An Array of Positioned Nucleosomes Potentiates Thyroid Hormone Receptor Action in Vivo*

Fyodor D. UrnovDagger § and Alan P. WolffeDagger

From the Dagger  Sangamo Biosciences, Point Richmond Tech Center, Richmond, California 94804 and § Laboratory of Molecular Embryology, NICHD, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, January 31, 2001, and in revised form, March 15, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The assembly of the genome into chromatin imposes a poorly understood set of rules and constraints on action by regulatory factors. We investigated the role played by chromatin infrastructure in enabling an acute response of the Xenopus TRbeta A gene to thyroid hormone receptor (TR), an extensively studied member of the nuclear hormone receptor superfamily. We found that in addition to the known TR response element (TRE) in the promoter, full range regulation required an upstream enhancer that contained multiple nonconsensus TREs and augmented ligand action at high receptor levels. An array of translationally positioned nucleosomes formed over the TRbeta A locus in vivo; unliganded TR engaged this array in linker DNA between two nucleosomes and via TREs on the surface of histone octamers. Remarkably, assembly of enhancer DNA into mature chromatin potentiated binding by TR to its target response elements and enabled a greater range of regulation by TR than was observed on immature chromatin templates. Because assembly of enhancer DNA into chromatin increased TR binding to the nonconsensus TREs, we hypothesize that chromatin disruption targeted by liganded TR to the enhancer may lead to receptor release from the template and to an attenuation of response to hormone.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The study of transcriptional regulation makes extensive use of artificial reporter constructs that contain several binding sites for a particular protein and are introduced into cells by transfection. This useful approach has two limitations. (i) Bona fide targets of eukaryotic transcription factors are assembled into chromatin, whereas nonreplicating episomes are not (1). (ii) The primary sequence of eukaryotic promoters is seldom a reiteration of binding sites for one protein. With only a few exceptions (2, 3), we are far from understanding how the regulatory behavior of a eukaryotic promoter emerges from the union of nonhistone protein-binding sites that it contains. In general, naturally occurring promoters studied in their native chromosomal context, e.g. the mouse mammary tumor virus long terminal repeat (MMTV LTR)1 (4-6), the Drosophila hsp26 gene (7-9), and the human pS2 (10, 11) and cathepsin D (12) promoters, offer a far more intricate picture of transcriptional regulation than assays with synthetic reporter constructs placed on nonreplicating episomes (13).

The Xenopus oocyte offers an attractive experimental system for studies of transcription; evolutionarily molded to contain sufficient histone protein and chromatin assembly machinery for several thousand somatic cells, it efficiently assembles microinjected DNA into physiologically spaced chromatin (14), which offers an excellent opportunity to investigate functional interactions between various regulatory factors and chromatin templates in vivo (15-20). The power of this model system is illustrated by data from our laboratory on action by the thyroid hormone receptor (TR); this extensively studied member of the nuclear hormone receptor (NHR) superfamily is exquisitely sensitive to nanomolar quantities of its ligand, thyroid hormone (T3) (21, 22). TR and T3-driven metamorphosis is a central phenomenon in anuran ontogeny (23, 24), and the frog oocyte contains vast stores of auxiliary components, i.e. corepressor and coactivators (25), to allow for transcriptional regulation by TR. By using the oocyte system, we showed that unliganded TR represses transcription via a chromatin-dependent pathway (20) that involves the targeting of a complex between the corepressor N-CoR and histone deacetylase 3 (19), and an auxiliary pathway of an undetermined mechanistic foundation (19), whereas in the presence of ligand, TR targets chromatin disruption (26) and the histone acetyltransferase CBP/p300 to activate transcription (27).

Significant progress in revealing the biochemical accomplices of the NHRs in effecting transcriptional control (25, 28) is in striking contrast to the paucity of structural data regarding their action on chromatin templates. In agreement with earlier chromatin-wide studies (29), our in vitro data showed that unliganded TR can engage a response element on the surface of a histone octamer (30). How TR, and other class II NHRs, accesses its binding sites within chromatin in vivo is unknown; we had earlier shown that TR can bind to a fully chromatinized, replicatively inert template in vivo (31), but whether such access is contingent on DNA assembly into a specific nucleoprotein architecture in vivo remains unresolved both for the promoter we studied and for every other class II NHR-responsive gene.

Because the regulatory properties of NHRs are significantly affected by the DNA sequence to which they bind (32-35), we studied TR signaling on one of its naturally occurring target sites in the Xenopus genome, the promoter for the TRbeta A gene (36). TR effects a >300-fold range of regulation on its own promoter (31), and this enables an autoregulatory loop that coordinates titers of hormone and receptor during Xenopus development (24). In the present study, we set out to map the structural determinants of primary DNA sequence of the TRbeta A gene promoter and of its assembly into chromatin that allows for such robust synergy with TR.

Our data reveal an upstream enhancer in the TRbeta A gene locus that contains several unusual TREs and is required for full range regulation of this gene by T3. We describe an array of translationally positioned nucleosomes over the promoter and enhancer in vivo that accommodates at least 4 TR/RXR heterodimer molecules both in linker DNA between adjacent nucleosomes and on the surface of histone octamers. In contradiction with commonly held notions that chromatin prevents access to DNA by regulatory factors, we show that chromatin assembly potentiates both binding to and action by TR/RXR on the enhancer. We hypothesize that structural distortion of the DNA assembled into a nucleosomal fiber creates a template that has a higher affinity for TR than naked DNA would be, and we suggest that chromatin disruption by liganded TR enables a novel mechanism of attenuating response to hormone by causing receptor release from the template.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Constructs for Transcription Analysis and in Vitro mRNA Synthesis-- Plasmid pTRbeta A (31) used as a reporter construct contains a fragment of the Xenopus laevis TRbeta A gene (36) (positions -1335 to +313 relative to the major transcription start site) subcloned into pBluescript II KS(-). The pTRbeta A-(Delta site 1) construct was generated by replacing the TRE at +276 with a unique NsiI recognition site using a combination of published techniques (39-41); pTRbeta A was denatured with NaOH as described (39) and used in a polymerase chain reaction with Pwo DNA polymerase (Roche Molecular Biochemicals) and the following primers (the NsiI site is underlined; these primers anneal immediately outside the TRE at +276): forward primer, 5'-AAAATGCATGCCCAGCGCCCTGGTGCACGATCAG-3', and reverse primer, 5'-AAAATGCATGCCTAGGGAGGACGAACAAGGATAG-3'.

Polymerase chain reaction settings were as follows: 94 °C (2 min) -10 cycles of 94 °C (15 s), 62 °C (30 s), 72 °C (3 min) -20 cycles with the same parameters, except 5 s added at each cycle extension step. This reaction generated a spurious 2.2-kb and a full-length 5-kb product that contained the entire starting plasmid with the 14 bp of the TRE replaced with the NsiI site; the 5-kb product was gel-purified, digested to completion with NsiI, recircularized with a Rapid Ligation kit (Roche Molecular Biochemicals), and transformed into E. coli following standard procedures. Clones were sequenced to confirm successful TRE replacement. Plasmid pTRbeta A-Delta enhancer (gift of Dr. Dmitry Guschin) contains a fragment of the TRbeta A locus (positions -154 to +313) subcloned into pCR2.1 (Invitrogen; Carlsbad, CA). Data identical to those shown here were obtained with a plasmid in which a more parsimonious deletion (a 350-bp NdeI-FseI fragment, positions -800 to -450]) eliminated just the TRE sequences from the TRbeta A enhancer (data not shown). Clones of TR and RXR were described previously (19, 31); these plasmids were linearized with EcoRI and in vitro synthesis of capped mRNA for microinjection was performed with an mMessage Machine kit (Ambion) exactly as described (19).

Preparation, Microinjection, and Maintenance of Oocytes and Primer Extension-- Oocytes were prepared and injected with mRNA and DNA exactly as described (19, 31). Total RNA was extracted using RNAzol (Tel-Test; Friendswood, TX) and assayed by primer extension with SuperScript reverse transcriptase (Life Technologies, Inc.) as described (19, 31). The reaction contained labeled primers that detected both mRNA synthesized from the reporter construct as well as an RNA stored in the oocyte that served as a loading control during analysis on 6% sequencing urea-polyacrylamide gel electrophoresis (19, 31).

Mapping of DNase I Hypersensitive Sites in Chromatin-- Oocytes were injected with 5 ng of RXRalpha mRNA and 5 ng of TR mRNA; following incubation for 10 h, 1 ng of double-stranded pTRbeta A DNA was injected. After 16 h of incubation, the oocytes were rinsed in MBSH medium, and 20 oocytes per experimental sample were homogenized in 10 mM Hepes, pH 7.6, 25 mM KCl, 5 mM MgCl2, 5% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT (42). The homogenate was placed in a 37 °C water bath for 1 min, and DNase I (DPRF grade; Worthington) was then added to 110-132 Worthington units/ml. Aliquots of 100 µl were removed from each sample at 1 and 3 min following addition of the enzyme and mixed vigorously with an equal volume of stop buffer (20 mM Tris, pH 7.4, 200 mM NaCl, 2 mM EDTA, 2% SDS). Proteinase K (Roche Molecular Biochemicals) was added to 700 µg/ml, and after incubation overnight at 37 °C, the DNA was extracted twice with an equal volume of phenol/chloroform/isoamyl alcohol (50:48:2, v/v/v), once with chloroform, precipitated with sodium acetate, and 0.7 volumes of isopropyl alcohol for 1 h on ice, and resuspended in TE buffer. RNA was removed by treatment with 100 µg/ml RNase A (Roche Molecular Biochemicals), and the DNA was digested to completion with EcoRI, followed by brief treatment with proteinase K, phenol/chloroform extraction, and precipitation with isopropyl alcohol as described above. The DNA was resuspended in TE buffer and fractionated (1 oocyte equivalent per sample) on a 2% high resolution blend (Amresco; Solon, OH) agarose gel in 1× TAE. Southern blotting with random prime-labeled probes was performed by transferring the DNA to 0.2 µm pore size Nytran Plus nylon membranes (Schleicher & Schuell), hybridizing, and washing as per the manufacturer's instructions. The final wash was performed in 0.2× SSC, 0.5% SDS, 65 °C. In Figs. 1C and 4A, the probe used for Southern blotting hybridized to the 3'-most portion of the TRbeta A gene construct and in Fig. 4B to the 5'-most portion. Filters were exposed to PhosphorImager screens or to x-ray film as described above.

Electromobility Shift Assays (EMSA)-- Proteins for EMSA were prepared by in vitro translation in a coupled transcription-translation system exactly as described (19). Equal volumes of reticulocyte lysate containing TR and RXR were incubated in binding buffer (43) in the presence of 1-2 µg poly(dI-dC)-poly(dI-dC) (Amersham Pharmacia Biotech) for 10 min on ice; 5 or 10 pmol of unlabeled competitor double-stranded oligonucleotide and 0.2 pmol 32P-labeled double-stranded oligonucleotide probe were then added, and the incubation was continued for an additional 35 min at room temperature prior to electrophoresis on a 5% polyacrylamide gel electrophoresis in 0.5× TBE at room temperature, drying, and autoradiography. All oligonucleotides used in the study were designed to have identical 4-bp 5' overhangs and to yield 28-bp fragments once the overhangs were filled in. The malic enzyme TRE (44) has the sequence GGGTTAggggAGGACA.

Mapping of MPE Cleavage Sites in Vivo-- A large group of oocytes was injected with 0.5 ng of single-stranded pTRbeta A DNA (gift of Dr. Trevor Collingwood); after 8 h, 5 ng each Xenopus TRbeta and RXRalpha mRNA was injected. After culture for 16 h in the presence or absence of 100 nM T3, chromatin was treated with methidiumpropyl EDTA as described (45, 46), with the following modifications: 7 oocytes were homogenized in 350 µl of room temperature 0.3 M sucrose, 10 mM Tris-Cl, pH 7.5, 60 mM KCl, 15 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 0.15 mM spermidine, 0.5 mM spermine. Methidiumpropyl EDTA (Sigma) was diluted to a final concentration of 5 mM in water and stored at -20 °C in single-use aliquots. Immediately prior to use, one such aliquot was diluted with water to a final concentration of 1.25 mM and mixed with an equal volume of 1.25 mM ferrous ammonium sulfate (Fe(NH4)2(SO4)2 (Sigma) prepared from a fresh 0.5 M aqueous stock. DTT was added to the MPE-Fe mixture to a final concentration of 10 mM from a 1 M stock prepared immediately prior to use. Freshly diluted hydrogen peroxide (Fisher) was added to the oocyte homogenate to 4 mM final, followed by the addition of 37.5 µl of the MPE-Fe-DTT reagent (final MPE concentration, 62.5 µM). The reaction was allowed to proceed at room temperature for 3 or 6 min and was stopped by addition of bathophenanthroline disulfonate (Fluka) to 5 mM final and 1/4th volume of stop buffer (2.5% SDS, 50 mM EDTA, 50 mM Tris-Cl, pH 7.5). DNA was purified by treatment with proteinase K and phenol extraction and analyzed by Southern blotting as described above.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The TRbeta A Promoter Contains Multiple in Vivo TR-binding Sites-- An outline of the experiment is shown in Fig. 1A; we first microinjected mRNAs for TR and RXR into the oocyte cytoplasm, and following receptor synthesis, we injected reporter DNA, the Xenopus TRbeta A promoter (36), into the nucleus. After an incubation period to allow chromatin assembly (14), we measured transcriptional activity of the reporter by primer extension. Because the oocyte lacks endogenous TR (31), high levels of basal transcription driven by the reporter were insensitive to T3 in the absence of added TR (Fig. 1B, lanes 3 and 4). In contrast, when TR/RXR was introduced into the oocyte, the promoter was very efficiently silenced (Fig. 1B, compare lane 5 and lanes 3 and 4), and addition of T3 to the medium activated transcription above basal levels (Fig. 1B, compare lanes 3 and 4 with lane 6). These data agree with published observations from our laboratory (31).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 1.   TR-dependent regulation of transcription and chromatin structure in the oocyte. A, outline of the experiment. B, uninjected oocytes (lanes 1 and 2), oocytes injected with double-stranded pTRbeta A DNA (lanes 3 and 4), or with TR/RXR mRNA and pTRbeta A DNA (lane 5 and 6) were cultured in the presence of T3 (where indicated), followed by RNA extraction and primer extension (see "Experimental Procedures"). Histone H4 mRNA that is stored in the oocyte was used as a loading control; transcriptional activity (txn) of the TRbeta A gene promoter (upper panel) was measured in each sample by normalizing against H4 mRNA levels (lower panel) in that sample and defining the average of transcriptional activity in lanes 3 and 4 (i.e. in the absence of injected mRNA) as 1 unit. C, oocytes left uninjected (lanes 2-5) or injected with TR/RXR mRNA (lanes 6-9) were cultured in the presence of T3 where indicated and treated with DNase I, and DNA was purified and analyzed by indirect end labeling (see "Experimental Procedures"). The arrow represents the TRbeta A gene, and the zigzag designates the receptor- and hormone-dependent disruption of chromatin upstream of the transcription start site. Lane "6 light" is a lighter exposure of lane 6, and the positions of the TR-dependent footprints (relative to +1) are shown to the right of the gel.

Like other NHRs (4), liganded TR targets disruption of histone-DNA contacts to the immediate vicinity of its bona fide binding sites in chromatin (26). We used DNase I footprinting coupled with indirect end labeling (47, 48) to map cis acting elements that allow the TRbeta A promoter to respond to receptor and hormone. As expected (30), TR-dependent binding and TR/T3-dependent disruption was observed over the previously characterized (49) TR response element (TRE) located at +276 relative to the transcription start site (open ellipse in Fig. 1C, compare lanes 2-5 with lanes 6-9).

In the presence of TR and T3, we reproducibly observed a disruption of histone-DNA contacts ~500 bp upstream of the transcription start site (zigzag between lanes 5 and 6 in Fig. 1C). In the absence of TR, this stretch of chromatin did not respond to T3 (compare lanes 2 and 3 with lanes 4 and 5 in Fig. 1C), indicating such disruption to be TR-dependent. Importantly, identical induction of this DNase I-hypersensitive site by T3 was observed in the presence of alpha -amanitin (data not shown), which abrogates all RNA polymerase II-dependent transcription in the oocyte, indicating such chromatin disruption to be a direct, rather than a secondary, consequence of TR-T3 action.

Within this ~300-bp DNA segment, at least 3 receptor footprints were reproducibly visualized both in the presence (open circles in Fig. 1C, compare lane "6 light" with lanes 4 and 5) and absence (lanes 8 and 9) of hormone, indicating that unliganded TR can access its binding sites within unremodeled chromatin. In multiple experiments (data not shown), even in the absence of T3 we observed three TR-dependent, alpha -amanitin-insensitive footprints in this area. We therefore conclude that in addition to the previously characterized TRE downstream of the transcription start site (49), the TRbeta A promoter contains an upstream TR- and T3-responsive segment of chromatin that can be bound by TR in vivo via 3 target sites. Furthermore, although we cannot formally exclude the possibility that chromatin structure alterations we describe (Fig. 1C) are due to secondary effects of unliganded TR, our data argue very strongly against this notion.

Noncanonical TREs in the TRbeta A Promoter-- The DNA sequence of the upstream stretch of the TRbeta A promoter bound by TR in vivo (Fig. 1C) is shown in Fig. 2A, and it contains three matches to a consensus TR response element (22). We used indirect end labeling with custom size markers and nucleotide resolution DNase I footprinting analysis to confirm that the footprints observed in vivo (Fig. 1C) coincide with these TREs (data not shown). The TRE located at -515 matched the canonical response element arrangement of a direct repeat of two hexameric half-sites spaced by 4 bp ("DR4"), whereas the TRE at -690 had a half-site spacing of 3 bp ("DR3"), and the TRE at -780 of 2 bp ("DR2"). The primary sequences of the half-sites diverged from the canonical "AGG(T/A)CA" consensus in precise agreement with in vitro data on TR/RXR binding to DNA (43, 50) (see "Discussion").


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 2.   Multiple TR-binding sites in the TRbeta A gene locus. A, sequence of the TRbeta A enhancer; location of partial sequence matches to TRE consensus and restriction sites used for custom markers in Fig. 4B and 5E are indicated. B, in vitro synthesized TR and RXR were incubated with a 32P-labeled double-stranded oligonucleotide probe corresponding to the Xenopus TRbeta A promoter exon B TRE (site 1). In lanes 2-15, a 25- or 50-fold molar excess (even- and odd-numbered lanes, respectively) of the indicated unlabeled double-stranded oligonucleotide was included in the reaction. The mal.enz. TRE competitor represents the well characterized TRE from the malic enzyme promoter (44); the mut. TRE is an oligonucleotide of identical length with point mutations in residues critical for binding by TR/RXR (50), and non-sp. is an oligonucleotide of identical length of random sequence. The reaction mixes were resolved by EMSA and the bound and free bands quantitated via PhosphorImager analysis. To quantify relative competitor potency, the intensity of the shifted band (TR/RXR-DNA) derived from receptor binding in the absence of competitor (lane 1) was set to 100%, and the signal obtained in the presence of the various competitors was expressed as a fraction thereof (shown below each lane as % bound).

We used an electromobility shift assay (EMSA) to confirm that these DNA sequences can bind TR/RXR. As shown in Fig. 2B, a 25- or 50-fold molar excess of these putative TREs competed with a canonical response element for binding by TR (lanes 5-11). Consistent with their divergence from a 4-bp half-site spacing, elements 2 and 3 bound TR/RXR with lower affinity than element 4 (compare lanes 8-11 with lanes 6 and 7) or a well defined TRE from the human genome (lanes 4 and 5). A "TRE" with point mutations in all DNA residues crucial for binding (50) (lanes 12 and 13) or a DNA segment of equal length but random sequence (lanes 14 and 15) both failed to compete for TR/RXR binding (compare with lane 1). We obtained identical results in experiments using all these TREs in labeled form rather than as competitors (data not shown).

We conclude that the putative upstream enhancer of the TRbeta A gene contains 3 binding sites for TR/RXR, 2 of which have altered half-site spacing relative to a canonical TRE (51). We show that these unusual TREs can bind TR/RXR in vitro in a sequence-specific manner, albeit with reduced affinity relative to canonical TREs.

The TRbeta A Enhancer Functions in Vivo to Augment Response to T3-- We used in vivo transcription assays to determine if the putative enhancer has a role in mediating response to T3. We hypothesized that its function may be to expand the range of regulation effected by hormone. If that were indeed the case, then a construct containing site 1 (Fig. 2A) as its sole TRE would be expected to exhibit a less prominent response to ligand than the wild-type promoter. We compared the full-length promoter to a truncated version in which all 3 enhancer TREs were deleted. In multiple experiments we reproducibly found that the Delta  enhancer construct was repressed by TR as efficiently as the wild-type promoter (compare lanes 2 and 5 in Fig. 3A). This indicated that TR binding to site 1 is sufficient to allow full repression by unliganded TR/RXR on this promoter. In contrast, activation by liganded TR/RXR was reproducibly impaired by deleting the enhancer (compare lanes 3 and 6 in Fig. 3A). These observations confirmed our hypothesis that the enhancer functions to augment transcriptional activation driven by liganded TR/RXR.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3.   The TRbeta A enhancer functions in vivo to augment regulation by T3. A, oocytes were injected with TR/RXR mRNA (lanes 2-6), followed by injection of pTRbeta A (lanes 1-3) or pTRbeta A-(Delta enhancer) (lanes 4-6) DNA, and culture in the presence or absence of T3 as indicated. Transcriptional activity of the TRbeta A promoter was measured as described in Fig. 1. A summary of data from 4 independent experiments is shown in the graph on the right; the S.E. for TR + T3 samples in lanes 3 (wild-type promoter) and 6 (enhancer deletion) were 10.8 ± 2.4 and 2.7 ± 0.5, respectively. B, oocytes were injected with increasing quantities of TR/RXR mRNA as indicated, followed by injection of pTRbeta A (lanes 1-7) or pTRbeta A-(Delta site 1) (lanes 8-14). Following culture in the presence or absence of T3, transcriptional activity of the TRbeta A promoter was measured as described in Fig. 1. A summary of data from 4 independent experiments is shown on the graph below (the S.E. for lane 12 is 2.45).

If the enhancer TREs are bona fide low affinity binding sites for TR, as indicated by our EMSA data (Fig. 2B), then a deletion of the highest affinity (i.e. site 1) TRE from the full-length TRbeta A promoter would be expected to impair the resulting response of the construct to low concentrations of TR/RXR, whereas a normal range of regulation by T3 in the presence of high quantities of the receptor should be observed. We microinjected several TR/RXR mRNA concentrations into the oocyte, and we used EMSA to confirm that the quantities of mRNA we inject fall within the range of a linear translational response (data not shown). We then injected the wild-type TRbeta A promoter, and a construct in which the major TRE at +276 was replaced by an NsiI site (pTRbeta A-(Delta site 1)) into oocytes, and we evaluated their transcriptional competence when challenged with a range of TR amounts and in the presence and absence of T3.

In agreement with our hypothesis that the putative enhancer can confer response to hormone, we reproducibly found that at intermediate and high concentrations of TR, the Delta site 1 construct responded to T3 in a manner identical to that of the wild-type promoter (Fig. 3B, compare lanes 4-7 with lanes 11-14). In addition, we reproducibly observed that at low concentrations of TR/RXR, the construct containing only the lower affinity enhancer TREs was impaired relative to the wild-type promoter in repression by unliganded TR (compare lane 2 with lane 9) and also moderately impaired in activation by liganded TR (compare lane 3 with lane 10). These data support our hypothesis that the enhancer contains low affinity in vivo binding sites for TR.

We conclude that the multiple TREs found upstream of the TRbeta A promoter transcription start site form a bona fide enhancer for TR/RXR action in vivo; we show that the enhancer augments the range of regulation affected by T3 (Fig. 3A) and that it can mediate response to T3 when sufficient quantities of receptor are present in the system (Fig. 3B, lanes 11-14).

A TR-bound Array of Translationally Positioned Nucleosomes within the Enhancer-- The TREs within the TRbeta A enhancer diverge from consensus and yet bind TR in vivo (Fig. 1C) and respond to T3 when bound (Fig. 3). We hypothesized that some feature of in vivo nucleoprotein organization of this DNA stretch potentiates TR action on the enhancer. Three NHR-controlled promoters offer examples of such potentiation; these are GR regulation of the MMTV LTR (4-6), and estrogen receptor (ER) regulation of the pS2 (10, 11) and the vitellogenin B1 (52) promoters. In all these cases, nonrandom histone octamer positions relative to the DNA within the target locus have been implicated in augmenting response to hormone.

We investigated directly whether the TRbeta A enhancer forms a specific chromatin organization in vivo by using the chemical agent methidiumpropyl-EDTA-iron(II) (MPE); it exhibits a marked preference for cleaving linker DNA while displaying negligible primary sequence bias (45, 46, 53). In contrast to MPE action on naked DNA, which generated a homogeneous smear (Fig. 4A, lanes 2 and 3), chromatin templates exhibited a marked and reproducible nonrandom sensitivity to MPE; over multiple experiments we consistently found that, even in the absence of TR, pronounced sites of cleavage by MPE spaced by ~180 bp were observed in chromatin (lanes 4 and 5). In the presence of unliganded TR no significant alteration was observed (compare lanes 4 and 5 with lanes 6 and 7), whereas addition of T3 lead to a localized destruction of this regular chromatin array (lanes 8 and 9; zigzag bar next to autoradiograph). Identical disruption was observed in the presence of alpha -amanitin (data not shown).


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 4.   The TRbeta A promoter and enhancer are bound into an array of translationally positioned nucleosomes in vivo. A, oocytes were injected with single-stranded pTRbeta A DNA (lanes 4-9), followed by injection TR/RXR mRNA (lanes 6-9), and culture in the presence or absence of T3 as indicated. Oocytes were treated with MPE (see "Experimental Procedures"), and MPE cleavage sites in chromatin were revealed by indirect end labeling exactly as in Fig. 1C. Lanes 2 and 3 contain sample of naked pTRbeta A DNA treated with MPE. Lane 10 contains custom size markers. A schematic representation of the nucleosomal organization of the locus (ellipses) relative to the transcription start site (arrow), TRE location (filled circles), and TR/T3-dependent chromatin disruption (zigzag) is included to the right of the autoradiograph. B, MPE-treated chromatin was analyzed by indirect end labeling with a probe that hybridizes to the upstream end of the TRbeta A gene. Custom size markers in lane 10 reveal the location of the enhancer TREs (see Fig. 2A for location of restriction enzyme recognition sites). Lane "7 dark" represents a longer exposure of the sample in lane 7. The schematic is annotated as above. C, a summary of the relative location of histone octamer positions and TR-binding sites in the TRbeta A promoter/enhancer in vivo.

These data indicate that the TRbeta A promoter is assembled into an array of "translationally" positioned nucleosomes in vivo (lanes 4 and 5; octamer position shown schematically to the right), that unliganded TR can bind to this promoter without disturbing this array (lanes 5 and 6; approximate location of TR footprints shown as filled circles), and that in the presence of ligand, TR destroys this positioned array over ~4 nucleosomes, while leaving other nucleosomes in this area intact. We note, however, that whereas these data reveal the predominant positions of histone octamers over this locus relative to the DNA, they cannot be used to determine nucleosome translational frames to nucleotide precision nor used as evidence for identical nucleosome positions over every template in the nucleus; thus, the existence of a frequency-biased distribution of multiple frames (54, 55) is possible.

To obtain a more precise estimate of the location of the new TREs we described in the enhancer relative to predominant nucleosome positions, we used custom size markers (4) that migrate at the exact location of the TR footprints (these are prepared by digesting genomic DNA with a restriction enzyme with a cleavage site close to the location of the TRE, see Fig. 2A). A sample of MPE-treated chromatin in the presence and absence of TR was analyzed by indirect end labeling along such markers (shown in lane 10). This analysis revealed that site 4 (the TRE at position -515 located over the SmaI site (Fig. 2A)) is in a linker region between two nucleosomes and that site 3 (the TRE at position -690) found just upstream of the EarI site (Fig. 2A) is also in a linker region, 175 bp away (Fig. 4B, lanes 6 and 7, and also "7 dark" which represents a longer exposure of the sample in lane 7). Thus, two molecules of TR/RXR bound to sites 3 and 4 straddle linker DNA entering and exiting the same translationally positioned histone octamer. In contrast, site 2 (the TRE at position -780 immediately downstream of the EagI site) is only 90 bp away from linker DNA and is, therefore, located on the surface of a histone octamer.

We reproducibly found an area of TR-independent MPE hypersensitivity immediately upstream of site 2; the resulting stretch of DNA resistant to MPE was ~110 bp in length, i.e. less than 1 nucleosome (see stretch of chromatin between EagI and ApaI sites in Fig. 4B, lanes 4-7). We do not understand the structural basis of this nucleoprotein assembly, and we have provisionally represented the adjacent DNA as a remodeled nucleosome (dotted ellipse); it is possible that factors endogenous to the oocyte exert some unilateral (i.e. from the upstream side only) disruption of histone-DNA contacts in the area.

Our data on nucleosome and TRE position in the area are summarized in Fig. 4C; we show that nucleosomes in this area exhibit a nonrandom distribution relative to the DNA and that the TRbeta A enhancer is assembled into 3 translationally positioned nucleosomes, with 2 molecules of TR/RXR bound to linker DNA, and a 3rd molecule occupying the surface of a nucleosome-like particle.

TR Binding and Action on the TRbeta A Enhancer Is Potentiated by Chromatin Assembly-- The nonrandom chromatin infrastructure over the enhancer (Fig. 4) suggested that its dynamic assembly might affect the ability of TR/RXR to act via this stretch of DNA. The Xenopus oocyte offers a unique opportunity to investigate this issue directly; the assembly of microinjected double-stranded DNA into chromatin occurs gradually over the course of several hours (14). For example, as shown in Fig. 5B, 1 h after injection into the oocyte nucleus, pTRbeta A DNA is highly sensitive to micrococcal nuclease, and nucleosomal density is low (lanes 2 and 3); after 3 h nucleosome density increases, but the bulk of the signal is derived from mono- and dinucleosomal particles, and the signal from a "seminucleosome" (0.5n), a chromatin assembly intermediate (56, 57), is conspicuous. By later time points (lanes 11 and 12), chromatin assumes a more mature configuration, and signal from assembly intermediates is undetectable.


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 5.   Chromatin assembly potentiates binding to and action by TR/RXR on the TRbeta A enhancer. A, schematic representation of the experiment. B, kinetics of assembly of double-stranded pTRbeta A DNA following injection into oocytes were monitored by micrococcal nuclease cleavage and Southern blotting exactly as described (19). The location of various cleavage intermediates and a scan of the indicated lanes is included to the right of the autoradiograph. C, levels of TR in the oocyte during the experiment shown in D were monitored by extracting protein from uninjected oocytes (lane 1) or oocytes injected with TR at the indicated time points, and Western blotting with an antibody against Xenopus TRbeta (20) was performed as described (19). The location of size markers is indicated to the left; the question mark designates a large polypeptide that cross-reacts with the antibody and serves as a loading control. D, oocytes were injected with TR/RXR mRNA as indicated. Double-stranded pTRbeta A or pTRbeta A-(Delta site 1) DNA was then injected as indicated, and oocytes were processed for primer extension 2.5 (lanes 1-6) or 16 h (lanes 7-12) following DNA injection. Transcriptional activity was measured as described in Fig. 1. E, oocytes left uninjected with mRNA (where indicated) or injected with TR/RXR mRNA were injected with double-stranded pTRbeta A and treated with DNase I exactly as described in Fig. 1C at 2.5 (lanes 1-7) and 16 h (lanes 8-10) following DNA injection. TR/RXR footprints were revealed by indirect end labeling using custom size markers that designate the location of TR footprints exactly as in Fig. 2B. A scan of the indicated lanes is included to the right of the autoradiograph. The position of the enhancer is indicated by the shaded bar, and the position of the TR/RXR-dependent footprints is indicated by the black ovals.

This interesting property of the oocyte, i.e. a kinetics of chromatin assembly that is markedly delayed relative to the 30-min replication-coupled pathway (58), allows for the experimental arrangement shown in Fig. 5A; oocytes are injected with mRNA and incubated for a set length of time to allow receptor synthesis. DNA is then injected, and oocytes are cultured in the presence or absence of T3; groups of oocytes are removed at specific time points following DNA injection, and transcription is measured (or chromatin structure is assayed, see below). Thus, the regulatory competence of TR/RXR can be examined on templates that are in various states of chromatin assembly.

In such experiments, we reproducibly found that the full-length TRbeta A promoter was regulated by T3 with equal efficiency at all states of template chromatinization (Fig. 5D, compare lanes 1-3 with lanes 7-9). In contrast, a construct that contained only the TRbeta A enhancer (i.e. no site 1) was robustly responsive to TR when DNA was fully chromatinized (lanes 10-12) but impaired in response to hormone at early time points (lanes 4-6), when DNA is in immature, partly assembled chromatin (Fig. 5B, lanes 2 and 3 and 5 and 6). We reproducibly found that at early time points following DNA injection, the Delta site 1 construct could not be repressed by unliganded TR/RXR (Fig. 5D, compare lane 4 with lane 5). In addition, on immature chromatin templates we reproducibly observed lower transcriptional activation by liganded TR on the Delta site 1 construct as compared with the wild-type promoter (Fig. 5D, compare lanes 4 and 6 with lanes 1 and 3).

The data shown in Fig. 5D had a trivial explanation; we had earlier shown that the Delta site 1 construct is impaired in responding to TR and T3 at low TR levels (Fig. 3B, lanes 8-10). It was possible that the difference we saw in TR action on the two reporter constructs (Fig. 5D, lanes 1-3 versus lanes 4-6) was due to low TR levels at the early time point (lanes 1-6) and an increase by the late time point (lanes 7-12). To address this issue, we examined the TR content of the oocyte during the course of the experiment by Western blotting (Fig. 5C); the antibody used recognized a ~240-kDa polypeptide (Fig. 5C, ?) in oocytes that were not injected with TR mRNA (Fig. 5C, lane 1), which served as a loading control. Such analysis showed that TR levels did not change in the oocyte over the course of the experiment (Fig. 5C, compare lanes 2 and 3; also compare TR levels in each lane to loading control), indicating that the difference in regulation of the Delta site 1 construct by TR at various time points (Fig. 5D, lanes 4-6 versus 10-12) was not due to a change in TR concentration over the course of the experiment.

The impaired ability of TR to act via response elements bound into immature chromatin (Fig. 5D, lanes 4-6) suggested that chromatin assembly might potentiate the physical interaction of TR/RXR with the enhancer. To investigate this issue, we performed genomic footprinting experiments identical to those shown in Fig. 1C, except we assayed TR/RXR binding to chromatin at various time points following DNA microinjection (the experimental outline is shown in Fig. 5A). As shown in Fig. 5E, at an early time point, no detectable change in chromatin architecture over the enhancer could be detected in the presence of TR/RXR (compare stretch of chromatin designated by the shaded bar, the enhancer, in lanes 2-4 with lanes 5-7). PhosphorImager-based comparison of samples containing and lacking TR yielded identical scans (scans of lanes 3 and 6 in Fig. 5E are shown to the right of the gel).

In contrast, after 16 h of chromatin assembly, 3 footprints were observed over this area in the presence (lane 9) but not in the absence (lane 8) of TR. A PhosphorImager scan of the sample is shown to the right of the gel (lane 9), and the location of the footprints, which was coincident with custom markers (lane 11) that designate the position of the TREs (Fig. 2A), is indicated by shaded ovals.

We conclude that action of TR/RXR on the enhancer element we have described is potentiated by assembly of the target DNA into chromatin (Fig. 5D). We show that the capacity of unliganded TR to bind the unusual TREs in the TRbeta A enhancer is regulated by the extent of template chromatinization, such that more mature chromatin templates are bound to a greater extent than those in the process of assembly (Fig. 5E). Importantly, this change in the functional properties of TR (Fig. 5D) and in its ability to bind the enhancer (Fig. 5E) occurs without any detectable alteration of TR protein levels (Fig. 5C).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The thyroid hormone receptor can regulate transcription in vitro on naked DNA templates, i.e. in the absence of any chromatin (59, 60). In the nucleus, however, the receptor establishes a poorly understood functional relationship with the chromatin infrastructure of its target promoters (21, 22). The present study describes a positive role for a structurally mature chromatin template in allowing maximal response to TR and T3; we show that a naturally occurring TR-regulated gene contains an upstream enhancer bound into an array of translationally positioned nucleosomes in vivo (Fig. 4), and we demonstrate that this nucleoprotein entity potentiates binding by TR/RXR to multiple nonconsensus TREs located in the enhancer (Figs. 1C, 2A and 5E), thereby expanding the range of regulation effected by the liganded NHR (Figs. 3A and 5D). To the best of our knowledge, these are the first data in the literature that a non-viral NHR target gene presents itself to the receptor in the form of a specifically structured chromatin template that is required for full range regulation by hormone.

The previously described (49) TR-binding site in the TRbeta A promoter ("site 1" at +276) is sufficient to make it responsive to T3 (Fig. 3A). We found, however, that its action cannot account for the entirety of T3 response (Fig. 3), and we present evidence that additional regulation is imparted by a segment of DNA located at positions -800 to -500. This element fits several criteria for an enhancer; it is bound by TR in vivo (Fig. 1C); it contains 3 sequence elements that can be bound by TR in vitro (Fig. 2B); it is sufficient to enable response to TR (Fig. 3B) and allows liganded TR to drive higher levels of transcription than can be achieved via a single TRE (Fig. 3A). Our data agree with findings of an earlier study (61), in which EMSA and transient transfection assays suggested the existence of TR-responsive elements upstream of the TRbeta A transcription start site.

Our footprinting (Fig. 1C), EMSA (Fig. 2B), and functional (Fig. 3A) data reveal the existence of 3 unusual TREs in the TRbeta A gene enhancer. The primary sequence of these response elements diverges from the established AGG(A/T)CA consensus; for example, the TR-bound right half-site (51) of element 4 is GGGTTA and of element 3 is GTGTCA. Both class I (62, 63) and class II NHRs (50) are known to tolerate variation in primary DNA sequence and half-site spacing of their target sites. The naturally occurring TREs in the TRbeta A gene locus conform to findings from several in vitro studies (43, 50, 64); for example an "A-G" transition at position 1 in the AGGTCA half-site was shown to enhance TR/RXR binding by ~40%, whereas a "C-T" transition in position 5 reduced it to ~60% of wild type (50). The resulting half-site, GGGTTA, is found in the TRbeta A enhancer and efficiently binds TR/RXR in vivo (Fig. 1C) while exhibiting only a moderate reduction in affinity in vitro (Fig. 2B). The 1.9-kb TRbeta A promoter fragment contains ~20 sequences that are partial (mismatch = 3 nucleotides) matches to the TRE "consensus" (data not shown), but only 3 are bound by TR in vivo (Fig. 1C); comparison of the remaining 17 to data presented by Judelson and Privalsky (50) indicates that the mismatches would be expected to significantly impair receptor binding. Our in vivo data thus fully support the notion that TR exhibits a restricted, rule-based promiscuity in DNA binding.

Our data indicate that binding and action by TR on the enhancer are potentiated by chromatin assembly (Fig. 5), a surprising finding considering that nucleosomes impede binding to DNA by most nonhistone factors (66, 67) with only a few exceptions (65). In the promoter we study, the translational frame of nucleosome occupancy is receptor-independent (Fig. 4A), and sites 3 and 4 are presented to the receptor in linker DNA surrounding the same nucleosome (Fig. 4C). It is possible, therefore, that the spatial proximity of DNA entering and exiting the histone core creates a "static loop" (7, 8, 10, 11, 52) that enables an interaction between two TR/RXR heterodimers. It is also possible that the unwinding and distortion of the DNA helix complexed with histones (68) potentiates binding by TR/RXR to site 2 (a motif of two direct repeats spaced by 2 bp) by increasing the distance between the two half-sites to a level more compatible with heterodimerization on the DNA of the two NHRs (which normally require a 4-bp spacer (51)).

Naturally occurring promoters frequently contain multiple binding sites for a particular regulator (69, 70), and many NHR-responsive genes are controlled via arrays of receptor response elements; for example, the MMTV LTR contains at least 6 GR response elements (71), and the androgen receptor (AR)-regulated prostate-specific antigen enhancer includes 4 AR response elements (72). Such promoter structure is thought to enable a sigmoidal response curve to a rise in regulator titer (69, 70, 73, 74). The TRbeta A promoter conforms to this paradigm, having evolved under selective pressure to coordinate ligand and receptor levels and allow for autoregulation by hormone of the titer for its own receptor during metamorphosis (75, 76). In addition, our data illuminate a novel role for chromatin assembly and primary promoter sequence in a general phenomenon, enabling a correlation between titers of a given NHR and its cognate ligand (76), which has been observed for GR (77-79), ER (80), as well as the androgen (81, 82), ecdysone (83), and retinoic acid (84) receptors.

Early in development, when TR levels are low, regulation of the TRbeta A gene likely occurs via the site 1 TRE, which has the highest affinity for TR (this is sufficient for repression (Fig. 3A) and moderate activation (Fig. 3B)). As hormone and receptor titers rise during ontogeny, binding by TR to the lower affinity enhancer TREs (Fig. 2B) is predicted to lead to graded transcriptional activation above basal levels (Fig. 3, A and B). Thus, one possible developmental function of the enhancer may be to mediate the positive, feed-forward branch of the autoregulatory loop between T3 and TR; the placement of lower affinity TREs in the enhancer ensures that it begins to function only when TR titer is sufficiently high.

An important component of an autoregulatory circuit is the attenuation of response to signal which, in the case of TR and T3, is partially effected by proteolysis of liganded TR (19, 85). We suggest that the unique structure of the TRbeta A enhancer may also contribute to such attenuation; binding by unliganded TR/RXR to the enhancer is potentiated by assembly of the DNA into a mature array of translationally positioned nucleosomes (Fig. 5E) and liganded TR disrupts this array (Figs. 1C and 4A), thereby creating a lower affinity template for itself. Thus, TR-targeted chromatin disruption may lead to receptor release from the enhancer and a lowering of transcription initiation rates (Fig. 3A). In vivo, non-NHR transcriptional regulators such as Swi5p (86), and such NHRs as GR (71, 87) and ER (12), bind chromatin only transiently, and it is quite possible, although unproven, that the large ATP-dependent chromatin remodeling complexes they target are partly responsible for such "hit-and-run" action. In our model system this issue can be studied with in vitro assembled chromatin templates that have been shown to recapitulate certain aspects of gene regulation by NHRs (37, 71, 88).

    ACKNOWLEDGEMENTS

We are very grateful to Dmitry Guschin for providing the pTRbeta A-Delta enhancer construct, Trevor Collingwood for a gift of single-stranded pTRbeta A, Keith Robertson for advice on polymerase chain reaction-based promoter mutagenesis, and Karen Chapman for suggesting references describing NHR gene autoregulation. F. D. U. sincerely thanks Paul Wade, GertJan Veenstra, Trevor Collingwood, Dmitry Guschin, Peter Jones, Alexander Strunnikov, Rohinton Kamakaka, and Daniel Weeks for insightful advice on experimental procedures and for helpful discussions throughout the course of this work.

    FOOTNOTES

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

To whom correspondence should be addressed: Sangamo Biosciences, Point Richmond Tech Center, 501 Canal Blvd., Suite A100, Richmond, CA 94804. Tel.: 510-970-6000, ex 255; Fax: 510-236-8951; E-mail: furnov@sangamo.com.

Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M100924200

    ABBREVIATIONS

The abbreviations used are: MMTV LTR, mouse mammary tumor virus long terminal repeat; TR, thyroid hormone receptor; TRE, TR response element; NHR, nuclear hormone receptor; T3, triiodothyronine; RXR, retinoid X receptor; bp, base pair; kb, kilobase pairs; MPE, methidiumpropyl-EDTA-iron(II); DTT, dithiothreitol; EMSA, electromobility shift assays.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Jeong, S., and Stein, A. (1994) Nucleic Acids Res. 22, 370-375[Abstract]
2. Thanos, D., and Maniatis, T. (1995) Cell 83, 1091-1100[Medline] [Order article via Infotrieve]
3. Agalioti, T., Lomvardas, S., Parekh, B., Yie, J., Maniatis, T., and Thanos, D. (2000) Cell 103, 667-678[Medline] [Order article via Infotrieve]
4. Zaret, K. S., and Yamamoto, K. R. (1984) Cell 38, 29-38[Medline] [Order article via Infotrieve]
5. Archer, T. K., Lefebvre, P., Wolford, R. G., and Hager, G. L. (1992) Science 255, 1573-1576[Medline] [Order article via Infotrieve]
6. Hager, G. L. (2001) Prog. Nucleic Acids Res. Mol. Biol. 66, 279-305[Medline] [Order article via Infotrieve]
7. Thomas, G. H., and Elgin, S. C. (1988) EMBO J. 7, 2191-2201[Abstract]
8. Lu, Q., Wallrath, L. L., and Elgin, S. C. (1995) EMBO J. 14, 4738-4746[Abstract]
9. Wallrath, L. L., Lu, Q., Granok, H., and Elgin, S. C. (1994) BioEssays 16, 165-170[Medline] [Order article via Infotrieve]
10. Sewack, G. F., and Hansen, U. (1997) J. Biol. Chem. 272, 31118-31129[Abstract/Free Full Text]
11. Sewack, G. F., Ellis, T. W., and Hansen, U. (2001) Mol. Cell. Biol. 21, 1404-1415[Abstract/Free Full Text]
12. Shang, Y., Hu, X., DiRenzo, J., Lazar, M. A., and Brown, M. (2000) Cell 103, 843-852[Medline] [Order article via Infotrieve]
13. Smith, C. L., and Hager, G. L. (1997) J. Biol. Chem. 272, 27493-27496[Free Full Text]
14. Almouzni, G., and Wolffe, A. P. (1993) Genes Dev. 7, 2033-2047[Abstract]
15. Kass, S. U., Landsberger, N., and Wolffe, A. P. (1997) Curr. Biol. 7, 157-165[Medline] [Order article via Infotrieve]
16. Landsberger, N., and Wolffe, A. P. (1995) Mol. Cell. Biol. 15, 6013-6024[Abstract]
17. Landsberger, N., and Wolffe, A. P. (1997) EMBO J. 16, 4361-4373[Abstract/Free Full Text]
18. Li, Q., Herrler, M., Landsberger, N., Kaludov, N., Ogryzko, V. V., Nakatani, Y., and Wolffe, A. P. (1998) EMBO J. 17, 6300-6315[Abstract/Free Full Text]
19. Urnov, F. D., Yee, J., Sachs, L., Collingwood, T. N., Bauer, A., Beug, H., Shi, Y. B., and Wolffe, A. P. (2000) EMBO J. 19, 4074-4090[Abstract/Free Full Text]
20. Wong, J., Patterton, D., Imhof, A., Guschin, D., Shi, Y. B., and Wolffe, A. P. (1998) EMBO J. 17, 520-534[Abstract/Free Full Text]
21. Collingwood, T. N., Urnov, F. D., and Wolffe, A. P. (1999) J. Mol. Endocrinol. 23, 255-275[Abstract/Free Full Text]
22. Zhang, J., and Lazar, M. A. (2000) Annu. Rev. Physiol. 62, 439-466[CrossRef][Medline] [Order article via Infotrieve]
23. Brown, D. D., Wang, Z., Kanamori, A., Eliceiri, B., Furlow, J. D., and Schwartzman, R. (1995) Recent Prog. Horm. Res. 50, 309-315[Medline] [Order article via Infotrieve]
24. Tata, J. R. (1999) Biochimie (Paris) 81, 359-366[CrossRef][Medline] [Order article via Infotrieve]
25. Xu, L., Glass, C. K., and Rosenfeld, M. G. (1999) Curr. Opin. Genet. & Dev. 9, 140-147[CrossRef][Medline] [Order article via Infotrieve]
26. Wong, J., Shi, Y. B., and Wolffe, A. P. (1997) EMBO J. 16, 3158-3171[Abstract/Free Full Text]
27. Li, Q., Imhof, A., Collingwood, T. N., Urnov, F. D., and Wolffe, A. P. (1999) EMBO J. 18, 5634-5652[Abstract/Free Full Text]
28. Robyr, D., Wolffe, A. P., and Wahli, W. (2000) Mol. Endocrinol. 14, 329-347[Free Full Text]
29. Samuels, H. H., Perlman, A. J., Raaka, B. M., and Stanley, F. (1982) Recent Prog. Horm. Res. 38, 557-599[Medline] [Order article via Infotrieve]
30. Wong, J., Li, Q., Levi, B. Z., Shi, Y. B., and Wolffe, A. P. (1997) EMBO J. 16, 7130-7145[Abstract/Free Full Text]
31. Wong, J., Shi, Y. B., and Wolffe, A. P. (1995) Genes Dev. 9, 2696-2711[Abstract]
32. Lefstin, J. A., Thomas, J. R., and Yamamoto, K. R. (1994) Genes Dev. 8, 2842-2856[Abstract]
33. Kurokawa, R., Soderstrom, M., Horlein, A., Halachmi, S., Brown, M., Rosenfeld, M. G., and Glass, C. K. (1995) Nature 377, 451-454[CrossRef][Medline] [Order article via Infotrieve]
34. Lefstin, J. A., and Yamamoto, K. R. (1998) Nature 392, 885-888[CrossRef][Medline] [Order article via Infotrieve]
35. Olson, D. P., Sun, B., and Koenig, R. J. (1998) J. Biol. Chem. 273, 3375-3380[Abstract/Free Full Text]
36. Shi, Y. B., Yaoita, Y., and Brown, D. D. (1992) J. Biol. Chem. 267, 733-738[Abstract/Free Full Text]
37. Liu, Z., Wong, J., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9485-9490[Abstract/Free Full Text]
38. Wong, C. W., and Privalsky, M. L. (1995) Mol. Endocrinol. 9, 551-562[Abstract]
39. Du, Z., Regier, D. A., and Desrosiers, R. C. (1995) BioTechniques 18, 376-378[Medline] [Order article via Infotrieve]
40. Jones, D. H., and Howard, B. H. (1991) BioTechniques 10, 62-66[Medline] [Order article via Infotrieve]
41. Zaret, K. S., Liu, J. K., and DiPersio, C. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5469-5473[Abstract]
42. Zaret, K. S., Milos, P., Lia, M., Bali, D., and Gluecksohn-Waelsch, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6540-6544[Abstract]
43. Chen, H., Smit-McBride, Z., Lewis, S., Sharif, M., and Privalsky, M. L. (1993) Mol. Cell. Biol. 13, 2366-2376[Abstract]
44. Petty, K. J., Desvergne, B., Mitsuhashi, T., and Nikodem, V. M. (1990) J. Biol. Chem. 265, 7395-7400[Abstract/Free Full Text]
45. Cartwright, I. L., Hertzberg, R. P., Dervan, P. B., and Elgin, S. C. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3213-3217[Abstract]
46. Belikov, S., Gelius, B., Almouzni, G., and Wrange, O. (2000) EMBO J. 19, 1023-1033[Abstract/Free Full Text]
47. Nedospasov, S., and Georgiev, G. (1980) Biochem. Biophys. Res. Commun. 29, 532-539
48. Wu, C. (1980) Nature 286, 854-860[Medline] [Order article via Infotrieve]
49. Ranjan, M., Wong, J., and Shi, Y. B. (1994) J. Biol. Chem. 269, 24699-24705[Abstract/Free Full Text]
50. Judelson, C., and Privalsky, M. L. (1996) J. Biol. Chem. 271, 10800-10805[Abstract/Free Full Text]
51. Rastinejad, F., Perlmann, T., Evans, R. M., and Sigler, P. B. (1995) Nature 375, 203-211[CrossRef][Medline] [Order article via Infotrieve]
52. Schild, C., Claret, F. X., Wahli, W., and Wolffe, A. P. (1993) EMBO J. 12, 423-433[Abstract]
53. Cartwright, I. L., and Elgin, S. C. (1989) Methods Enzymol. 170, 359-369[Medline] [Order article via Infotrieve]
54. Fragoso, G., John, S., Roberts, M. S., and Hager, G. L. (1995) Genes Dev. 9, 1933-1947[Abstract]
55. Fragoso, G., and Hager, G. L. (1997) Methods 11, 246-252[CrossRef][Medline] [Order article via Infotrieve]
56. Tse, C., Fletcher, T. M., and Hansen, J. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12169-12173[Abstract/Free Full Text]
57. Adams, C. R., and Kamakaka, R. T. (1999) Curr. Opin. Genet. & Dev. 9, 185-190[CrossRef][Medline] [Order article via Infotrieve]
58. Verreault, A. (2000) Genes Dev. 14, 1430-1438[Free Full Text]
59. Fondell, J. D., Roy, A. L., and Roeder, R. G. (1993) Genes Dev. 7, 1400-1410[Abstract]
60. Fondell, J. D., Ge, H., and Roeder, R. G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8329-8333[Abstract/Free Full Text]
61. Machuca, I., Esslemont, G., Fairclough, L., and Tata, J. R. (1995) Mol. Endocrinol. 9, 96-107[Abstract]
62. Zilliacus, J., Wright, A. P., Carlstedt-Duke, J., and Gustafsson, J. A. (1995) Mol. Endocrinol. 9, 389-400[Medline] [Order article via Infotrieve]
63. Nelson, C. C., Hendy, S. C., Shukin, R. J., Cheng, H., Bruchovsky, N., Koop, B. F., and Rennie, P. S. (1999) Mol. Endocrinol. 13, 2090-2107[Abstract/Free Full Text]
64. Harbers, M., Wahlstrom, G. M., and Vennstrom, B. (1998) J. Steroid Biochem. Mol. Biol. 67, 181-191[CrossRef][Medline] [Order article via Infotrieve]
65. Cirillo, L. A., and Zaret, K. S. (1999) Mol. Cell 4, 961-969[Medline] [Order article via Infotrieve]
66. Imbalzano, A. N., Kwon, H., Green, M. R., and Kingston, R. E. (1994) Nature 370, 481-485[CrossRef][Medline] [Order article via Infotrieve]
67. Wolffe, A. P. (1998) Chromatin: Structure and Function , 3rd Ed , pp. 240-314, Academic Press, San Diego
68. Hayes, J. J., Tullius, T. D., and Wolffe, A. P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7405-7409[Abstract]
69. Meyer, B. J., Maurer, R., and Ptashne, M. (1980) J. Mol. Biol. 139, 163-194[Medline] [Order article via Infotrieve]
70. Dynan, W. S., and Tjian, R. (1983) Cell 35, 79-87[Medline] [Order article via Infotrieve]
71. Fletcher, T. M., Ryu, B.-W., Baumann, C. T., Warren, B. S., Fragoso, G., and Hager, G. (2000) Mol. Cell. Biol. 20, 6466-6475[Abstract/Free Full Text]
72. Huang, W., Shostak, Y., Tarr, P., Sawyers, C., and Carey, M. (1999) J. Biol. Chem. 274, 25756-25768[Abstract/Free Full Text]
73. Driever, W., and Nusslein-Volhard, C. (1989) Nature 337, 138-143[CrossRef][Medline] [Order article via Infotrieve]
74. Ptashne, M. (1992) A Genetic Switch: Phage lambda  and Higher Organisms , 2nd Ed , pp. 16-31, Cell Press, Cambridge, MA, 85-96
75. Yaoita, Y., and Brown, D. D. (1990) Genes Dev. 4, 1917-1924[Abstract]
76. Tata, J. R. (2000) Insect Biochem. Mol. Biol. 30, 645-651[CrossRef][Medline] [Order article via Infotrieve]
77. Kalinyak, J. E., Dorin, R. I., Hoffman, A. R., and Perlman, A. J. (1987) J. Biol. Chem. 262, 10441-10444[Abstract/Free Full Text]
78. Dong, Y., Poellinger, L., Gustafsson, J. A., and Okret, S. (1988) Mol. Endocrinol. 2, 1256-1264[Abstract]
79. Eisen, L. P., Elsasser, M. S., and Harmon, J. M. (1988) J. Biol. Chem. 263, 12044-12048[Abstract/Free Full Text]
80. Barton, M. C., and Shapiro, D. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7119-7123[Abstract]
81. Lin, M. C., Rajfer, J., Swerdloff, R. S., and Gonzalez-Cadavid, N. F. (1993) J. Steroid Biochem. Mol. Biol. 45, 333-343[CrossRef][Medline] [Order article via Infotrieve]
82. Grad, J. M., Dai, J. L., Wu, S., and Burnstein, K. L. (1999) Mol. Endocrinol. 13, 1896-1911[Abstract/Free Full Text]
83. Thummel, C. S. (1996) Trends Genet. 12, 306-310[CrossRef][Medline] [Order article via Infotrieve]
84. de The, H., Vivanco-Ruiz, M. M., Tiollais, P., Stunnenberg, H., and Dejean, A. (1990) Nature 343, 177-180[CrossRef][Medline] [Order article via Infotrieve]
85. Dace, A., Zhao, L., Park, K. S., Furuno, T., Takamura, N., Nakanishi, M., West, B. K., Hanover, J. A., and Cheng, S. Y. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8985-8990[Abstract/Free Full Text]
86. Cosma, M. P., Tanaka, T., and Nasmyth, K. (1999) Cell 97, 299-311[Medline] [Order article via Infotrieve]
87. McNally, J. G., Muller, W. G., Walker, D., Wolford, R., and Hager, G. L. (2000) Science 287, 1262-1265[Abstract/Free Full Text]
88. Kraus, W. L., and Kadonaga, J. T. (1998) Genes Dev. 12, 331-342[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.