transcription Start Point The probe used for analysis, SPTK-CAT, has been previously de- scribed (Cato et al., 1986). Transcription start sites for the TK-CAT fu- sion mRNA were determined using the RFrAase protection procedure and uniformly labeled antisense RNA probes (Zinn et al., 1983; Melton et al., 1984). T47D cells were cotransfected with the plasmids pTK-CAT- 5A, RSV-CAT (Gorman et al., 1982a), and RSV.GR (Miesfeld et al., 1986). Correctly initiated TK-CAT mRNA protects 210 nucleotides of the probe. RSV-CAT gives rise to a band of 150 nucleotides. Following transfection the cells were treated for 10 hr without steroids or with lo-’ M dexamethasone or 10-s M R5020. Total RNA was extracted by the guanidinium-cesium chloride method as previously described (Maniatis et al., 1982). Seventy-five microgram aliquots of total cellular RNA were hybridized for IO hr at 45% with 10 fmol (3 x 10s cpm) of probe in 20 PI of hybridization buffer (80% formamide, 0.4 M NaCI, 40 mM PIPES [pH 6.41, 1 mM EDTA). Following hybridization samples were chilled, diluted with 300 frl of buffer (0.3 M sodium acetate, 10 mM I-is, 5 mM EDTA [pH 7.01) containing IO Kg/ml RNAase A and 150 U/ml RNAase Tl, and digested for 20 min at 30°C. Reactions were adjusted to 0.2% SDS and 80 pglml proteinase K, followed by incubation for 10 min at 37°C phenol extraction, and ethanol precipitation in the pres- ence of carrier tRNA. The samples were then denatured and analyzed on a 6.5% acrylamide-8 M urea sequencing gel (Maxam and Gilbert, 1980).

Computer Graphics The computer graphic representation of the receptor binding sites was carried out with an Evans and Southerland Colour Multipicture System and a Digital Equipment Corp. VAX 11/780 computer. For display and manipulation of B-DNA double helices, the UCSF MIDAS molecular modeling software was used (Langridge et al., 1981; Bash et al., 1983).

Acknowledgments

We thank P Skroch, A. C. B. Cato, and H. Ponta(Karlsruhe) for making the oligonucleotide mutants available to us, and Hans Postma and H. Bosshard (EMBL, Heidelberg) for help in the generation of the com- puter graphic pictures. We also thank Rick Mikcisek (Heidelberg) for the SPTK-CAT probe used in RNAase mapping experiments, and Mrs. H. Ballach for help in typing the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft and from the Fonds der Chemischen Industrie.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘~adverfisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 8, 1987; revised February 19, 1988.

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