Two-Step Synergism between the Progesterone Receptor and the DNA-Binding Domain of Nuclear Factor 1 on MMTV Minichromosomes

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1 Molecular Cell, Vol. 4, 45 54, July, 1999, Copyright 1999 by Cell Press Two-Step Synergism between the Progesterone Receptor and the DNA-Binding Domain of Nuclear Factor 1 on MMTV Minichromosomes Luciano Di Croce,* Ronald Koop,* Patrizia Venditti,* Hannes M. Westphal,* Karl P. Nightingale, Davide F. V. Corona, Peter B. Becker, and Miguel Beato* * IMT, Institut für Molekularbiologie und Tumorforschung Philipps-Universität Marburg Emil-Mannkopff-Straße 2 D Marburg EMBL Meyerhofstraße 1 D Heidelberg Germany Summary In contrast to its behavior as naked DNA, the MMTV promoter assembled in minichromosomes can be activated synergistically by the progesterone receptor and NF1 in a process involving ATP-dependent chro- matin remodeling. The DNA-binding domain of NF1 is required and sufficient for stable occupancy of all receptor-binding sites and for functional synergism. Activation of purified minichromosomes is observed in the absence of SWI/SNF and can be enhanced by recombinant ISWI. Receptor binding to minichromosomes recruits ISWI and NURF38, but not brahma. We propose a two-step synergism in which the receptor triggers a chromatin remodeling event that facilitates access of NF1, which in turn stabilizes an open nucleosomal conformation required for efficient binding of further receptor molecules and full transactivation. Introduction et al., 1990). In addition, the two proteins do not synergize in transcription assays with free DNA as template (Kalff et al., 1990; Brüggemeier et al., 1991), suggesting that chromatin is required for efficient activation of the promoter (Chávez and Beato, 1997). To study the interaction between PR and NF1 under conditions allowing dynamic transitions of nucleosome structure, we assembled MMTV minichromosomes using Drosophila embryo extracts (Becker and Wu, 1992). These extracts contain an excess of core histones, the machinery required for chromatin assembly, and several ATP-dependent chromatin remodeling activities, such as a SWI/SNF-related brahma complex (Elfring et al., 1998), NURF (Tsukiyama and Wu, 1995), ACF (Ito et al., 1997), and CHRAC (Varga-Weisz et al., 1997). Several studies have shown that chromatin assembled in these extracts behaves as a dynamic substrate for transcription factor binding and is an efficient template for cellfree transcription studies (see, for instance, (Sandaltzo- poulos and Becker, 1998). We have previously shown that MMTV minichromosomes assembled in Drosophila embryo extracts exhibit nucleosomes positioned over the MMTV promoter in a pattern similar to that described in vivo (Venditti et al., 1998). Here we show that the interaction of PR and NF1 with the MMTV promoter in minichromosomes is synergistic in terms of DNA binding and transcriptional activation, thus mirroring the in vivo situation. This in vitro system allows us to describe two mechanistically distinct and ordered steps in the recip- rocal synergism between the two factors. First, PR facili- tates binding of NF1 by an ATP-dependent process that results in marked reduction of the linking number of chromosomal DNA. Second, stable PR binding is enhanced by bound NF1 in a process that does not require the known transactivation domain of NF1, suggesting that bound NF1 acts as a wedge that keeps the nucleosome in a conformation compatible with access of PR to all HREs. Preliminary experiments suggest that the ATP-dependent machinery responsible for the PR- induced chromatin remodeling contains ISWI and the inorganic pyrophosphatase NURF38 (Gdula et al., 1998), but not SFN-2-related ATPases. Recombinant Drosoph- ila ISWI, but not purified Drosophila CHRAC (Varga- Weisz et al., 1997) or purified yeast SWI/SNF complex (Quinn et al., 1996), enhances transcriptional activation of MMTV minichromosomes depleted of endogenous chromatin remodeling activities. The MMTV promoter is organized into positioned nucleosomes and is silent in the absence of hormones (Hager et al., 1993; Beato, 1996). Apart from the core promoter elements, it encompasses within 150 bp five hormoneresponsive elements (HREs), which are bound by either the glucocorticoid receptor (GR) or the progesterone receptor (PR), upstream of a binding site for nuclear factor 1 (NF1) and two octamer motifs, all of which are required for optimal transcription (Beato, 1996). Hor- mone induction in vivo leads to chromatin remodeling (Cordingley et al., 1987), simultaneous occupancy of all the cis-acting elements on the surface of a nucleosomelike particle (Truss et al., 1995), and functional synergism Results between the corresponding trans-acting factors (Brügbut Not on Free DNA PR and NF1 Synergize on MMTV Minichromosomes gemeier et al., 1991; Chávez et al., 1995). However, on either naked DNA or purified mononucleosomes, horhuman progesterone receptor isoform B and NF1 in bac- For these experiments, we expressed histidine-tagged mone receptors and NF1 do not cooperate for binding to their respective sites in the promoter (Brüggemeier ulovirus-infected Sf9 cells and purified the recombinant proteins through column chromatography (Figure 1a). The partially purified proteins were both active as transcription factors in HeLa nuclear extracts but did not To whom correspondence should be addressed ( beato@ imt.uni-marburg.de). synergize on chromatin-free templates, confirming ear- These authors contributed equally to this work. lier observations with partially purified native factors

2 Molecular Cell 46 Figure 1. Recombinant Proteins Are Differentially Active in Transcription of Free DNA Templates and Minichromosomes (a) Recombinant PR and NF1 expressed and purified from baculovirus-infected Sf9 cells: SDS gels stained with Coomassie blue are shown. The lanes labeled PR and NF1 show recombinant PR and NF1, respectively (arrows). The molecular weight of the markers (M) are indicated in kilodaltons. (b) Influence of mutating the NF1-binding site on transcription activation on naked DNA templates by endogenous NF1 and recombinant PR in a HeLa nuclear extract. The transcripts from the wild type (wt), the mutant NF1 ( NF1), and the 77 truncated MMTV promoters are shown. Note that the NF1 transcript is 20 nt longer. (c) Effect of NF1 and PR on transcription from naked DNA templates in NF1-depleted extracts. Quantification yields a 2-fold effect of added NF1 on the wt template (lane 3), a 100- fold effect of PR alone (lane 2), and a 90-fold effect of both factors together (lane 4). (d) Effect of NF1 and PR on transcription from MMTV minichromosomes in NF1-depleted HeLa nuclear extract. Quantification yields a synergism between PR and NF1 of 20-fold at the lowest concentration of PR (25 nm) (compare lanes 3 and 6), and 4-fold at the intermediate (50 nm) and high (100 nm) receptor concentrations (compare lanes 4 and 5 with lanes 7 and 8, respectively). (e) Influence of preincubation with different factors on transcription of MMTV minichromosomes. The chromatin templates incubated with either buffer, NF1 (90 nm), PR (50 nm), or both factors together for 30 min at 26 C, prior to addition of the remaining factors or buffer and start of transcription. The time of addition of the factors is indicated on the top. (f) Influence of ATP on transcription of MMTV minichromosomes centrifuged through spin columns. The ATP-free minichromosomes were incubated with the indicated factors in the absence or presence of 4 mm ATP and used as templates for cell-free transcription in the HeLa nuclear extract. (Kalff et al., 1990; Brüggemeier et al., 1991). Transcrip- 1d). This finding supports the notion that the chromatin tion of naked DNA in the absence of added PR was structure plays a repressive role prior to hormone induc- detectable, albeit weak. Significantly, this basal tran- tion (Piña et al., 1990; Archer et al., 1991; Eisfeld et al., scription was reduced by mutation of the NF1-binding 1997). Addition of recombinant PR selectively enhanced site (Figure 1b) or by NF1 depletion of the HeLa nuclear transcription from MMTV minichromosomes in a concentration-dependent extract (Möws et al., 1994). Addition of recombinant NF1 manner, but the overall level of to the depleted extract slightly increased basal tran- transcription was lower than with naked DNA (Figure scription (Figure 1c). Recombinant PR added to the 1d, lanes 3 5). Low concentrations of PR resulted in NF1-depleted extract specifically activated transcrip- an over 10-fold induction with free DNA templates, yet tion from the wild-type MMTV promoter (wt), but not virtually no induction was detectable with MMTV mini- from a truncated promoter ( 77) lacking the hormone chromosomes (Figure 1d, lane 3). This likely reflects the responsive elements (Figure 1c). The stimulation by PR reduced accessibility of the HREs in MMTV chromatin was independent of added NF1 (Figure 1c) and was not (Piña et al., 1990; Perlmann, 1992; Chávez and Beato, affected by mutation of the NF1 site (Figure 1b). At low 1997). However, when both PR and NF1 were present, concentrations of receptor, addition of NF1 reduced the a synergistic activation of MMTV minichromosome transcription extent of transactivation (data not shown). Thus, recombinant was observed (Figure 1d, compare lanes 3 5 PR and NF1 cannot synergize on naked MMTV with lanes 6 8). This effect was particularly evident at promoter DNA, in agreement with previous results the lowest PR concentration, which alone was almost (Brüggemeier et al., 1990; Kalff et al., 1990; Möws et al., inactive but yielded a significant induction in the presence 1994). of NF1 (Figure 1d, compare lanes 3 and 6). Syner- Since the transcriptional synergism between hormone gism was dependent on the presence of the HREs (Figure receptors and NF1 depends on the nucleosome density 1d, 77 transcript) and was abolished by a mutation (Chávez and Beato, 1997), we assembled MMTV pro- of the NF1 site (data not shown). Thus, we have reproduced moter plasmids in minichromosomes and used them in a cell-free system the synergistic activation of as templates for in vitro transcription. These templates the MMTV promoter by hormone receptors and NF1 exhibit a positioned nucleosome over the MMTV promoter characteristic of the in vivo situation (Cordingley et al., (Venditti et al., 1998) and are transcriptionally re- 1987; Chávez et al., 1995; Truss et al., 1995). pressed even in the presence of added NF1 (Figure Synergistic activation of MMTV chromatin required a

3 Two-Step Activation of MMTV Minichromosomes 47 Figure 2. Differential Binding of PR and NF1 to Naked DNA and Minichromosomes Assayed by DNase I Footprinting The figure shows autoradiograms of the sequencing gels illustrating the binding of factors to naked promoter DNA (left panel), and to MMTV minichromosomes at high (400 nm, central panel) and low (100 nm, right panel) concentrations of PR. The position of the relevant cis elements is indicated on the left. The regions protected by the factors are indicated by dashed lines. The histogram shows the quantitation of the protection over each entire cis element in lanes 10, 11, and 12 as the percentage of the control lane 9. The band marked by an asterisk was used to normalize the loading of lanes. 30 min incubation of the preassembled minichromo- described for mono- and dinucleosomes, no binding of somes with either both recombinant factors or with PR NF1 to MMTV minichromosomes could be found (Figure alone prior to the start of transcription. In contrast, prein- 2, compare lanes 5 and 8; see also Piña et al., 1990; cubation with NF1 alone or addition of both factors immediately Archer et al., 1991; Eisfeld et al., 1997; Venditti et al., prior to the start of transcription was insuffi- 1998). High concentrations of PR lead to occupancy not cient to activate the chromatin templates (Figure 1e). only of the distal HRE1 but also to the central HREs The effect of PR during the preincubation required ATP, (Figure 2, lane 7), which are not accessible in mononucleosomes as demonstrated by separating the minichromosomes (Piña et al., 1990). At these concentrations, from low molecular weight molecules by centrifugation PR enabled binding of NF1 to its cognate site, and an through a spin column prior to incubation with receptor extended footprint was found in the presence of both in the presence or absence of ATP. Transcription from factors (Figure 2, lane 6). This effect required ATP, as these centrifuged minichromosomes was reduced, but no binding of NF1 was observed when apyrase was the synergism between PR and NF1 was completely added during the preincubation with PR (data not dependent on the presence of ATP during the incubation shown). Thus, PR facilitates binding of NF1 to its cog- with PR (Figure 1f). nate site on the promoter in minichromosomes, in contrast to what was observed for either naked MMTV DNA PR and NF1 Bind Cooperatively or in isolated mononucleosomes. This is likely due to to MMTV Minichromosomes the presence of ATP-dependent chromatin-remodeling Next we compared the binding of PR and NF1 to the machines in the Drosophila embryo extract (Elfring et MMTV promoter presented either as free DNA or in mini- al., 1994; Tsukiyama and Wu, 1995; Varga-Weisz et al., chromosomes. When the factors were added separately, 1995; Ito et al., 1996). These activities could be either each bound efficiently to its target sequence on recruited to the minichromosomes or activated by the the naked promoter DNA (Figure 2, compare lanes 3 receptors (Yoshinaga et al., 1992; Muchardt and Yaniv, and 4 with lane 1). However, simultaneous addition of 1993) and could facilitate NF1 binding. both factors resulted in a reduction of NF1 binding (Figure We also analyzed the binding behavior of the receptor 2, compare lanes 2 and 4) and in little if any PR at a lower and more physiological concentration, which binding (Figure 2, compare lanes 2 and 3). The effect alone does not activate transcription of chromatin tem- of NF1 on the protection of distal HREs is due to the plates (Figure 1d, lane 3). At this PR concentration, we cooperative binding of PR to the MMTV HREs (Truss et did not observe significant protection over the HREs al., 1992). Thus, NF1 competes with PR for binding to (Figure 2, compare lanes 9 and 11; see also Figure 1d, the naked promoter DNA (Brüggemeier et al., 1990). As lanes 1 and 3). However, this amount of PR was sufficient

4 Molecular Cell 48 to help NF1 access its binding site (Figure 2, compare lanes 9 and 10 with lane 12; see also the histogram with the quantitation of lanes 10 12), most likely by recruiting chromatin remodeling activities. Unexpectedly, in the presence of NF1 and low levels of PR, significant protection over the central HREs was also detected, as is particularly evident for HRE2 (Figure 2, compare lanes 11 and 12; histogram). This finding suggests not only that PR is necessary to permit NF1 binding but also that bound NF1 in turn facilitates occupancy of the HREs at limiting receptor concentrations. Therefore, there is a reciprocal and sequential synergism between PR and NF1 on MMTV promoter chromatin. Receptor Binding Does Not Alter Nucleosome Positioning but Changes the Linking Number of the Minichromosomal DNA To investigate the influence of receptor-mediated activation of the MMTV minichromosomes on chromatin structure, we analyzed the translational positioning of nucleosomes over the MMTV promoter by indirect end labeling (Nedospasov and Georgiev, 1980). In the absence of added factors, we detected a regular cleavage pattern indicative of positioned nucleosomes (Venditti et al., 1998), which was not altered by incubation with either PR alone or with PR and NF1 (Figure 3a). These experiments were performed under conditions leading to functional synergism and clear footprints over the HREs and the NF1 site. Thus, we conclude that, as reported in vivo (Truss et al., 1995), full binding of receptors and NF1 does not completely disrupt the nucleoso- mal structure and does not modify nucleosome phasing. Another method that has been used to measure chromatin remodeling for circular DNA molecules is a supercoiling assay. Each nucleosome constrains one negative superhelical turn, and nucleosome disruption or histone acetylation can lead to changes in DNA topology (Norton et al., 1989; Wong et al., 1997). Following incuba- tion of the minichromosomes with high concentrations of PR, we detected a shift in the distribution of topoisomers equivalent to the loss of several superhelical turns (Figure 3b, compare lanes 1 and 3). This effect was reproducible, but the reduction in the linking number varied between 3 and 5 among various experiments, likely reflecting the different extent of PR binding to the minichromosomes. NF1 per se did not change the distribution of topoisomers (Figure 3b, compare lanes 1 and 4) and did not alter the shift induced by high PR binding (Figure 3b, compare lanes 2 and 3). At lower concentrations of PR, the receptor alone had a weak effect on the DNA linking number, but PR and NF1 resulted in a significant change in the distribution of topoisomers (Figure 3c). This confirms the results of binding experiments in which low concentrations of PR required the presence of NF1 for stable binding to the MMTV minichromosomes. The effect of PR was not influenced by exogenously added topoisomerase I (data not shown) and required the HREs, as it was not observed with the MMTV promoter mutant truncated upstream of the NF1-binding site (Figure 3d). In addition, the effect was ATP dependent since it was abolished by addition of apyrase (Figure 3b, compare lanes 5 and 6). Therefore, we conclude that PR binding is accompanied by an Figure 3. Influence of Activation by PR on the Nucleosome Positioning and DNA Topology (a) Reconstituted wt MMTV minichromosomes were incubated with the indicated factors under conditions similar to those described in the legend to Figure 2 (central panel), digested with MNase, and phenol extracted. The isolated DNA was restricted overnight with EcoRI, resolved on a 1.2% agarose gel, and subjected to indirect end labeling analysis using a radiolabeled EcoRI-BglII probe span- ning 263 nucleotides in the CAT region, immediately downstream of the MMTV promoter (Venditti et al., 1998). Two different extents of nuclease digestion are shown. The positions of MMTV nucleo- somes are indicated on the left. (b) Reconstituted wt MMTV minichromosomes were incubated for 30 min with 100 nm PR and/or NF1. The DNA was extracted, and aliquots of 25 ng were subjected to electrophoresis on 1.3% agarose gels containing 4 M chloroquine (Krajewski and Becker, 1998). The gel was blotted onto a Quiabrane nylon-plus membrane (Quiagen), and the topoisomer distribution was revealed by hybridization with the radiolabeled EcoRI-BglII probe. Lanes 5 and 6 show samples treated with apyrase (2 U/ml) before incubation with PR. (c) Experiment similar to that described in (b) but performed with lower concentrations of PR (25 nm). The picture shows an ethidium bromide stained agarose gel. (d) Experiment similar to that described in (b) but performed with the MMTV promoter truncated upstream of the NF1-binding site ( 77). ATP-dependent remodeling of the chromatin structure manifested in a reduction of the linking number of the minichromosomal DNA. The DNA-Binding Domain of NF1 Is Sufficient for Synergism with PR To approach the mechanism by which NF1 contributes to the synergism with PR, we used a truncated NF1 protein (NF1-DBD, Figure 4a), which consists essentially

5 Two-Step Activation of MMTV Minichromosomes 49 Figure 4. The DNA-Binding Domain of NF1 Is Sufficient for Synergism with PR on Minichromosomes (a) Recombinant NF1-DBD: Coomassie-stained SDS gel with two different amounts of the recombinant protein (lanes 2 and 3) compared with markers (M) of the indicated molecular weights (kda). (b) Comparison of NF1-DBD and NF1 in terms of binding to an NF1 oligonucleotide in a gel shift assay. Quantification of the DNA in the retarded complexes as the percentage of the total DNA. (c) Synergistic activation of transcription from MMTV minichromosomes. PR was added at 100 nm and NF1-DBD at 125 nm (lane 3), 250 nm (lane 4), and 500 nm (lane 5). (d) Cooperative binding of NF1-DBD and PR to minichromosomes as determined by DMS protection. Minichromosomes were incubated with NF1-DBD (250 nm), PR (150 nm), or both factors together and subjected to DMS footprinting analysis. The histogram shows the quantitation of the protection over the relevant guanine residues in lanes 2, 3, and 4 as the percentage of the control lane 1. In HRE1, one guanine on each half of the palindrome was protected (Piña et al., 1990). In HRE5, two adjacent guanines were quantified, of which only one was protected. The bands marked by asterisks were used to normalize the loading of lanes. The ATP-Dependent Remodeling Activity Is Likely NURF To initiate the characterization of the ATP-dependent remodeling activity involved in PR activation of MMTV minichromosomes, we centrifuged the minichromosomes through a glycerol cushion. These isolated mini- chromosomes retained 15% 20% of the ISWI immunoreactive complexes present in the extract and exhibited intact chromatin structure (Di Croce et al., 1999; Figure 5a). The conditions of centrifugation lead to complete removal of brahma-containing complexes as demonstrated with antibodies to Drosophila brahma (Figure 5a) or with antibodies to rat Brg1, which cross-reacts with Drosophila brahma (Di Croce et al., 1999). In tran- scription experiments, the purified minichromosomes were still able to respond to PR and NF1 with synergistic transcriptional activation, though a relatively high background was observed (Figure 5b). These results exclude SWI/SNF-like complexes as the only activities involved in mediated transcriptional activation of MMTV mini- chromosomes by PR and NF1. To further characterize the remodeling activities in the purified minichromosomes, we treated the assembly reactions with 0.05% sarkosyl for 5 min, as this deter- gent has been shown to inactivate ISWI-containing remodeling complexes (Tsukiyama et al., 1994; Varga- Weisz et al., 1997). After removal of the detergent by centrifugation through spin columns, these minichromosomes exhibited a high background in the transcription assay but were almost completely inactive in terms of specific transcription from the MMTV promoter (Figure of the DNA-binding domain and lacks the previously described transcription activation functions (Meisterernst et al., 1989; Mermod et al., 1989). This recombinant NF1-DBD bound to the NF1 site of the MMTV promoter DNA in vitro with approximately half the affinity of the complete NF1 (Figure 4b). Similar to intact NF1, the truncated form did not bind cooperatively with PR to the naked MMTV promoter (data not shown) and was unable to access the chromatin-assembled MMTV promoter in the absence of PR (Figure 4d, lane 2). However, in combination with low concentrations of PR, the truncated NF1 did bind to its cognate site in chromatin, as demonstrated by protection against methylation by dimethyl sulfate (Figure 4d, compare lanes 1 and 4) or by DNase I footprinting (data not shown). This shows that the first step of the reciprocal synergism does not require the transactivation function of NF1. In addition, NF1-DBD facilitated binding of low levels of PR to the central HREs (Figure 4d, compare lane 1 with lanes 3 and 4; histogram). The extent of these effects was less pronounced than with the intact NF1, likely reflecting the lower DNA affinity of the truncated NF1. This finding suggests that the second step in the reciprocal synergism is also independent of the transactivation domain of NF1. Consistent with these binding studies, in vitro transcription assays show that NF1-DBD did not activate transcription or synergize with PR on naked DNA templates, and it was inactive on its own on chromatin templates (data not shown). Strikingly, however, NF1- DBD did stimulate transcription from chromatin templates in the presence of PR, only 2.5-fold less efficiently than intact NF1 (Figure 4c, lanes 3 5). We conclude that the transactivation domain within the C-terminal half of NF1 is not essential for the functional synergism with PR on chromatin templates.

6 Molecular Cell 50 Figure 5. Characterization of the ATP-Dependent Chromatin Remodeling Activity (a) Western blots of minichromosomes purified through a glycerol cushion after incubation with either PR alone or PR and NF1. The blots were incubated with the following antibodies: upper panel, Brm; middle panel, ISWI; lower panel, NURF-38. I, aliquot of the chromatin assembly reaction. C, purified minichromosomes in the absence of added factors. PR, minichromosomes purified after incubation with PR (200 nm). PR/NF1, minichromosomes purified after incubation with PR (200 nm) and NF1 (90 nm). In several experiments, incubation with NF1 alone did not change the amount of immunoreactive material (data not shown). (b) Transcription of minichromosomes isolated through a glycerol gradient. Preincubation was performed in the presence of increasing concentrations of PR (12.5, 25, 50, and 100 nm) in the absence or in the presence of NF1 (90 nm). (c) Transcription of minichromosomes treated with 0.05% sarkosyl for 5 min and centrifuged through a spin column. The minichromosomes were preincubated with ISWI (8 nm), PR (50 nm), and NF1 (90 nm) for 45 min followed by addition of the transcription mix. 5c, lanes 1 4). We used these detergent-treated mini- chromosomes for complementation studies with various ATP-dependent remodeling activities. Addition of SWI/ SNF complexes purified from yeast (Quinn et al., 1996) did not improve the transcriptional response of these minichromosomes (data not shown), further supporting the notion that SWI/SNF is not the activity supporting MMTV activation. Similarly, addition of CHRAC purified from Drosophila extracts did not improve the synergistic activation of transcription in detergent-treated minichromosomes (data not shown), suggesting that CHRAC is not able to mediate the synergism between PR and NF1. Interestingly, addition of recombinant Drosophila ISWI to the detergent-treated minichromosomes resulted in a significant and reproducible enhancement of transcriptional activation by PR and NF1, though the transcription levels were lower than with minichromosomes not treated with detergent (Figure 5c, lanes 5 8). Collectively, these results suggest that an ISWI-containing complex distinct from CHRAC is likely to mediate synergistic transactivation by PR and NF1. In addition, the complementation assays suggest that this function can be partly fulfilled by isolated ISWI. To test whether PR is able to recruit chromatin remodeling complexes to the minichromosomes, we incubated the assembly reactions with PR, alone or in combination with NF1, centrifuged the minichromosomes through a glycerol cushion, and analyzed them for the presence of remodeling factors. Incubation with PR or with PR and NF1 led to a significant and reproducible increase in the amount of ISWI bound to the minichromosomes while brahma remained undetectable (Figure 5a). The effect on ISWI content was not observed with minichromosomes assembled on the 77 MMTV promoter, which lacks the HREs (data not shown). Thus, PR is able to recruit ISWI-containing complexes to the MMTV minichromosomes through its interaction with the HREs. Since CHRAC was excluded by the functional data (see above), we were left with NURF and ACF as the two known ISWI-containing complexes in Drosophila embryos that could mediate PR-induced remodeling. As no antibodies against ACF components were available to us, we used an antibody against the p38 subunit of NURF (Gdula et al., 1998). Western blotting showed that NURF-38 is also recruited to the MMTV minichromosomes upon incubation with PR alone and that this re- cruitment is enhanced by simultaneous incubation with PR and NF1 (Figure 5a, bottom panel). These data sug- gest that one of the complexes recruited by PR to the MMTV minichromosomes is NURF, although we cannot exclude that other ISWI-containing complexes are also recruited. Discussion Our results demonstrate a chromatin requirement for the synergistic activation of the MMTV promoter by PR and NF1, which bind and function synergistically on the surface of a positioned nucleosome. Our data support the existence of at least two mechanistically distinct steps in the synergism between PR and NF1 during transactivation of MMTV chromatin (Figure 6). First, PR binding to the accessible HREs on the surface of the promoter nucleosome recruits an ATP-dependent remodeling activity. This activity changes the structure of the nucleosomal array leading to an unstable or reversible open state, which is compatible with NF1 binding. At high concentrations of PR, this open conformation allows binding of PR to the central HREs, which are masked in regular nucleosomes. At physiological con- centrations of PR, however, bound NF1 plays an important role in a second step of the synergism by stabilizing the open conformation of the nucleosome, which in turn facilitates binding of further PR molecules to the central HREs. As this effect can be obtained with the NF1-DBD, we hypothesize that complete occupancy of the HREs is sufficient for promoter activation without participation of the characterized NF1 transactivation domains. The first step in the synergism between PR and NF1 is mechanistically different from the intrinsic synergistic binding of various transcription factors to nucleosomes on artificial promoters (Adams and Workman, 1995; Polach and Widom, 1996). Similar to what we report here

7 Two-Step Activation of MMTV Minichromosomes 51 Figure 6. Two-Step Model The nucleosome core particle is shown with a central H3/H4 tetramer as a rectangle and the two H2A/H2B dimers as ellipses. RA, ISWI- containing chromatin remodeling activity. It is not known whether RA remains attached to the nucleosome during the second step. with minichromosomes in the absence of ATP, hormone receptors and NF1 do not bind synergistically to their respective sites on positioned MMTV mononucleosomes (Piña et al., 1990). The permissive action of PR on NF1 binding requires ATP-dependent activities pres- ent in the Drosophila embryo extract. It remains to be established whether the enhancing effect of the NF1- DBD on PR binding, the second step in the synergism, is dependent on ATP or simply reflects the intrinsic syn- ergism between transcription factors described with artificial promoters organized in nucleosomes (Adams and Workman, 1995; Polach and Widom, 1996). The nature of the chromatin changes taking place during PR-induced remodeling are subtle in terms of nucleosome structure, but quite dramatic in terms of DNA topology. No change in nucleosome positioning and no indication for nucleosome disruption were found, in agreement with our analysis of the chromatin changes following MMTV induction in intact cells (Truss et al., 1995). However, the topology of the minichromosomal DNA was changed upon incubation with PR and ATP, leading to a significant reduction in DNA linking number. Recent experiments in hormone-treated intact cells suggest that, in addition to the change in DNase I sensitivity around the dyad axis of nucleosome B (Truss et al., 1995), broader alterations in chromatin structure take place over the MMTV promoter (Fragoso et al., 1998), in agreement with the topological changes reported here. These changes are reminiscent of those reported for the Xenopus TR A gene promoter assembled in minichromosomes, which responds to the binding of a heterodimer of liganded thyroid hormone receptor and retinoic acid X receptor with an extensive change in DNA topology (Wong et al., 1997). Though these changes are equivalent to the disruption of six nucleosomes, the underlying molecular mechanism is unknown. The remodeled MMTV nucleosome could be a particle completely or partially depleted of histone H2A/H2B dimers, since removal of histone H2A/H2B dimers should preserve the nucleosomal structure and nucleosome positioning (Spangenberg et al., 1998) while reducing the negative supercoiling of minichromosomal DNA (Zucker and Worcel, 1990). In agreement with this idea, the MMTV promoter assembled on a positioned tetramer of histones H3/H4 can bind NF1 and PR (Spangenberg et al., 1998; unpublished data). A nucleosome-dependent functional synergism has been described with other transcription factors and promoters in cell-free transcription assays. On HIV chromatin templates, there is a synergism between the p65 subunit of NF- B and Sp1, but this synergism is associated with disruption of a nucleosome, and the implicated remodeling activities are unknown (Pazin et al., 1996). A similar synergism has been recently reported between the sterol-responsive element binding protein SREBP- 1a and Sp1 using an artificial promoter in a defined transcription system (Naar et al., 1998). This synergism also requires TFIID and a multiprotein complex containing CBP, which interacts directly with SREBP-1a, but ATP dependence has not been reported, and the proposed model implies nucleosome disruption. In a purified system, transcription of chromatin templates, but not of naked DNA, requires in addition to FACT (Orphanides et al., 1998) a novel remodeling and spacing factor, RSF, which remodels nucleosomes in an ATP- dependent process and is composed of hsfn2h and a 325,000 kda subunit (LeRoy et al., 1998). The chromatin remodeling activity mediating the synergism between PR and NF1 does not seem to be related to the SWI/SNF complex (Elfring et al., 1998), as syner- gistic transactivation was observed with minichromosomes centrifuged through a glycerol cushion, which are free of brahma-containing complexes (Di Croce et al., 1999). Moreover, immunodepletion of the SWI/SNF- related complexes with immobilized antibodies to brahma or Brg-1 did not influence the transcriptional activation of the MMTV minichromosomes by PR and NF1 (data not shown). This was unexpected since a direct interaction between steroid hormone receptors and components of the SWI/SNF complex has been documented (Yoshi- naga et al., 1992; Muchardt and Yaniv, 1993; Cairns et al., 1996; Fryer and Archer, 1998), and bound receptors are known to facilitate SWI/SNF-mediated disruption of MMTV mononucleosomes (Östlund-Farrants et al., 1997). In the Drosophila embryo extract, the activity in- volved in mediating the chromatin-dependent synergism between PR and NF1 seems to be an ISWI-con- taining complex, as the level of transcriptional activation correlated with the level of ISWI associated with the minichromosomes. Experiments with CHRAC and SWI/ SNF have shown that the core subunit containing the DNA- or nucleosome-activated ATPase activity can partly fulfill the function of the complex (Corona et al., 1999; Phelan et al., 1999). Indeed, recombinant purified ISWI was able to enhance the transcriptional synergism between PR and NF1 on minichromosomes treated with

8 Molecular Cell 52 sarkosyl, which are free of endogenous ISWI-containing DNA Binding Assays activities. Plasmid DNA or assembled chromatin was incubated for 30 min in buffer containing 110 mm NaCl without added factors or with PR, Experiments performed with minichromosomes iso- NF1, or both factors together. Where indicated, apyrase was added lated through a glycerol cushion suggest that incubation 10 min prior the addition of factors, at a concentration of 2 U/ml. with PR results in recruitment of ISWI to the minichromo- After incubation, the samples were treated with DNase I or DMS/ somes. As ISWI is only found in complexes with other piperidine and analyzed by linear PCR with [ - 32 P]-labeled primer proteins (Corona et al., 1999), we assume that PR re- A25 complementary to the region between 25 and 50 of the cruits an ISWI-containing complex to the MMTV mini- MMTV-LTR as described (Venditti et al., 1998). Due to the lack of PEG and the higher salt concentration, 4-fold higher concentrations chromosomes. Three such complexes have been deof PR than in the transcription assay were required. DNA NF1 comscribed in Drosophila embryo extracts. One of them, plexes were analyzed by electrophoretic mobility shift assay using CHRAC (Varga-Weisz et al., 1997), was tested in comple- a 20 bp oligonucleotide containing an NF1-binding site (5 mentation assays with minichromosomes deprived of CCTTTGGCACTGTGCCAAAG-3 ). endogenous ATP-dependent remodeling activities and was shown to be unable to facilitate synergistic activa- Chromatin Structure and Topology tion of transcription by PR and NF1. Of the other two The analysis of translational nucleosome positioning by indirect end labeling was performed as previously reported (Venditti et al., 1998). complexes, ACF (Ito et al., 1997) and NURF (Tsukiyama When required, topoisomerase I (Amersham Inc.) was added (3 U/ g and Wu, 1995), we could study only NURF, as a specific DNA). The topological analysis of minichromosomal DNA was perantibody directed against its p38 subunit was made formed in the presence of 4 M chloroquine as described (Krajewski available to us (Gdula et al., 1998). Incubation with PR and Becker, 1998). Quantification of the topoisomer distribution was and NF1 led to an increase in the amount of NURF-38 performed using NIH image software. associated with MMTV minichromosomes, compatible with the notion that one of the complexes recruited by Purification of Minichromosomes PR is NURF, or a NURF-related complex containing ISWI Minichromosomes were purified by 30 min centrifugation through a cushion of 47% glycerol in a table top ultracentrifuge, as published and p38. NURF has been shown to assist GAL4 in the elsewhere (Di Croce et al., 1999). early steps of chromatin transcription in vitro and could fulfill a similar function in our assays (Mizuguchi et al., Western Analysis 1997). Since the ISWI genes are highly conserved in Samples were separated on an 8% SDS gel and transferred to nylon evolution (Eisen et al., 1995), a NURF-like complex could membrane. After blocking with TBS containing 5% milk, proteins mediate hormone induction of MMTV in mice cells. were detected with the specific antibodies and peroxidase-conju- gated goat anti-rabbit IgG. The blots were washed with TBS containing Experimental Procedures 0.1% Tween 20 and developed by enhanced chemilumines- cence (ECL) reactions (Amersham). Recombinant Proteins and Plasmids The histidine-tagged human PR B (Russmann et al., 1997) was cloned Acknowledgments in a baculovirus vector and expressed in Sf9 cells. The cytosolic fraction was labeled with [ 3 H]R5020, and the receptor was purified We thank B. Gross for preparation of PR and NF1; Craig Peterson, by phosphocellulose and nickel column chromatography and stored Worcester, for purified yeast SWI/SNF complex; Carl Wu, NIH, for at 85 C. Recombinant pig NF1-C2 (Meisterernst et al., 1989; Rupp NURF38; Christian Muchardt, Pasteur Institute, for Brm; and Örjan et al., 1990) tagged with six histidines at the N terminus was similarly Wrange, Karolinska Institute, for Brg1. The work reported here purified from baculovirus-infected Sf9 cells. The DNA-binding dowas supported by grants from the European Union, the Deutsche main of rat NF1 (Dekker et al., 1996) (amino acids 1 236) was ex- Forschungsgemeinschaft, and the Fond der chemischen Industrie. pressed in E. coli and purified through chromatography on SP- L. D. C. and P. V. were fellows of the Fondazione Pasteur/Cenci- Sepharose, phenyl-sepharose, and DNA affinity chromatography Bolognetti. using a concatemer of the wild-type binding site from the adenovirus origin of replication. 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