Retinoid Receptor-Induced Alteration of the Chromatin Assembled on a Ligand-Responsive Promoter in Xenopus Oocytes

Saverio Minucci1, Jiemin Wong, Jorge C. G. Blanco, Yun-Bo Shi, Alan P. Wolffe and Keiko Ozato

Laboratory of Molecular Growth Regulation (S.M., J.C.G.B., K.O) Laboratory of Molecular Embryology (J.W, Y.-B. S, A.P.W) National Institutes of Child Health and Human Development Bethesda, Maryland 20892


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoic acid (RA) stimulates transcription from the retinoic acid receptor ß2 (RARß2) promoter in mammalian embryonal cells. Evidence by in vivo deoxyribonuclease I (DNase I) hypersensitivity assay indicates that RA treatment of these cells results in an alteration of chromatin structure in and near the promoter. To study the role of chromatin in RA-activated transcription, we assembled the RARß2 promoter into chromatin in Xenopus oocytes. Ectopic expression of RAR and retinoid X receptor (RXR) enhanced transcription without ligand, irrespective of whether chromatin was assembled in a replication-dependent or -independent manner, although ligand addition led to a further, marked increase in transcription. Moreover, expression of RAR and RXR, without ligand addition, induced DNase I-hypersensitive sites in the chromatin-assembled promoter. Futhermore, expression of RAR and RXR in oocytes led to local disruption of chromatin assembled over the promoter without ligand. Similar ligand-independent, but RXR/RAR-dependent nucleosomal disruption was observed in an in vitro chromatin reconstitution system using Drosophila embryonic extracts. Thus, unliganded receptors expressed in oocytes are capable of accessing to the chromatin-assembled promoter and activating transcription without ligand, indicating that chromatin assembly per se is not sufficient to reproduce ligand-dependent chromatin changes and promoter activation seen in mammalian cells. The oocyte system may serve as a model to study mechanisms of RA-dependent alterations of chromatin structure.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoic acid receptor ß2 (RARß2) is one of the immediate early genes induced by retinoic acid (RA) in mouse and human embryonal carcinoma cells (1, 2, 3, 4, 5) and is thought to play a role in subsequent elicitation of biological effects (6, 7). The promoter is active in early mammalian embryos (8) and myeloid cells (9). Analyses of cells with disrupted RAR genes indicate that transcription of the RARß2 promoter is mediated by multiple RARs (10). Both the human and mouse RARß2 promoters contain, within a 150-bp upstream region, a canonical RA-responsive element of the DR-5 type, to which the retinoid X receptor (RXR)/RAR heterodimer binds (1, 2, 4, 5). Additional elements present within this region are a cAMP-responsive element and an auxiliary retinoic acid response element (RARE), both of which affect RA-responsive promoter activity (3, 4, 11). We have recently studied RA-induced chromatin structure alterations in and near the RARß2 promoter in P19 embryonal carcinoma cells and found that this region of chromatin undergoes a rapid change after RA addition, as evidenced by induction of restriction site accessibility and deoxyribonuclease I (DNase I) hypersensitivity, which coincides with transcriptional activation of the promoter (12). These changes also correlated with ligand-induced in vivo footprinting of this promoter (4, 5, 9). These findings suggested that the chromatin structure formed in and around the RARß contributes to transcriptional activity. Of further interest, RA-induced restriction site accessibility and DNase I hypersensitivity seen in this promoter are remarkably similar to glucocorticoid- and progesterone-induced chromatin changes in the mouse mammary tumor virus (MMTV) promoter, which have been extensively studied in various cells both with integrated and episomal promoters (13, 14, 15, 16). The similarity is extended to ligand-dependent appearance of footprints in vivo (17). Although transcriptional activation coincides with extensive nucleosomal disruption in some genes (18, 19, 20), ligand-dependent changes in the MMTV and RARß2 promoters do not seem to accompany gross reorganization or disruption of nucleosomes over the promoter (12, 16, 21).

The Xenopus oocyte chromatin assembly system (22, 23, 24, 25) offers a versatile model to study how chromatin affects transcription. A significant advantage of this system over in vivo cell systems is that the chromatin assembly and transcription can be studied with exogenously injected templates and transcription factors expressed from injected RNAs. Additionally, with this system, one can study the activity of chromatin that had been assembled during or after template replication (22). The oocyte system has been used for analysis of chromatin assembly and transcription of the Xenopus TRßA gene (23, 24). These studies demonstrated that in the absence of thyroid hormone (T3), RXR/(thyroid hormone receptor (TR) heterodimers efficiently bind to the promoter assembled in nucleosomes and repress transcription. T3 addition results in transcriptional activation and extensive disruption of nucleosomes, although chromatin disruption by itself is not sufficient for transcriptional activation (24). These findings provided a valuable reference to the present study, which addressed RA-responsive transcription by RXR/RAR in the oocyte injection system. In this study we asked 1) whether ectopically expressed RXR/RAR heterodimers can access to a chromatin-assembled RARß2 promoter and induce changes in its structure, and 2) whether ectopically expressed RXR/RAR heterodimers repress and enhance transcription depending on ligand addition, as observed with the TRßA promoter (23, 24).

Our results show that unliganded RXR/RAR heterodimers are capable of accessing to the chromatin-assembled promoter and inducing DNase I hypersensitivity in a position comparable to that induced in P19 cells after ligand addition. Furthermore, expression of unliganded receptors led to disruption of nucleosomes as detected by altered sensitivity to micrococcal nuclease (MNase) digestion. In accordance, unliganded heterodimers stimulated transcription from the RARß2 promoter without requiring ligand, although ligand addition markedly increased levels of transcription. No evidence for transcriptional repression was observed in the RA-responsive promoter. Thus, the RARß2 promoter can be efficiently assembled into chromatin to direct transcription by the unliganded heterodimer, suggesting that ligand requirement observed in the natural environment is ascribed to another level of regulation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression and Function of Retinoid Receptors in Xenopus Oocytes
As a first step in the analysis of the function of retinoid receptors in the chromatin assembly system, oocytes injected with capped mRNAs encoding the Xenopus RXR{alpha} and RAR{alpha}1 (27) were tested for RARE-binding activity by electrophoretic mobility shift assays (EMSA). As shown in Fig. 1Go, A and B, extracts from uninjected oocytes showed no detectable binding activity for the RARE derived from the RARß2 gene (1, 2, 4, 5). Injection of increasing amounts of mRNAs for RAR and RXR led to a dose-dependent increase in the formation of a DNA-protein complex (Fig. 1AGo, lanes 2–4), which was competed by excess unlabeled RARE (Fig. 1AGo, lane 5). The complex was composed of RXR/RAR heterodimers, since it was detected after injection of mRNAs for both RAR and RXR, but not of a single mRNA for either receptor alone (Fig. 1BGo, compare lanes 2 and 3 with lanes 4 and 5). The heterodimer-RARE complex was observed even when oocyte extracts injected with single mRNA were later admixed (Fig. 1BGo, lane 5). Thus, injection of the two mRNAs into Xenopus oocytes leads to the formation of heterodimers that bind to the RARE.



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Figure 1. Expression of RXR and RAR in Xenopus Oocytes after mRNA Injection

A and B, EMSA analysis: in panel A, oocyte extracts from uninjected oocytes (lane 1) or oocytes injected with 0.02, 0.2, and 2 ng of mRNAs for RXR and RAR (lanes 2–4) were subjected to EMSA using the 188-bp RARß2 promoter fragment as a probe, in the absence (lanes 1–4) or in the presence (lane 5) of 50-fold excess unlabeled RARE oligomers. In panel B, extracts from uninjected oocytes (lane 1) or oocytes injected with 2 ng of single mRNA for RXR (lane 2) or RAR (lane 3) or 1 ng each of both mRNAs (lane 4) were tested. In lane 5, extracts from oocytes injected with a single mRNA for RAR or RXR were mixed and tested as above. The arrow indicates the position of heterodimers. The identity of the faster migrating band of weak intensity has not been determined. C and D, Transcription analysis: oocytes’ nuclei were injected with 0.2 or 2 ng of the ds-RARß2/GL3 plasmid harboring the wild-type RARß2 promoter (nt -65 to nt +14, containing one RARE), or xRARE/GL3 (the promoter with a point mutation in the RARE that abolishes receptor binding (C) and G6PD-luc (harboring the murine G6PD promoter) (D), followed by intracytoplasmic injection of mRNAs for RXR and RAR (0.2 ng RNA for lanes 1 and 4 in panel C, 0.02 ng RNA for lane 2 in panel C, and 2 ng RNA for lanes 3 and 4 in panel D). The oocytes were incubated with all-trans-RA at 10 µM for 12–15 h, and luciferase transcripts were detected by primer extension.

 
To determine whether exogenously expressed receptors stimulate transcription, oocytes were injected with the receptor mRNAs and the double-stranded (ds) plasmid DNA harboring the RARß2 promoter connected to the luciferase gene (4, 5). Oocytes were then treated with 10 µM all-trans-RA for 12 h, and the levels of luciferase RNA were measured by primer extension (Fig. 1CGo). In the mock injection, only very low levels of luciferase RNA were detected (Fig. 1CGo, lane 3). However, injection of receptor RNAs led to a dose-dependent, 20- to 30-fold increase in reporter expression (Fig. 1CGo, lanes 1 and 2). In contrast, the mutated RARß2 promoter containing a point mutation in the RARE that abolished binding of RXR/RAR heterodimer (5, 28) failed to increase reporter expression (Fig. 1CGo, lanes 4 and 5). Dot-blot analysis of the DNA extracted from the oocytes shows that similar amounts of reporter DNA were injected, confirming the validity of transcriptional activation after receptor mRNA injection (Fig. 1CGo). To establish the specificity of transcriptional stimulation, an RA-nonresponsive glucose-6-phosphate dehydrogenase (G6PD) promoter (29) connected to the luciferase gene was also tested. As shown in Fig. 1DGo, levels of luciferase RNA were unaffected by injection of the mRNAs, either in the presence or absence of RA. These results show that the RXR/RAR heterodimer expressed in Xenopus oocytes binds to the RARE and enhances transcription from an RA-responsive promoter.

RXR/RAR Heterodimers Stimulate Transcription from Both the Single-Stranded (ss) and ds RARß2 Templates: The Absence of Repression by Unliganded Heterodimer
One of the advantages of the oocyte system is that ss-DNA templates undergoing replication can be assembled into nucleosomes (22). Chromatin assembly on a ss-template follows kinetics faster than those on a ds-template, and the resultant template represses basal transcription more efficiently than a ds-DNA template. Previous work (23, 24) showed that transcription from the TRßA promoter derived from the ss-DNA template is strongly repressed by unliganded TR and RXR, and the repression is relieved by addition of T3. Thus, it was of interest to test whether RAR and RXR expressed in oocytes act in a comparable manner on the RARß2 promoter. In Fig. 2AGo, transcription from the RARß promoter was examined on the ss-template. To test the effect of the timing of receptor expression, the order of injection was varied with respect to the template DNA and receptor mRNAs; in protocol a, oocytes were first injected with the ss-template, incubated for 6 h (allowing replication-coupled chromatin assembly to complete) (22), and then they were injected with the receptor mRNAs. In protocol b, oocytes were first injected with the receptor RNAs, incubated for 6 h (allowing sufficient levels of RXR and RAR expression), and then injected with the DNA template. In experiment a, newly synthesized receptors would have to access to the RARE on a fully assembled template to exert their activity, whereas in experiment b, presynthesized receptors might have access to the RARE during chromatin assembly. As shown in Fig. 2AGo (lanes 1 and 2), without injected receptor mRNAs, ss-DNA template produced very low levels of transcripts. Addition of RA did not significantly increase transcription; depending on the batches of oocytes, a low level of RA inducibility was occasionally found, presumably due to the low level expression of endogenous receptors (data not shown). However, injection of receptor mRNAs significantly increased transcription even in the absence of ligand (lanes 3 and 5), which was observed regardless of whether the template was injected before or after mRNA injection. Transcriptional repression was not observed by the unliganded receptors under these conditions. Furthermore, addition of RA caused a marked increase in transcription (Fig. 2AGo, lanes 4 and 6), again regardless of the order of injection (compare lanes 3 and 4 with lanes 5 and 6). These results show that unliganded RAR and RXR stimulate RARß2 transcription from the template that was assembled into chromatin during replication, and addition of ligand only increases levels of transcription. Transcription of the G6PDH gene (23), measured as a control, was comparable in these oocyte samples (Fig. 2AGo). The effect of ligand was also tested on transcription from a ds-RARß2 template. As shown in Fig. 2BGo, without receptor mRNA injection, transcription was minimal in the absence of ligand, but addition of RA led to some transcription in these samples, which could be attributed to the low level expression of endogenous receptors in oocytes (lanes 1 and 2). Similar to the results with the ss-DNA template, injection of receptor mRNAs stimulated transcription from the ds-template in the absence of ligand, and addition of RA further increased transcription (Fig. 2BGo, lanes 3–6). Again, no transcriptional repression was seen by unliganded receptors. The outcome was the same, irrespective of whether receptor mRNAs were injected before or after the injection of the template. The transcript levels of the endogenous histone (H4) gene (23), tested as a control, were comparable in the these oocyte samples (Fig. 2BGo).



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Figure 2. Functional Analysis of RXR/RAR Heterodimers in Xenopus Oocytes

A, Oocytes were injected with the ss-plasmid RARß2/GL2 containing the wild-type RARß2 promoter (nt -104 to nt +14) and the ds-G6PD-luc plasmid. In lanes 3–4, oocytes were injected with 0.2 ng of receptor mRNAs 6 h before injection of plasmid DNA. In lanes 5–6, oocytes were injected with DNA 4 h before injection of the mRNAs. Oocytes were incubated with (+) or without (-) 10 µM all-trans-RA for overnight and luciferase RNA was detected by primer extension. B, Oocytes were injected with the ds-plasmid RARß2/GL2. In lanes 3–4, oocytes were injected with 0.2 ng of receptor mRNAs 6 h before injection of plasmid DNA. In lanes 5–6, receptor mRNAs were injected 6 h after plasmid DNA injection. Luciferase transcripts were detected as above. As a control, transcripts for endogenous H4 were detected by primer extension. C, Oocytes were injected with the ds-plasmid RARE2tk-luc (containing two copies of the RARE in front of the tk promoter fused to the luciferase gene) 6–8 h before injection of 0.2 ng of receptor mRNAs.

 
To examine whether transcription mediated by RXR and RAR is influenced by the context of the basal promoter, we tested another ds-template in which two copies of the RARE (DR-5) were fused to the herpes simplex virus thymidine kinase (TK) promoter (5, 28). As shown in Fig. 2CGo, in the absence of receptor mRNA injection, transcription was low but was increased after addition of RA, analogous to the RARß2 promoter above. Similarly, injection of receptor RNAs greatly increased transcription even in the absence of RA, although RA gave an additional increase in transcription. As in Fig. 2BGo, transcript levels of control histone H4 were comparable in these samples. These results indicate that both unliganded and ligand-bound RXR/RAR heterodimers stimulate transcription from RA-responsive promoters, regardless of whether the chromatin was assembled during or after DNA replication. As detailed in Discussion, these results are similar to the observations made in an in vitro transcription system (11).

Binding of RXR/RAR Heterodimers to the Nucleosome-Associated RARß2 Promoter in Vitro
The above experiments suggest that RXR/RAR heterodimers are capable of accessing to the retinoid-responsive promoters assembled in oocyte chromatin. To ascertain the accessibility of the heterodimers to the chromatin-associated template in vitro, EMSAs were performed with a 188-bp RARß2 promoter fragment complexed with monosomes and recombinant RXR/RAR heterodimers. The fragment prepared by PCR encompassed from nucleotide (nt) -128 to nt +60 of the RARß2 promoter, which includes the main RARE (-51 to -33) and the auxiliary RARE (-79 to -61) (11). This fragment was assembled into nucleosomes using histone octamers prepared from chicken erythrocytes (30). As shown in Fig. 3AGo (lanes 1–4), incubation of free DNA fragment with increasing concentrations of heterodimers led to the formation of multiple complexes representing binding to the two RAREs. Two bands seen in lane 3 likely represent heterodimer binding to one or two RAREs. As shown in Fig. 3AGo (lanes 5–9), the monosome-associated DNA fragment without receptors migrated to position b (compare free DNA that migrated to position a). Addition of increasing amounts of RXR/RAR heterodimer led to the formation of more slowly migrating bands (shown as d–f). The formation of receptor-DNA complexes in monosome-assembled promoter was observed with approximately 10-fold higher amounts of heterodimers than in free DNA. Bands d–f probably represent heterodimers complexed with the monosome-assembled promoter, whereas band c may represent heterodimers bound to a small amount of residual free DNA. The heterodimer-monosome DNA complexes (d and e) were eliminated by excess RARE oligomers of the DR-5 type, but not control oligomer (Fig. 3BGo). As expected from EMSA data with an RARE probe (Fig. 1Go), addition of RA did not affect the complex formation (data not shown). These results show that the monosome-bound RARß2 promoter permits binding of the RXR/RAR heterodimer through the RAREs in the absence of ligand.



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Figure 3. EMSA Analysis with a Monosome-Bound RARß2 Promoter in Vitro

An end-labeled 188-bp RARß2 promoter fragment (-128/+60) generated by PCR was assembled into nucleosomes in vitro. A, Increasing amounts of purified recombinant RXR and RAR were incubated with a free RARß2 fragment (lanes 1–4) or with a fragment reconstituted into a monosome (lanes 5–9) and analyzed on a 4% native polyacrylamide gel. The free fragment is marked by a; monosome-bound DNA fragment without receptors is marked by b. Heterodimer/DNA complexes are shown as c–f. B, Competition analysis. A free (lane 1) or monosome-bound RARß2 fragment was incubated with RXR and RAR in the absence (lane 2) or presence of 34-bp RARE oligomers (lane 3) or control oligomers of the YY1-binding site (lane 4). b Represents monosome-bound DNA. c Is likely heterodimers bound to free DNA. d–f Represent heterodimer-monosome-DNA complexes.

 
Heterodimer-Induced Chromatin Alterations in Xenopus Oocytes
Oocyte injection data in Figs. 1Go and 2Go and binding data in Fig. 3Go indicate that the chromatinized template is accessible to receptors both in the absence and in the presence of the ligand. Previously, however, an alteration of chromatin structure was observed in and around the RARß2 promoter in a RA-dependent manner in mouse embryonal cells (12). A similar hormone-dependent chromatin structure transition has been noted for the MMTV promoter in various cells (13, 14, 16, 17). Thus, it was important to assess whether the chromatin assembled in Xenopus oocytes undergoes a structural alteration after heterodimer expression and whether such changes, if observed, are ligand dependent. Oocytes were injected with the ss-RARß2 template before or after injection of receptor mRNAs. Oocytes were then homogenized and digested with increasing concentrations of DNase I. DNA was purified, cut with EcoRI in vitro, and subjected to Southern blot hybridization with a 32P-labeled, 600-bp HindIII-EcoRI fragment as a probe. As shown in Fig. 4Go (lanes 1–8), injection of the template alone generated several diffuse bands of low intensity, and the addition of RA did not affect the digestion pattern. However, injection of receptor mRNAs led to the appearance of two hypersensitive bands, which mapped within the RARß2 promoter (arrows in Fig. 4Go). The appearance of the hypersensitive bands was observed both when template was injected before and after mRNA injection and was not affected by addition of RA. Digestion of purified plasmid DNA presented in lanes 25 and 26 shows a uniform distribution, indicating that the patterns observed with the oocyte samples are not due to intrinsic sensitivity of the promoter to DNase I. Thus, heterodimer expression leads to the generation of DNase I-hypersensitive sites in the RARß2 promoter, indicating that the chromatin structure undergoes a marked change upon binding of receptors to the template. Similar sites in the RARß2 promoter were found to be sensitive to DNase I in P19 cells, but were detected in a RA-dependent manner (12)



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Figure 4. Induction of DNase I Hypersensitivity by RXR/RAR Heterodimers

Oocytes were injected with a ss-RARß2/Gl2 plasmid. In lanes 9–16, they were injected with 0.2 ng of receptor mRNAs before injection of plasmid DNA. In lanes 17–24, oocytes were injected with plasmid DNA before injection of receptor mRNAs. After overnight incubation with 10 µM all-trans-RA, oocyte homogenates were digested with DNAseI (0.2 to 2 U). Purified DNAs were digested with EcoRI in vitro, resolved on an agarose gel, blotted, and hybridized with an end-labeled EcoRI/HindIII probe (shown in the bottom). Plasmid DNA (lanes 25 and 26) was digested with DNaseI and EcoRI and run as a control. The arrows indicate the position of the hypersensitive sites induced after receptor expression.

 
Heterodimer-Induced Nucleosomal Disruption in Xenopus Oocytes
Previous studies with TR/RXR heterodimers showed that nucleosomes organized over the TRßA promoter are extensively disrupted after T3 addition even in the absence of transcription (23, 24). Nucleosomal disruption coincidental with gene activation has been shown to occur in other promoters as well (18, 19, 20). However, for some ligand-responsive promoters, nucleosome disruption does not seem to be readily observed (12, 16, 21). Here we studied whether receptor expression changes configuration of nucleosomes over the RARß2 promoter assembled in oocytes. Oocytes were injected first with the ss-RARß2 template, followed by second injection with receptor RNAs 6 h later. Oocyte homogenates were digested with increasing amounts of MNase, DNA purified, and subjected to Southern blot analysis with a fragment encompassing the RARß2 promoter (-128/+60). As shown in Fig. 5AGo, oocyte samples injected with the template alone without receptor generated a MNase digestion pattern indicative of ordered nucleosomal organization (with the position of mono-, di-, and trisomes shown by arrowhead). This pattern was not affected by addition of RA. Oocyte samples injected with receptor RNAs appeared to be more extensively digested than samples without receptor injection and produced a distinct band below the size of a monosome (lanes 4–6, arrow in Fig. 5AGo). Very similar digestion patterns were observed after treatment with RA (lanes 7–9). However, when these samples were probed with a control fragment derived from the plasmid vector without the RARß2 promoter (Fig. 5BGo), the MNase digestion patterns were essentially the same among uninjected oocytes and those injected with receptor mRNAs (compare lanes 1–3 with lanes 4–6), and RA treatment did not alter the digestion pattern (lanes 7–9). These results suggest that expression of RXR/RAR heterodimers leads to disruption of local nucleosomes, which is unaffected by ligand.



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Figure 5. Nucleosomal Disruption Induced by Unliganded RXR/RAR: Analysis with the Xenopus Oocyte System

Oocytes were injected with the ss-RARß2SK plasmid 3 h before injection of mRNAs (2 ng) and incubated overnight in the presence or absence of 10 µM all-trans-RA. Oocyte homogenates were digested by MNase (2, 6, or 30 U), and purified DNAs were analyzed by Southern blot hybridization using a probe corresponding to the RARß2 promoter (nt -128 to nt +60, panel A) or with the vector plasmid (panel B).

 
Heterodimer-Induced Nucleosome Disruption in an in Vitro Reconstitution System
Nucleosomal disruption by RAR and RXR observed over the RARß2 promoter in oocytes seemed to differ somewhat from that by TR and RXR, where addition of T3 dramatically increased disruption of nucleosomes assembled over the TRßA gene (23, 24).

To further study the possible nucleosomal disruption by RXR/RAR heterodimers, and the effect of ligand, we used an in vitro chromatin reconstitution system developed by Tsukiyama et al. (31) that utilizes Drosophila embryo extracts. This cell-free system has been investigated for the nucleosomal organization of the hsp70 gene, which led to recent isolation of nucleosome-remodeling factors (32). In the experiments shown in Fig. 6Go, the RARß2 gene fragment from nt -630 to nt +570 in the pSK plasmid was reconstituted into chromatin with the Drosophila extracts to which recombinant RXR and RAR were added before (lanes 4–6) or after chromatin reconstitution (lanes 7–12). Reaction mixtures were digested with MNase and subjected to Southern blot analysis with a probe corresponding to the RARß2 promoter (Fig. 6AGo) or to a region farther upstream from the promoter, tested as a control (Fig. 6BGo). Similar to the previous observations with the hsp70 promoter (31, 32), incubation with the Drosophila embryo extracts, without receptors, led to the assembly of an ordered nucleosomal array on the RARß2 template (lanes 1–3 in Fig. 6Go, A and B). Addition of RAR and RXR drastically altered MNase digestion pattern in the RARß promoter region; it generated extensive subnucleosomal fragments with a concomitant reduction in the oligosomal and monosomal complexes, which was seen in a MNase dose-dependent manner (Fig. 6AGo, lanes 4–12). Similar subnucleosomal fragments were reported to be produced after factor binding to the hsp70 gene (32). Essentially the same pattern of alterations in the MNase digestion was observed after RA addition (compare lanes 4–5 with lanes 7–9 in Fig. 6AGo), irrespective of whether receptors were added before or after chromatin assembly. In contrast, the MNase digestion pattern for the farther upstream region (Fig. 6BGo) was not affected by addition of heterodimers and/or of RA. These results are consistent with the data in Fig. 5Go and show that RXR/RAR heterodimers are capable of disrupting local nucleosomes assembled over the promoter, which is not influenced by RA.



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Figure 6. Nucleosomal Disruption Induced by Unliganded RXR/RAR: Analysis with the in Vitro Chromatin Reconstitution System

The plasmid RARß2SK containing the RARß2 promoter (from nt -630 to nt +570) was assembled into chromatin in vitro using Drosophila embryo extracts (Materials and Methods). In lanes 4–6, purified RXR/RAR heterodimers were added 6 h before assembly, whereas in lanes 7–12 RXR/RAR was added 6 h after assembly and incubated in the presence or absence of 10 µM all-trans- RA for 40 min. Reactions were digested with 25 U of MNase at room temperature. Purified DNAs were analyzed by Southern blot hybridization using an oligonucleotide probe corresponding to the RARE in the RARß2 promoter (nt -58 to nt -28, panel A) or to the region nt -550 to nt -575 (panel B). The positions of mono-, di-, and trisomes are marked with open arrowheads.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The present study found that the RARß2 promoter assembled into chromatin in Xenopus oocytes drives transcription through ectopically expressed RXR and RAR. This process coincides with ligand-independent alteration of local chromatin structure. The transcription and chromatin changes caused by unliganded RAR/RXR heterodimers revealed in this system share some features with, but differ in other aspects from, those observed by TR/RXR heterodimers in the oocyte model (23, 24) and those in P19 embryonal cells (12).

Transcriptional Activation by Unliganded RXR/RAR Heterodimers: The Lack of Transcriptional Repression
Expression of unliganded RXR and RAR led to stimulation of transcription from both the RARß2 and RARE-TK promoters under all conditions tested, i.e. where the template was preassembled before receptor expression, or assembled in the presence of receptors, and where the injected template was a ss- or ds-plasmid (Figs. 1Go and 2Go). Nevertheless, addition of RA led to a further increase in transcription in all of these cases. A notable aspect of these findings is the lack of transcriptional repression by unliganded RXR/RAR, which might have been anticipated as unliganded TR/RXR repressed transcription from the TRßA promoter, particularly strongly when the ss-DNA template was assembled into chromatin, and more weakly with a ds-template (23, 24). In all cases, repression was found neither with the RARß2 nor with the RARE-Tk promoter (Figs. 1Go and 2Go). Repression by RXR/RAR might also have been expected, based on the previous study showing that TR and RAR repress transcription from chimeric promoters transfected into mammalian cells (33, 34).

Although detailed information on retinoid levels in Xenopus oocytes is not available to us, the lack of repression is not easily explained by the possible presence of endogenous retinoids, since addition of RA caused marked transcriptional stimulation, occasionally even in oocytes that had not been injected with receptor mRNAs (Fig. 2BGo), where receptor concentrations were much lower than in the presence of ectopic receptors. These observations indicate that transcriptional repression is not an obligatory function of unliganded receptors, but may depend on specific promoter architecture. Moreover, results of transcription analysis obtained in the oocyte system bear a remarkable resemblance to those observed by an in vitro transcription system described by Valcarcel et al. (11), in which transcription from the RARß2 promoter was stimulated by the RXR/RAR even in the absence of ligand, although ligand addition increased levels of transcription. Thus, the RARß2 promoter assembled in the oocyte system (and the in vitro transcription system) appears to obliterate ligand requirement imposed in the native cellular environment, presumably because the template assembled in this (and the in vitro transcription) system is readily accessible to the unliganded RXR/RAR heterodimer, while that in the native environment requires a ligand-dependent step to bind to heterodimers (4). Unliganded heterodimers, once gaining access to the template, may moderately activate transcription perhaps through the AF-1 domain, and/or ligand independent interaction with basal transcription factors even in the absence of coactivator recruitment.

Ligand-Independent Alteration of Chromatin: DNase I Hypersensitivity
In several models, induction of DNase I hypersensitivity is associated with transcriptional activation (35, 36, 37). DNase I hypersensitivity is induced in a hormone-dependent manner in the MMTV promoters and is accompanied by factor occupancy (14, 15, 16). Thus, it is reasonable to assume that induction of DNase I hypersensitivity by unliganded RXR/RAR seen in the present work is a consequence of their binding to the template. Similarly, DNase I-hypersensitive sites are shown to be induced in the TRßA promoter by unliganded TR/RXR (24); however, although T3 enhances the levels of hypersensitivity in the TRßA promoter, RA had no effect on the RARß2 promoter. In contrast to the apparent lack of ligand effects, previous analyses of the RARß2-luciferase gene integrated into P19 embryonal cells showed ligand-dependent induction of DNase I hypersensitivity (12). Interestingly, the hypersensitive sites mapped in the present study are very close, if not identical, to those mapped in P19 cells (12). Since the promoter and reporter fragment analyzed here are the same as that in Ref. 12, and since DNase I hypersensitivity coincided with ligand-dependent occupancy of the receptors to the promoter in P19 cells, it is likely that in the oocyte system unliganded receptors are capable of binding to the template, bypassing the ligand requirement present in P19 cells. It is possible that the oocyte system may not contain sufficient levels of a cofactor(s) that governs ligand-dependent accessibility of receptors. The absence of ligand dependence may alternatively be attributed to the properties of histones assembled in this system, such as levels of acetylation or deacetylation of nucleosomal histones, which are shown to influence accessibility of transcription factors to the template (26, 38), or to the availability of linker histones (39, 40). In either case we feel that chromatin changes found in this system are unlikely to be attributed to the architecture of the promoter, i.e. the mini-chromosome rather than an integrated sequence, since chromatin changes comparable to integrated counterparts have been reported in the MMTV promoter in the episomal plasmids (14).

Nucleosomal Disruption Induced by Unliganded RXR/RAR
Expression of RXR/RAR heterodimers markedly increased MNase sensitivity over the RARß2 promoter (Fig. 5Go), indicating that binding of unliganded heterodimers to the promoter leads to nucleosomal disruption in a locally restricted manner. A comparable local nucleosomal disruption was observed by unliganded RXR/RAR heterodimers in an in vitro model of chromatin reconstitution using Drosophila extracts. This in vitro system has been extensively analyzed by Tsukiyama and co-workers (31, 32), who isolated a nucleosome-remodeling factor, NURF, which disrupts nucleosomes assembled over the hsp70 promoter. It is possible that such a nucleosome-remodeling factor(s) is recruited by unliganded RXR/RAR in the oocyte system as well as the in vitro system. Although RA had no detectable effects on nucleosomal disruption over the RARß2 promoter, T3 dramatically increased chromatin disruption over the TRßA promoter in the oocyte system (23, 24). At present, the basis of the different behavior of ligands seen for the RARß and TRßA promoter is not clear. However, it is interesting to note that despite conspicuous nucleosomal disruption observed for this and the TRßA promoter in the oocyte system (23, 24), ligand-dependent nucleosomal remodeling/disruption has not been readily demonstrated for the MMTV and RARß2 promoters in mammalian cells in vivo (12, 16). Considering extensive nucleosomal disruption observed in other promoters (18, 20), it may be reasonable to assume that nucleosomal disruption is an integral part of chromatin changes associated with gene activation, but can be obscured by other causes for some promoters such as the MMTV and RARß2 in P19 cells: in vivo nucleosomal disruption may occur only transiently and may not be easily detected by the standard MNase digestion assay. It is also possible that other factors mobilized to the promoter may reduce sensitivity to MNase digestion. In any event, although ligand addition had no apparent effect on the extent of nucleosome disruption and of DNase I hypersenstivity, it did enhance transcription from the RARß2 promoter. These results suggest that nucleosomal disruption is not a consequence of transcription, although it may be a prerequisite for transcription. Consistent with this notion, nucleosomal disruption occurred even in the absence of transcription (23). Thus, in the oocyte system RA might exert its effects on an event that follows receptor- induced chromatin alterations, but which affects transcription.

In summary, the Xenopus oocyte system employed in this work shows that unliganded RXR/RAR heterodimers can induce extensive chromatin alterations over the RARß2 promoter and stimulation of transcription, bypassing the ligand requirement present in the native environment. This system might serve as a valuable model to investigate the molecular mechanisms underlying receptor-induced chromatin alterations.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
Plasmids RARß2SK, RARß2GL2, RARßs/GL3, xRAREs/GL3, RARE(2)tk-luc, containing the murine RARß2 promoter, and G6PD-luc have been described (4, 5, 28, 29). The ss-plasmids were prepared from the phagemids induced with VCS M13 as described (23). mRNAs encoding Xenopus RXR{alpha} and Xenopus RAR{alpha}1 mRNAs were prepared from plasmids kindly provided by Bruce Blumberg (27) using the Message Machine kit (Ambion, Austin TX) according to the manufacturer’s protocol.

Microinjection of Xenopus Oocytes
These procedures were carried out as described (22, 23). The DNAs (1.1 ng per oocyte for ss-DNA, 2.2 ng per oocyte for ds-DNA) were injected into the oocyte nuclei, whereas RNAs (amounts indicated in each assay) were injected into the oocyte cytoplasm. After injection, the oocytes were incubated at 18 C overnight in buffer supplemented with antibiotics (22). The amounts of injected DNA were quantified by dot-blot or Southern blot hybridization of purified DNA (23).

Transcription Analysis
RNAs were prepared from 15–20 injected oocytes (15, 16, 17, 18, 19, 20) using the RNAzol (Tel-test, Inc., Friendswood, TX) as described (23). Luciferase transcripts were detected by primer extension using the primer 5'-CCAGGGCGTATCTCTTCATAGCC-3', corresponding to nt position +126 to +149 using the amounts of RNA equivalent to two oocytes. Histone H4 transcripts, measured as an internal control, were detected with the primer described in Ref. 24.

EMSA Assay with a Nucleosome-Reconstituted RARß2 Probe
A 188-bp fragment from the RARß2 promoter (-128/+60) was generated by PCR with one of the two primers end-labeled with 32P. The PCR products were purified and reconstituted with the octamer exchange method as described, using histone octamers prepared from chicken erythrocytes (23, 30). The monosome fraction was then purified by sucrose gradient separation as described (30). Recombinant murine RXRß and human RARß with a histidine tag at the N-terminal end were produced in a baculovirus vector and purified on nickel-affinity columns. Their ability to heterodimerize with each other and to specifically bind to RARE oligomers was verified for each receptor preparation: the details of vector construction, receptor purification, and RARE binding activities will be described elsewhere (J. C. G. Blanco, S. Minucci, X.-J. Yang, K. K. Walker, H. Chen, R. M. Evans, Y. Nakatani, and K. Ozato, unpublished). EMSA analysis was performed as described (23).

DNAse I Hypersensitivity Analysis
These analyses were performed essentially as described (24). Briefly, 20 injected oocytes were homogenized in digestion buffer (10 mM HEPES, pH 8.0, 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.1% NP40, and 5% glycerol). Equal aliquots of the extracts (5 oocyte equivalent) were digested with 2.5, 5, 10, and 20 U of DNAse I (GIBCO-BRL, Bethesda, MD) for 2.5 min at room temperature. DNAs were purified, digested in vitro with 10 U of EcoRI and 2 µg RNase A, and then resolved on a 1% agarose gel, blotted to Hybond N membranes (Amersham, Arlington Heights, IL), and hybridized with a 32P-labeled HindIII-EcoRI fragment corresponding to the luciferase gene. As a control for deproteinated DNA, DNAs purified from injected oocytes were digested with DNase I at a 100- to 200-fold lower concentration than that used for oocyte samples.

MNase Digestion Assay in Xenopus Extracts
These assays were performed as described (23). Briefly, 20 injected oocytes were homogenized in digestion buffer as above. Equal aliquots of the extracts (6.7 oocyte equivalents) were digested with 2, 6, and 30 U of MNase (Boehringer Mannheim, Indianapolis, IN) for 20 min at room temperature. DNAs were purified and resolved on a 1.3% agarose gel, blotted to Hybond N membranes, and hybridized with the indicated probes.

MNase Digestion Assay with in Vitro Reconstituted Nucleosomes
Chromatin assembly extracts were prepared from pre-blastoderm stage Drosophila embryos as described (31, 41). The extracts were incubated with 100 ng RARß2SK plasmid DNA and 650 ng FX174 DNA for 6 h at 26 C. One hundred to 200 ng of purified recombinant RXR/RAR heterodimers (see above) were added to the reaction at the indicated times and incubated for 30 min at 26 C. MNase digestion was performed with 25 U. DNAs were then purified, resolved on a 1.3% agarose gel, and hybridized with indicated probes.


    ACKNOWLEDGMENTS
 
The authors thank Drs. Ben-Zion Levi, Nicoletta Landsberger, Toshio Tsukiyama, Carl Wu, Igor Dawid, Giuseppe Martini, and Hong Chun for valuable reagents and advice and Robin Kahn for technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Keiko Ozato, Laboratory of Molecular Growth Regulation, Building 6, Room 2A01, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2753.

1 Present address: European Institute of Oncology, Department of Experimental Oncology, 20141, Milan Italy. Back

Received for publication October 3, 1997. Revision received November 20, 1997. Accepted for publication December 9, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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