An interaction between the 5' flanking distal and proximal regulatory domains of the rat prolactin gene is required for transcriptional activation by estrogens.

An Interaction between the 5" Flanking Distal and Proximal Regulatory Domains of the Rat Prolactin Gene Is Required for Transcriptional Activation by Estrogens

Mark A. Seyfred* and Jack Gorski Department of Biochemistry (J.G.) University of Wisconsin Madison, Wisconsin 53706 Department of Molecular Biology (M.A.S.) Vanderbilt University Nashville, Tennessee 37235

mine the transcriptional response of the PRL gene to E2. (Molecular Endocrinology 4:1226-1234,1990)

In vitro studies have demonstrated that the estrogen receptor (ER) can bind to the rat PRL estrogen response element (ERE) located 1700 basepairs upstream of the transcriptional start site. However, the mechanism by which the receptor-DNA complex influences the activity of RNA polymerase located in the promoter region is not understood. To begin investigating this process, we developed cell lines derived from GH3 cells that contain steroid-responsive bovine papillomavirus minichromosomes. Within these minichromosomes is a hybrid gene composed of the 5' flanking region of the PRL gene, driving the expression of the Tn5 gene. The episomal PRL DNA sequences responded to 17/?-estradiol (E2) by increasing the rate of Tn5 gene transcription. Nucleosome mapping experiments using micrococcal nuclease demonstrated that nucleosome-like structures were assembled on the minichromosome in an ordered array separated by 150-200 basepairs of DNA. Novel S1 nuclease as well as DNase-lhypersensitive sites in the chromatin of the promoter and distal regulatory regions of the episomal PRL gene were detected by indirect end-labeling studies. The nuclease hypersensitive sites in the distal region containing the ERE were modified after treatment of the cells with either E2 or the antiestrogen 4-hydroxytamoxifen. However, only E2 treatment of cells resulted in an increase in the nuclease hypersensitivity of the promoter region and induced gene expression, while antiestrogen treatment had no effect on either parameter. This suggests that complex interactions between factors located at the distal and proximal regulatory regions ultimately deter-

INTRODUCTION

DNA sequences involved in the modulation of transcription have been localized to the 5'- and 3'-flanking regions of genes as well as within intervening sequences. Although many of the regulatory elements reside within 100-200 basepairs (bp) of the start site of transcription, a number of these elements have been located in regions more than 1 kilobase (kb) away from the promoter elements. It is not clear how proteins that bind to DNA sequences 1 kb away from the promoter influence the activity of RNA polymerase, although a number of mechanisms, such as "looping," have been postulated to explain this "action at a distance" phenomenon (1-4). Steroid hormone receptors have been shown to interact at distal (>1 kb) sites from the promoter and to modulate the transcriptional activity of the gene (5-9). These proteins contain specific trans-activating domains (10-14), but it is not clear how these domains interact with other components of the transcriptional machinery located in the promoter region to modulate the transcription of the target gene. Rat PRL gene transcription is regulated by a number of different polypeptide and steroid hormones. These hormones mediate the expression of PRL by acting through two separate regions of DNA located in its 5' flanking sequences. The proximal regulatory region is contained within the first 400 bp of 5' up-stream sequences, whereas the distal region lies between 15001800 bp 5' up-stream of the start site of transcription. The proximal region confers enhanced transcriptional

0888-8809/90/1226-1234$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society

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The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2014. at 07:08 For personal use only. No other uses without permission. . All rights reserved.

Estrogen Effects on PRL Chromatin

activity to cAMP, phorbol esters, epidermal growth factor (EGF), and TRH (15) and reduced activity to glucocorticoids (16). The distal element contains a tissue-specific enhancer (17) and a second element that confers a positive transcriptional response to cAMP, EGF, and TRH (18). LT 3 also influences the transcription rate of the PRL gene, but its response (induce or inhibit) as well as its site of action (proximal or distal elements) are controversial and appears to depend on the type of pituitary cells examined (19-22). Induction of PRL transcription by estrogen [17/3-estradiol (E2)] has been shown to be mediated through an estrogen response element (ERE), also located in the distal element. The estrogen receptor (ER) can bind to the PRL ERE in vitro; however, the affinity of this interaction has not been quantified (8, 9). Although an active hormonereceptor complex is required to observe the hormone response, the role of the hormone in the binding of receptor to DNA is not clear. Kumar and Chambon (23) reported that occupation of the ER by E2 is required for the binding of receptor to the ERE of the vitellogenin (Vit) gene. In contrast, the binding affinity of thyroid hormone and glucocorticoid and progesterone receptors for target DNA sequences is largely not affected by ligand binding (24-26). Other nonhistone proteins have been shown to modulate the binding of the E2receptor (E2R) complex to the Vit ERE (27) and to be involved in the steroid hormone response (28, 29). Although much progress has been made in describing the components involved in E2-regulated gene expression, the mechanism by which these components interact over the large distances that separate the ERE and the promoter in the PRL gene and affect the transcriptional state of the gene remains largely unknown. In an attempt to better understand the molecular events involved in the E2-induced activation of PRL gene expression, we examined the chromatin structure surrounding the two up-stream regulatory elements of the PRL gene after treatment of cells with E2 or the antiestrogen 4-hydroxytamoxifen (MHT). MHT binds to the ER with an affinity similar to that of E2 (30) and converts the ER to a form that is tightly bound to the nucleus. The MHT-ER complex has also been shown to bind to the ERE of the Vit gene in vitro (23). However, based on differences in extractability from isolated nuclei, the MHT-ER complex interacts quite differently with general chromatin compared to the E2-ER complex (31-33). In this study we used an amplified E2-responsive PRL-minichromosome system (34) to examine changes in the S1 nuclease and DNase-l hypersensitivity of the 5' flanking region of the PRL chromatin in response to hormone. Both E2 and MHT were found to modify the chromatin structure in the distal region of the episomal PRL gene. In contrast, only E2 induced hypersensitivity in the chromatin structure of the promoter region and stimulated gene expression, while MHT had no effect on the chromatin structure of the promoter region or on the transcription rate of the PRLTn5 gene. This suggests that although the ER can bind to its response element when it is complexed with either

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E2 or anti-E2, only the E2-receptor complex can interact productively with the transcriptional machinery located in the proximal promoter region and induce transcription.

RESULTS Nucleosome-Like Structures Are Assembled on PRL-Tn5-BPV Episomes It is unclear how steroid hormone receptors interact and regulate the expression of specific target genes complexed within a network of chromatin proteins in the cell nucleus. To begin investigating this process, we created cell lines derived from the rat pituitary cell line GH3 that contain steroid-responsive minichromosomes. These bovine papillomavirus (BPV)-based minichromosomes replicate episomally at an amplified copy number that ranges from 25-100 copies/cell. Within the BPV vector is a hybrid gene composed of the 5' up-stream regulatory elements of the rat PRL gene fused to the antibiotic resistance gene, Tn5 (Fig. 1). This hybrid gene responds to E2 by increasing the rate of transcription of the PRL-Tn5 hybrid gene 2- to 3-fold (34) (Fig. 2). Although Southern analysis had previously demonstrated that the PRL-minichromosomes exist in the cells as supercoiled structures, these studies could not ascertain if nucleosomes were complexed to the supercoiled DNA. To determine if nucleosome-like structures were assembled onto the PRL-minichromosomes, nuclei were isolated from G11 cells and digested with increasing concentrations of micrococcal nuclease (MNase), which cuts DNA spanning the linker region between nucleosomes. The DNA was purified and di-

SV40 poly-A

BPV Late Region

Fig. 1. The PRL-Tn5-BPV Vector The vector contains the transcriptional regulatory elements (•) of the rat PRL gene (-1960 to - 1 0 bp), which drives the expression of the reporter gene Tn5 P ) . The entire BPV genome is present in the vector (•); however, only the BPV69 genes are expressed in transfected cells. Further details on the construction of the vector and characterization of the cell lines containing the PRL-Tn5-BPV minichromosomes have been described (34).


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Fig. 2. Effects of E2 and MHT on PRL-Tn5 Transcription in G11 Cells Transcription run-on assays were performed on nuclei isolated from cells cultured in medium containing either 10% fetal bovine serum (FBS) and 10 nM E2 (FE) or 10% newborn calf serum and 10 nM E2 (E), 10 nM MHT (T), or ethanol vehicle (C) for 18 h. Rates of RNA synthesis (parts per million/30-min reaction) are given for both the Tn5 and BPV69 genes. The results are given as the mean and SE of four separately treated samples from two independent experiments.

gested with Pst\, and the sites in the PRL 5' up-stream region accessible to MNase digestion were analyzed by indirect end labeling. Figure 3 demonstrates that MNase digestion created a series of regularly spaced PRL DNA fragments, separated by 150-200 bp. Similar positioning of nucleosomes on the PRL DNA was also detected using a 32P-labeled probe corresponding to the pBR322 region immediately 5' of the PRL sequences (data not shown). This pattern of MNase cutting indicates that nucleosome-like structures are phased along the PRL 5' flanking sequences in PRLTn5 minichromosomes. MHT has been shown to be able to bind to the ER and interact with the ERE of the Vit gene in vitro. However, the transcription of reporter gene constructs containing the Vit ERE was not activated by MHT (23). Similarly, MHT had no effect on the transcription rate of the PRL-Tn5 gene in G1I cells, while E2 treatment increased the rate of transcription approximately 2-fold (Fig. 2). These results would suggest that other events, in addition to binding of the ER to the ERE, must occur to activate PRL gene transcription. To begin identifying these events, we examined the chromatin structure of the 5' up-stream regulatory region of the episomal PRL gene for alterations in response to E2 and MHT. Treatment of cells with either MHT or E2 did not appreciably affect the phasing pattern of the nucleosomes on the 5' up-stream sequences of PRL, nor did it affect the level of any specific nucleosome-protected DNA fragments (Fig. 3). This latter result indicates that the amount of any particular nucleosome associated with specific distal sequences of PRL DNA is not affected by hormone treatment. If hormone treatment resulted in the displacement of a nucleosome, there would be a decrease in the level of the corresponding DNA frag-

837

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Fig. 3. Nucleosome-Like Structures Positioned at Specific Sites on the 5' Flanking Region of the PRL Gene in the PRLMinichromosomes of G11 Nuclei were isolated from PRL-minichromosome containing G1I cells treated, as described in Fig. 2, with ethanol vehicle (C), E2 (E), or MHT (T). The nuclei were digested with increasing concentrations of micrococcal nuclease (MNase) for 10 min at 37 C, and the DNA was isolated. The purified DNA was digested to completion with Pst\ and subjected to indirect endlabeling analysis using a 255-bp 32P-labeled Pst\ (-1953 bp) to Hini\ (-1698 bp) PRL DNA fragment. DNA fragment sizes listed on the left were obtained by hybridization to a partial H/nfl digestion of a Pst\ (-1953 bp) to Pst\ (-10 bp) DNA fragment.

ment due to MNase digestion of the DNA in the nucleosome-unprotected region. S1 Nuclease Hypersensitivity of PRL-Tn5 Chromatin Although the nucleosome arrangement on the PRL-Tn5 gene was not affected by hormone treatment, subtle changes in the PRL-Tn5 chromatin structure were observed in response to E2 or MHT. These changes were detected by probing the PRL-Tn5 chromatin structure with the single stranded endonuclease, S1. When the sensitivity of the PRL-Tn5 chromatin to S1 nuclease was examined, two distinct regions of the PRL-Tn5 chromatin were found to be hypersensitive (Fig. 4, A and B). A broad region (HSII) of approximately 200 bp was located in the far 5' up-stream region of the gene nearly 1750 bp from the start site of transcription. HSII contains two sites of strong S1 hypersensitivity centered at -1580 and -1720 bp. These sites were not


1229


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located in a nucleosome-free region, but, rather, within DNA sequences bounded by nucleosomes II and III (Fig. 3). The second site (HSI) encompasses the transcriptional start site and includes the promoter elements of the PRL-Tn5 hybrid gene (34, 35). These S1 -hypersensitive sites in PRL chromatin are not intrinsic to the PRL DNA sequences or to the supercoiled nature of the plasmid. S1 nuclease did not cleave purified supercoiled plasmid preparations of PRL-Tn5-BPV in this region. However, a site mapping to -828 bp up-stream of the start site of transcription was susceptible to cleavage by S1 (Fig. 4C). A (dC-dT)n • (dA-dG)n region is located in the 5'-flanking region between -866 and -845 bp. Such sequences have been shown to be S1 sensitive in the adenovirus late promoter (36) and could potentially adopt the triple helical conformation of H-DNA (37). After treating the cells with E2 for 18 h, the HSII region became more susceptible to S1 nuclease than the control (E vs. C; Fig. 4A). The increase in hypersensitivity was seen as early as 5 h after hormone treatment and paralleled the time course of E2-induced PRL-Tn5 transcription (data not shown). Both regions (-1580 and -1720 bp) within HSII were affected equally. The S1 hypersensitivity of these same two regions in HSII was also induced by treatment with MHT (T vs. C; Fig. 4A), although MHT had no effect on the rate of Tn5 transcription (Fig. 2). Cells grown in medium containing 10% fetal bovine serum and 2 nM E2 (FE) not only had the highest rate of PRL-Tn5 transcription (Fig. 2), but the HSII chromatin in these cells were also the most sensitive to S1 nuclease (Fig. 4A). The down-stream region (HSI) of the PRL-Tn5 gene hypersensitive to S1 nuclease spans a region from 50 Fig. 4. Effects of E2 and MHT on the S1 Nuclease-Hypersensitive Sites Present in the 5' Flanking Region of the PRL Gene in PRL-Minichromosomes A, Nuclei were isolated from PRL-minichromosome-containing cells treated, as described in Fig. 2, with FBS and E2 (FE), ethanol vehicle (C), E2 (E), or MHT (T). They were then digested with increasing concentrations of S1 nuclease for 30 min at 37 C. The DNA was isolated, digested to completion with Ava\, and subjected to indirect end-labeling analysis using a 1042-bp 32P-labeled Ava\ to 8amHI BPV DNA fragment. DNA fragment sizes listed on the left were obtained by hybridization to a partial Pst\ digestion of a pBr322-BPV69 plasmid. B, Similar to A, except the isolated DNA was digested with SamHI and then probed with a 923-bp 32P-labeled Tn5 DNA fragment from Pst\ to Pstl. S1 nuclease-hypersensitive fragments more than 2500 bp in size could not be mapped to their position in PRL-Tn5 due to the location of the probe. Standard DNA fragments (lane S) were obtained by hybridization to a partial flsal digest of a plasmid containing the Tn5-SV40 DNA sequences. C, Purified supercoiled pPRL-Tn5-BPV plasmid DNA was digested with increasing concentrations of S1 nuclease for 30 min at 37 C, and the DNA was then isolated. The purified DNA was digested to completion with Pst\ and subjected to indirect end-labeling analysis using an 800-bp 32Plabeled Sat/3 A to Sau3A PRL DNA fragment to probe the blot. Standard DNA fragments (lane S) were obtained by hybridization to a partial H/nfl digestion of a Pst\ (-1953 bp) to Pst\ (-10 bp) PRL DNA fragment.


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bp 3' to approximately 200 bp 5' of the start site of transcription (Fig. 4, A and B). This region became more hypersensitive to S1 nuclease after treating the cells for 18 h with E2 compared to the control (Fig. 4, A and B; E vs. C). In contrast to the results obtained when HSII was examined, MHT had no effect on the susceptibility of HSI chromatin for S1 nuclease compared to the control (Fig. 4, A and B; T vs. C). Cells grown in FE medium were more sensitive to S1 nuclease attack of HSI (Fig. 4A) than even the E2-treated cells grown in calf serum. Therefore, the degree of S1 nuclease hypersensitivity in the promoter region of the PRL-Tn5 chromatin correlates with the rate of Tn5 transcription. DNase-l Hypersensitivity of PRL-Tn5 Chromatin Similar regions of the PRL-Tn5 chromatin that were sensitive to S1 nuclease were also hypersensitive to the endonuclease DNase I (Fig. 5). These hypersensitive regions were specific for PRL-Tn5 DNA assembled into chromatin, since these regions in isolated supercoiled plasmids were not hypersensitive to DNase-l (data not shown). Durrin et al. (38) previously reported that the HSI and HSII regions of the endogenous PRL gene in pituitary tumors of Fisher 344 rats were hypersensitive to DNase-l. However, their studies did not detect any effect of E2 on the hypersensitive sites (39). In contrast, we found that the susceptibility of HSII to DNase-l digestion increased when cells were treated

G1I

with either E2 or MHT compared to the control. As in the S1 nuclease studies, neither treatment exposed the chromatin as much as growing the cells in FE medium. Similar results were seen when the endogenous PRL gene in G1I cells was examined (data not shown). Careful examination of this (Fig. 5) and two other studies using different cell preparations reveals that MHT treatment actually causes the chromatin of HSII to be more susceptible to DNase-l attack compared to E2 treatment. This was most readily observed at 4 and 8 U/ml DNase-l concentrations. This strongly suggests that although the ER may interact in the same region of the PRL-Tn5 chromatin regardless of the ligand it is complexed with, the MHT-ER complex interacts in a slightly different manner compared to E2-ER, which leads to enhanced DNase-l susceptibility. Kumar and Chambon (23), using gel shift assays, also observed a slower migrating MHT-ER-Vit ERE complex compared to E2-ER-Vit ERE. E2 treatment also increased the DNase-l hypersensitivity of the HSI region compared to that of controls, yet E2-induced hypersensitivity remained below the level observed when the cells were grown in FE medium (Fig. 5). In contrast, MHT had little effect on DNase-l susceptibility to the promoter (HSI) region of the PRLTn5 chromatin compared to that of controls. As was observed when using S1 nuclease to probe the chromatin structure, the extent of DNase-l hypersensitivity of HSI, but not HSII, correlates with the rates of PRLTn5 transcription.

Chromatin

DISCUSSION 8 FE C E

DNase I (U/ml) T

Treatment

bp

Fig. 5. Effects of E2 and MHT on the DNase-l-Hypersensitive Sites Present in the 5' Flanking Region of the PRL Gene in PRL-Minichromosomes Nuclei were isolated from PRL-minichromosome-containing cells that were treated, as described in Fig. 2, with FBS and E2 (FE), ethanol vehicle (C), E2 (E), or MHT (T). They were then digested with increasing concentrations of DNase-l for 10 min at 37 C and subjected to indirect end-labeling analysis, as described in Fig. 4A.

Treatment of cells with E2 or MHT altered the chromatin structure of the distal regulatory element of the PRL gene, as detected by S1 nuclease or DNase-l hypersensitivity. This is in contrast to studies using the antiglucocorticoid dexamthasone 21-mesylate, where occupation of the receptor with the antihormone failed to induce DNase-l-hypersensitive sites in the glucocorticoid response element of the mouse mammary tumor virus long terminal repeat (40). The nuclease hypersensitive sites in the 5' flanking region of PRL were not located in the spacer regions between nucleosomes, but, rather, were centered within nucleosomes II and III. This region, located nearly 1750 bp up-stream of the transcriptional start site, contains the DNA sequences required for E2 induction of transiently expressed PRL reporter gene constructs and has been shown to bind ER in vitro (8, 9). Therefore, the increase in nuclease hypersensitivity after treatment of cells with E2 or MHT was probably a result of the hormone-occupied ER interacting either directly with the DNA in this distal region or possibly indirectly through a cascade of DNAbinding proteins. Richard-Foy et al. (40) demonstrated that the glucocorticoid receptor can apparently displace a nucleosome from the 5' up-stream regulatory region of the mouse mammary tumor virus long terminal re-



peat. Once the nucleosome was displaced, other transcription factors were allowed to bind to the promoter region, leading to activation of transcription. Treatment of G1I cells with E2 or MHT did not affect the location of nucleosomes on the 5' distal region of PRL, nor did it affect the amount of the DNA fragments protected by nucleosomes II or III (Fig. 3). However, it would be difficult to detect small changes in the level of a particular nucleosome-associated DNA fragment by the MNase assay. Although both E2-ER and MHT-ER appear to interact with the ERE region, only E2 treatment induces the expression of the PRL-Tn5 gene. Similar results have been seen in studies of the Vit gene (11, 23). We showed previously that the E2-induced increase in PRL transcription was not blocked by protein synthesis inhibitors; therefore, the E2 effect is not likely to be mediated through the synthesis of new proteins (34). These observations suggest that specific protein-protein interactions required for transcription activation are blocked by MHT binding to the receptor. Hansen and Gorski (41) reported that the conformation of the receptor is notably different when bound by E2 than when bound by MHT. Perhaps only the E2-dependent alteration in ER conformation exposes a transcription-activating domain in the protein, whereas the protein conformations of either the E2- or MHT-bound receptors can recognize the ERE. This is analogous to the properties of the thyroid hormone receptor, in which the primary effect of the hormone is to trigger a conformational change in the DNA-bound receptor that releases the repressor activity and reveals or induces the activation function (24). Although both E2- and MHT-bound ER recognize the same DNA sequences, the receptorchromatin complexes may be slightly different. We have shown that the chromatin structure surrounding the ERE is slightly more hypersensitive to DNase-l after treatment with MHT than after E2 treatment (Fig. 5). Similarly, Kumar and Chambon (23) observed that the MHT-ER-Vit DNA complex had a slower mobility in gel retardation assays than the E2-ER-Vit DNA complex. The functional significance of this difference in ER-DNA interactions remains obscure. More detailed analysis on the interaction of ER occupied with either MHT or E2 with the ERE in chromatin awaits in vivo footprinting analysis. In contrast to the events that occurred in the distal regulatory region, changes in both the DNase-l and S1 nuclease hypersensitivity of the gene's promoter region did correlate with rates of PRL gene expression. E2 induced an increase in S1 and DNase-l hypersensitivity in the promoter region compared to that of the control, while MHT had no effect on the nuclease hypersensitivity of the promoter region. It could be argued that the change in the chromatin structure was the result of an increase in the number of RNA polymerase molecules that traversed the Tn5 DNA template, disrupting the chromatin structure. However, since the hypersensitivity did not extend through the entire Tn5 gene and was observed 100-200 bp 5' of the transcription start site,

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it is doubtful that the hypersensitivity was caused by the movement of RNA polymerase through HSI. Since the promoter elements have no intrinsic response to E2 and do not bind the ER, the E2-induced alteration in the chromatin structure of the promoter region was probably due to interactions between the E2-ER complex on the distal element with chromatin proteins or transcription factors bound to the promoter region. These interactions then led to an activation of the RNA polymerasetranscription complex and an increase in PRL-Tn5 transcription. Even though the MHT-ER was also bound to the distal regulatory element, this complex did not result in a productive interaction between the distal and promoter regions, and thus, there was no change in the basal transcription rate of the Tn5 gene. This study also suggests that ER interaction with the ERE in the PRL gene induced a change in the chromatin to form a novel structure that was more sensitive to the single stranded nuclease, S1. S1 nuclease-hypersensitive structures have been found in the transcriptional regulatory regions of a number of genes. However, the same region of DNA that was S1 hypersensitive in chromatin was also shown to be S1 hypersensitive in isolated plasmid DNA (36, 42). In contrast, our results indicate that distal and proximal S1 nuclease-hypersensitive sites in the PRL gene were unique to PRL DNA assembled into chromatin and were not present in isolated supercoiled plasmids. Furthermore, the S1-sensitive site in plasmid DNA, perhaps due to the formation of H-DNA (37), was not sensitive to S1 nuclease in situ. Assembly of the DNA into nucleosomes may prohibit the formation of H-DNA. The S1-sensitive structure in the distal region of PRL-minichromosomes was formed regardless of whether the ligand that was attached to the receptor induced or had no effect on PRL gene expression. It is interesting to note that Lannigan and Notides (43) recently reported that in vitro the E2-ER complex binds preferentially to the coding strand of two regions of PRL DNA from -1530 to -1589 and from -1698 to -1784 bp compared to the noncoding strand or to double stranded DNA of this region. These same two regions at -1580 and -1720 bp were the sites of strongest S1 hypersensitivity in the PRL-Tn5 chromatin under the assay conditions for S1 nuclease. Even when cells were grown in medium conditions that resulted in basal Tn5 transcription rates, the chromatin in the HSI and HSII regions remained more sensitive to S1 than to other parts of the PRL-Tn5 chromatin (Fig. 4, A and B). This suggests that either these regions in the PRL-Tn5 chromatin did not immediately become tightly complexed with nucleosomes or the torsional stress localized in these regions of the supercoiled minichromosome was not completely relieved. Although approximately 90% of the unoccupied receptor is easily extracted from the nucleus of cells grown under these medium conditions (data not shown) (44), it is not known whether the unoccupied receptor or other tissue-specific factors that also interact in the HSII region (45, 46) remain associated with HSII, maintaining this


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S1 hypersensitivity. The distal S1-hypersensitive region is flanked by alternating purine-pyrimidine tracts (-1910 to -1850 and -1430 to -1260 bp) that could form ZDNA. Junctions between Z-DNA and B-DNA have been shown to be S1 sensitive (47). The unoccupied receptor or other cell-specific proteins may help in stabilizing the Z-conformation. Although it is tempting to speculate that the ER has a role in forming a novel single stranded DNA structure in the ERE, it may be the low ionic strength and low pH conditions of the assay and not the binding of the ER that forces the chromatin in this region to form an S1 -sensitive structure. The promoter region was also hypersensitive to S1, and this site does not bind ER, although other tissue-specific factors have been shown to interact in this region (46, 48, 49). In addition, the DNA sequences in the promoter region do not suggest a propensity for forming S1 -sensitive structures. Further investigations into the possibility that the ERE in HSII chromatin is unpaired would require the use of single stranded endonucleases that are active at physiological ionic strength and pH, such as Bal 31, or reagents that recognize unpaired bases, such as OsO4 (50). Although E2 induces the transcription rate of the PRLTn5 gene of cells grown in 10% calf serum, the E2induced rate of transcription remains 2-fold less than the rate observed when cells are grown in FE medium (Fig. 2). A number of components within the serum may be responsible for the additional induction. Day and Maurer (18) showed that cAMP, TRH, and EGF can all act synergistically with E2 in activating PRL gene expression. These factors can also act through the distal response element. Regardless of the identity of the activating factors, the same proximal and distal regions of the PRL gene as those observed with only E2 induction were strongly hypersensitive to S1 nuclease (Fig. 4, A and B) and DNase-l (Fig. 5). This suggests that activation of PRL gene transcription may proceed by a general mechanism independent of the type of activating factors.

Chromatin Structure Analysis S1 Nuclease The isolated nuclei were resuspended in a volume of S1 digestion buffer [30 ITIM NaOAc (pH 4.5), 30 mM NaCI, 3 mM ZnCI2, 0.2 mM EDTA, and 5% glycerol] (42) to give an absorbance of 15-20 A260 units and then incubated with increasing concentrations of S1 nuclease (Bethesda Research Laboratories, Gaithersburg, MD) for 30 min at 37 C. The reactions were stopped with sodium dodecyl sulfateproteinase-K-EDTA, and the DNA was purified by phenolchloroform extractions and ethanol precipitation. S1 nucleasespecific cleavages in the PRL-Tn5 chromatin were detected by indirect end-labeling analysis (52). Fifteen micrograms of DNA were digested with either Ava\ or BamHI and separated on 1.2% Tris, acetate, EDTA-agarose gels. The DNA fragments were transferred to nitrocellulose and hybridized (34) with either a 1042-bp Ava\ to SamHI BPV DNA probe or a 923-bp Pst\ to Pst\ Tn5 DNA probe labeled with 32P by random oligolabeling (53). S1 nuclease-sensitive sites in purified supercoiled plasmid pPRL-Tn5-BPV DNA were analyzed by digesting 10 ng plasmid DNA and 1 ^9 carrier herring sperm DNA in 50 fi\ S1 nuclease digestion buffer with increasing concentrations of S1 nuclease (Bethesda Research Laboratories) for 30 min at 37 C. The reactions were stopped with sodium dodecyl sulfateproteinase-K-EDTA, and the DNA was purified by phenolchloroform extractions and ethanol precipitation. The purified DNA was digested to completion with Pst\ and analyzed by indirect end labeling, as described above, using a 32P-labeled 800-bp Sau3A (-2334 bp) to Sau3A (-1534 bp) PRL DNA fragment. DNase-l The methods used were as described for S1 nuclease analysis, except the isolated nuclei were resuspended in nuclease digestion buffer [10% glycerol, 60 mM KCI, 15 mM NaCI, 20 mM Tris-CI (pH 7.5), and 0.5 mM dithiothreitol]. MgCI2 and CaCI2 was added to 5 and 2.5 mM, respectively. The G1I chromatin was then digested with increasing concentrations of DNase-l (Boehringer Mannheim Biochemicals, Indianapolis, IN) for 10 min at 37 C, and the DNA was isolated and analyzed, as described above, by indirect end labeling. Micrococcal Nuclease The methods used were the same as those described for DNase-l digestion, except only 2.5 mM CaCI2 was added. The G1I chromatin was digested with increasing concentrations of S7 nuclease (micrococcal nuclease; BMB) for 10 min at 37 C, the DNA was purified, and specific MNase digestion sites on the PRL-Tn5 chromatin were mapped by indirect end labeling. Fifteen micrograms of isolated DNA were digested with Pst\, and the fragments were separated on a 1.5% Tris, acetate, EDTA-agarose gel. The DNA was transferred to nitrocellulose and the membrane was probed with a 255-bp Pst\ (-1953 bp) to H/nfl (-1698 bp) PRL DNA fragment labeled with 32P by random oligolabeling (53).

MATERIALS AND METHODS Transcription Rates Cell Culture Conditions The creation and characterization of GH3 cell lines that carry the PRL-Tn5-BPV minichromosomes have been described (34). Clone G1I, used in the studies described here, contains unrearranged episomal PRL-Tn5-BPV DNA at a level of 4 0 50 copies/cell. To examine the effects of E2 or MHT, G11 cells were plated in Dulbecco's Modified Eagle's medium containing 10% fetal bovine serum, 0.6 ng/m] insulin, and 2 nM E2 (DFIE) at 3-5 x 106 cells/75-cm2 flask. After 18 h, the cells were rinsed and incubated in either DFIE or Dulbecco's Modified Eagle's medium containing 10% newborn calf serum and 0.6 ^g/n\\ insulin (DCI). After 30 h, all cells were refed their respective medium, and the DCI-fed cells were challenged with 10 nM E2 (E), 10 nM MHT (T), or ethanol vehicle (C) for 18 h. The cells were then harvested, and the nuclei prepared (51).

G11 cells were cultured, and nuclei were isolated as described above. Rates of Tn5 and BPV69 gene transcription were determined by run-on transcription assays, as previously described (34). Acknowledgments The authors thank Michael Kladde for his helpful discussions and critical review of this manuscript and Kathryn Holtgraver for assistance in preparing this manuscript.

Received April 9, 1990. Revision received May 17, 1990. Accepted May 17,1990. Address requests for reprints to: Dr. Mark Seyfred, Depart-



ment of Molecular Biology, Vanderbilt University, Box 1820 Station B, Nashville, Tennessee 37235. This work was supported by NIH Grants HD-0819 and HD07259, and NCI Grant CA-18110 (to J.G.). * Recipient of NIH Fellowship HD-06973.

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