Endocrine disruptors of sex hormone activities.

Sex hormones, such as androgens, estrogens and progestins are naturally occurring compounds that tightly regulate endocrine systems in a variety of living organisms. Uncontrolled environmental exposure to these hormones or their biological and synthetic mimetics has been widely documented. Furthermore, water contaminants penetrate soil to affect flora, fauna and ultimately humans. Because endocrine systems evolved to respond to very small changes in hormone levels, the low levels found in the environment cannot be ignored. The combined actions of sex hormones with glucocorticoids and other nuclear receptors disruptors creates additional level of complexity including the newly described "dynamic assisted loading" mechanism. We reviewed the extensive literature pertaining to world-wide detection of these disruptors and created a detailed Table on the development and current status of methods used for their analysis.

Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter.

We have examined the relationship between transcription and chromatin structure using a tandem array of the mouse mammary tumor virus (MMTV) promoter driving a ras reporter. The array was visualized as a distinctive fluorescent structure in live cells stably transformed with a green fluorescent protein (GFP)-tagged glucocorticoid receptor (GR), which localizes to the repeated MMTV elements after steroid hormone treatment. Also found at the array by immunofluorescence were two different steroid receptor coactivators (SRC1 and CBP) with acetyltransferase activity, a chromatin remodeler (BRG1), and two transcription factors (NFI and AP-2). Within 3 h after hormone addition, arrays visualized by GFP-GR or DNA fluorescent in situ hybridization (FISH) decondensed to varying degrees, in the most pronounced cases from a approximately 0.5-microm spot to form a fiber 1-10 microm long. Arrays later recondensed by 3-8 h of hormone treatment. The degree of decondensation was proportional to the amount of transcript produced by the array as detected by RNA FISH. Decondensation was blocked by two different drugs that inhibit polymerase II, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin. These observations demonstrate a role for polymerase in producing and maintaining decondensed chromatin. They also support fiber-packing models of higher order structure and suggest that transcription from a natural promoter may occur at much higher DNA-packing densities than reported previously.

The glucocorticoid receptor: rapid exchange with regulatory sites in living cells.

Steroid receptors bind to site-specific response elements in chromatin and modulate gene expression in a hormone-dependent fashion. With the use of a tandem array of mouse mammary tumor virus reporter elements and a form of glucocorticoid receptor labeled with green fluorescent protein, targeting of the receptor to response elements in live mouse cells was observed. Photobleaching experiments provide direct evidence that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment. Thus, the interaction of regulatory proteins with target sites in chromatin is a more dynamic process than previously believed.

Transcription factor loading on the MMTV promoter: a bimodal mechanism for promoter activation.

The mouse mammary tumor virus (MMTV) promoter attains a phased array of six nucleosomes when introduced into rodent cells. This architecture excludes nuclear factor 1/CCAAT transcription factor (NF1/CTF) from the promoter before glucocorticoid treatment and hormone-dependent access of nucleolytic agents to promoter DNA. In contrast, when the promoter was transiently introduced into cells, NF1/CTF was bound constitutively and nucleolytic attack was hormone-independent. Thus, induction at this promoter was a bimodal process involving receptor-dependent remodeling of chromatin that allows NF1/CTF loading and direct receptor-mediated recruitment of additional transcription factors.

Hormone-dependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter.

Activation of gene transcription involves chromatin remodeling by coactivator proteins that are recruited by DNA-bound transcription factors. Local modification of chromatin structure at specific gene promoters by ATP-dependent processes and by posttranslational modifications of histone N-terminal tails provides access to RNA polymerase II and its accompanying transcription initiation complex. While the roles of lysine acetylation, serine phosphorylation, and lysine methylation of histones in chromatin remodeling are beginning to emerge, low levels of arginine methylation of histones have only recently been documented, and its physiological role is unknown. The coactivator CARM1 methylates histone H3 at Arg17 and Arg26 in vitro and cooperates synergistically with p160-type coactivators (e.g., GRIP1, SRC-1, ACTR) and coactivators with histone acetyltransferase activity (e.g., p300, CBP) to enhance gene activation by steroid and nuclear hormone receptors (NR) in transient transfection assays. In the current study, CARM1 cooperated with GRIP1 to enhance steroid hormone-dependent activation of stably integrated mouse mammary tumor virus (MMTV) promoters, and this coactivator function required the methyltransferase activity of CARM1. Chromatin immunoprecipitation assays and immunofluorescence studies indicated that CARM1 and the CARM1-methylated form of histone H3 specifically associated with a large tandem array of MMTV promoters in a hormone-dependent manner. Thus, arginine-specific histone methylation by CARM1 is an important part of the transcriptional activation process.

Nucleoprotein structure influences the response of the mouse mammary tumor virus promoter to activation of the cyclic AMP signalling pathway.

Recent studies have provided evidence of crosstalk between steroid receptors and cyclic AMP (cAMP) signalling pathways in the regulation of gene expression. A synergism between intracellular phosphorylation inducers and either glucocorticoids or progestins has been shown to occur during activation of the mouse mammary tumor virus (MMTV) promoter. We have investigated the effect of 8-Br-cAMP and okadaic acid, modulators of cellular kinases and phosphatases, on the hormone-induced activation of the MMTV promoter in two forms: a transiently transfected template with a disorganized, accessible nucleoprotein structure and a stably replicating template with an ordered, inaccessible nucleoprotein structure. Both okadaic acid and 8-Br-cAMP synergize significantly with either glucocorticoids or progestins in activating the transiently transfected MMTV template. In contrast, 8-Br-cAMP, but not okadaic acid, is antagonistic to hormone-induced activation of the stably replicating MMTV template. Nuclear run-on experiments demonstrate that this inhibition is a transcriptional effect on both hormone-induced transcription and basal transcription. Surprisingly, 8-Br-cAMP does not inhibit glucocorticoid-induced changes in restriction enzyme access and nuclear factor 1 binding. However, association of a complex with the TATA box region is inhibited in the presence of 8-Br-cAMP. Thus, cAMP treatment interferes with the initiation process but does not inhibit interaction of the receptor with the template. Since the replicated, ordered MMTV templates and the transfected, disorganized templates show opposite responses to 8-Br-cAMP treatment, we conclude that chromatin structure can influence the response of a promoter to activation of the cAMP signalling pathway.

Two regions of the mouse mammary tumor virus long terminal repeat regulate the activity of its promoter in mammary cell lines.

In vivo expression of the mouse mammary tumor virus (MMTV) is restricted to a few organs, with the highest rate of transcription found in the mammary gland. Using a series of mammary and nonmammary murine cell lines, we have identified two regulatory elements, located upstream of the hormone responsive element, that specifically regulate the MMTV promoter. The first element displays an enhancerlike activity and is coincident with the binding of a nuclear factor (designated MP4; position -1078 to -1052 in the long terminal repeat) whose presence is apparently restricted to mammary cell lines. The second regulatory region mediates a repressive activity and is mapped to the long terminal repeat segment from -415 to -483. This repression is specific for a particular subtype of mammary cells (RAC cells) able to grow under two differentiation states (A. Sonnenberg, H. Daams, J. Calafat, and J. Hilgers, Cancer Res. 46:5913-5922, 1986). The MMTV promoter in mammary cell lines thus appears to be modulated by two cis-acting elements that are likely to be involved in tissue-specific expression in vivo.

Transcription factor access is mediated by accurately positioned nucleosomes on the mouse mammary tumor virus promoter.

A fragment of the mouse mammary tumor virus (MMTV) promoter was reconstituted from pure histones into a dinucleosome with uniquely positioned octamer cores. Core boundaries for the in vitro-assembled dinucleosome corresponded to the observed in vivo phasing pattern for long terminal repeat nucleosomes A and B. Nuclear factor 1 (NF1), a constituent of the MMTV transcription initiation complex, was excluded from the assembled dinucleosome, whereas the glucocorticoid receptor was able to bind. During transcription of MMTV in vivo, displacement of nucleosome B was necessary to permit assembly of the initiation complex. These results indicate that the nucleoprotein structure of the promoter can provide differential access to sequence-specific DNA-binding proteins and that active chromatin remodeling can occur during transcription activation.

Mouse mammary tumor virus chromatin in human breast cancer cells is constitutively hypersensitive and exhibits steroid hormone-independent loading of transcription factors in vivo.

We have stably introduced a reporter gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) into human T47D breast cancer cells to study the action of the progesterone receptor (PR) on transcription from a chromatin template. Unexpectedly, the chromatin organization of the MMTV LTR in these human breast cancer cells differed markedly from what we have observed previously. The region adjacent to the transcription start site (-221 to -75) was found to be constitutively hypersensitive to restriction enzyme cleavage in the absence of hormone. This region is normally encompassed within the second nucleosome of a phased array of six nucleosomes that is assembled when the MMTV LTR is stably maintained in mouse cells. Characteristically, in these rodent cells, the identical DNA sequences show increased restriction enzyme cleavage only in the presence of glucocorticoid. The increased access of restriction enzymes observed in the human PR+ cells was not observed in adjacent nucleosomes and was unaffected by treatment with the progesterone antagonist RU486. In addition, exonuclease III-dependent stops corresponding to the binding sites for nuclear factor 1 and the PR were observed before and after hormone treatment. These results indicate that MMTV chromatin replicated in these cells is organized into a constitutively open architecture and that this open chromatin state is accompanied by hormone-independent loading of a transcription factor complex that is normally excluded from uninduced chromatin.

Independent glucocorticoid induction and repression of two contiguous responsive genes.

Specific DNA sequence elements which contain binding sites for the glucocorticoid receptor mediate the action of glucocorticoid hormones on gene transcription. In glucocorticoid-inducible genes, these glucocorticoid-responsive elements behave as hormone-inducible enhancers of transcription. We have taken advantage of the bovine papillomavirus (BPV) system to test the stringency of glucocorticoid regulation of transcription. BPV episomes were constructed to contain two hormone-regulated transcription units in close proximity; one transcription unit is under control of a glucocorticoid-inducible promoter (mouse mammary tumor virus) while the other is under control of a glucocorticoid-inhibited promoter (pro-opiomelanocortin). Glucocorticoids independently regulated transcription of the two physically linked transcription units, irrespective of their relative orientation and of their proximity on the BPV episomes. This result contrasts with the so-called position-independent activity of enhancers and suggests that the multicomponent organization of eucaryotic promoters restricts the action of hormone-responsive regulatory elements to a specific transcription unit, thus accounting for the stringency of hormonal regulation observed in vivo.

The position and length of the steroid-dependent hypersensitive region in the mouse mammary tumor virus long terminal repeat are invariant despite multiple nucleosome B frames.

Stimulation of the mouse mammary tumor virus with steroids results in the generation of a DNase I-hypersensitive region (HSR) spanning the hormone responsive element (HRE) in the long terminal repeat. Restriction enzymes were used to characterize the accessibility of various sites within the HSR of mouse mammary tumor virus long terminal repeat-reporter constructions in four different cell lines. The glucocorticoid-dependent HSR was found to span minimally 187 bases, a stretch of DNA longer than that associated with histones in the core particle. Although the 5'-most receptor binding site within the HRE is downstream of -190, hypersensitive sites were found further upstream to at least -295. The relationship in the accessibility between pairs of sites in the vicinity of the HSR was further examined in one cell line by a two-enzyme restriction access assay. In the uninduced state, the accessibilities at these sites were found to be independent of each other. In contrast, when stimulated with hormone, the accessibilities at these sites were observed to become linked. That is, once a distinct promoter was activated, all of the sites within the HSR of that molecule became accessible. The HSR formed along an invariant stretch of DNA sequence despite the multiplicity of nucleosome frames in the nucleosome B region, where the HRE is located. The results indicate that the macroscopic length of the HSR does not arise from core length-remodeling events in molecules containing Nuc-B in alternative positions.

Glucocorticoid regulation of transcription at an amplified, episomal promoter.

The mouse mammary tumor virus long terminal repeat (MMTV LTR) has been introduced into cultured murine cells, using the 69% transforming fragment of bovine papilloma virus type 1 (BPV). Transformed cells contain up to 200 copies of the chimeric molecules per diploid genome. The restriction endonuclease map of the acquired recombinants, as well as the physical structure of the DNA, indicates that the LTR-BPV molecules present in these cells occur exclusively as unintegrated, extrachromosomal episome. When a 72-base pair direct repeat "enhancer" element (derived from the Harvey sarcoma retrovirus) was included in the MMTV LTR-BPV chimeric plasmids, DNA acquired through transfection, with a single exception, was integrated or rearranged or both. The transcriptional potential of the episomal MMTV promoter present in these cells was tested in two ways. First, steady-state levels of MMTV-initiated RNA were measured by quantitative S1 mapping. Second, the relative number of transcription complexes initiated in vivo was determined by using a subnuclear fraction highly enriched for MMTV-BPV minichromosomes in an in vitro transcription extension assay. Both approaches showed that the MMTV LTR present in the episomal state was capable of supporting glucocorticoid hormone-regulated transcription. We have therefore demonstrated the hormone response for the first time in a totally defined primary sequence environment. Significant differences both in the basal level of MMTV-initiated transcription and in the extent of glucocorticoid induction were observed in individual cell lines with similar episomal copy numbers. These phenotypic variations suggest that epigenetic structure is an important component of the mechanism of regulation.

A ligand binding domain mutation in the mouse glucocorticoid receptor functionally links chromatin remodeling and transcription initiation.

We utilized the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) in vivo to understand how the interaction of the glucocorticoid receptor (GR) with a nucleosome-assembled promoter allows access of factors required for the transition from a repressed promoter to a derepressed, transcriptionally competent promoter. A mutation (C644G) in the ligand binding domain (LBD) of the mouse GR has provided information regarding the steps required in the derepression/activation process and in the functional significance of the two major transcriptional activation domains, AF1 and AF2. The mutant GR activates transcription from a transiently transfected promoter that has a disordered nucleosomal structure, though significantly less well than the wild-type GR. With an integrated, replicated promoter, which is assembled in an ordered nucleosomal array, the mutant GR does not activate transcription, and it fails to induce chromatin remodeling of the MMTV LTR promoter, as indicated by nuclease accessibility assays. Together, these findings support a two-step model for the transition of a nucleosome-assembled, repressed promoter to its transcriptionally active, derepressed form. In addition, we find that the C-terminal GR mutation is dominant over the transcription activation function of the N-terminal GR activation domain. These findings suggest that the primary activation function of the C-terminal activation domain is different from the function of the N-terminal activation domain and that it is required for derepression of the chromatin-repressed MMTV promoter.

Structure and dynamic properties of a glucocorticoid receptor-induced chromatin transition.

Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W. D. Pennie, S. John, and G. L. Hager, Mol. Cell. Biol. 18:3633-3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a "hit-and-run" mechanism for GR action in living cells (J. G. McNally, W. G. Müller, D. Walker, and G. L. Hager, Science 287:1262-1264, 2000).

Direct visualization of the human estrogen receptor alpha reveals a role for ligand in the nuclear distribution of the receptor.

The human estrogen receptor alpha (ER alpha) has been tagged at its amino terminus with the S65T variant of the green fluorescent protein (GFP), allowing subcellular trafficking and localization to be observed in living cells by fluorescence microscopy. The tagged receptor, GFP-ER, is functional as a ligand-dependent transcription factor, responds to both agonist and antagonist ligands, and can associate with the nuclear matrix. Its cellular localization was analyzed in four human breast cancer epithelial cell lines, two ER+ (MCF7 and T47D) and two ER- (MDA-MB-231 and MDA-MB-435A), under a variety of ligand conditions. In all cell lines, GFP-ER is observed only in the nucleus in the absence of ligand. Upon the addition of agonist or antagonist ligand, a dramatic redistribution of GFP-ER from a reticular to punctate pattern occurs within the nucleus. In addition, the full antagonist ICI 182780 alters the nucleocytoplasmic compartmentalization of the receptor and causes partial accumulation in the cytoplasm in a process requiring continued protein synthesis. GFP-ER localization varies between cells, despite being cultured and treated in a similar manner. Analysis of the nuclear fluorescence intensity for variation in its frequency distribution helped establish localization patterns characteristic of cell line and ligand. During the course of this study, localization of GFP-ER to the nucleolar region is observed for ER- but not ER+ human breast cancer epithelial cell lines. Finally, our work provides a visual description of the "unoccupied" and ligand-bound receptor and is discussed in the context of the role of ligand in modulating receptor activity.

Understanding nuclear receptor function: from DNA to chromatin to the interphase nucleus.

The regulation of gene expression by steroid receptors is the fundamental mechanism by which these important bioregulatory molecules exert their action. As such, mechanisms utilized by receptors in the modulation of genetic expression have been intensively studied since the first identification of hormone-binding proteins. Although these mechanisms include both posttranscriptional (1) and posttranslational (2) components, the primary level of control involves direct modulation of the rate of transcription, and it is this process that has been the major focus of research in the field.

Role of the TATA box in transcription of the mouse mammary tumor virus long terminal repeat.

An in vitro transcription system from mammary cells was established to study transcription of the long terminal repeat (LTR) of the mouse mammary tumor virus (MMTV). Experiments with progressive 5'-deletion constructs of the MMTV LTR revealed that a 19-base pair (bp) region from -41 to -23 bp, encompassing the TATA box and flanking DNA sequence, was as transcriptionally active as larger promoter constructs, both in nuclear extracts from human mammary cell lines (T47D and MCF7) and a nonmammary cell line (HeLa). The cell-free system was capable of supporting transcriptional induction by factors binding upstream of the TATA box, however, since purified glucocorticoid receptor-induced transcription in larger promoter constructs encompassing the MMTV hormone-responsive elements. Transcription from two other promoters, the adenovirus major late promoter and the human immunodeficiency virus LTR, also revealed a significant transcriptional contribution of upstream elements. The 19-bp TATA region from the MMTV LTR was shown to have considerably more activity in this transcription system than comparable TATA regions from other promoters. Sequences critical to the MMTV TATA region were evaluated by single base pair mutagenesis and found to comprise a consensus TATA box sequence, TATAAAA, as well as a single A just upstream of the TATAAAA sequence. Thus, the high level of basal transcription observed with the TATA region from MMTV is due to a perfect consensus TATA box sequence and a single base immediately 5' adjacent. It is likely that the high basal rate of transcription observed with this TATA box region on histone-free templates represents an inappropriate level of basal expression and that a complete evaluation of transactivation mechanisms in this system will require the recapitulation in vitro of the chromatin-mediated repressive state that exists in vivo.

Mechanism of dexamethasone 21-mesylate antiglucocorticoid action: I. Receptor-antiglucocorticoid complexes do not competitively inhibit receptor-glucocorticoid complex activation of gene transcription in vivo.

The actions of dexamethasone 21-mesylate (DM) have been studied in two recently developed cultured murine cell lines containing approximately 200 copies of episomal minichromosome. This minichromosome contains the glucocorticoid regulatory element in the mouse mammary tumor virus long terminal repeat fused upstream of v-rasH sequences in a totally defined primary sequence environment. The levels of v-rasH mRNA were measured as an index of glucocorticoid regulated expression of this chimeric gene. In addition, expression of the endogenous single copy mouse metallothionein I (MT-I) gene was monitored simultaneously. DM was found to be an essentially pure antagonist of dexamethasone (dex)-stimulated expression of both the episomal chimeric gene and the endogenous MT-I gene. The covalent labeling efficiency by DM of glucocorticoid receptors in intact cells approached 100%, surpassing previously observed whole cell DM labeling efficiencies. These results strengthen the hypothesis that covalent complex formation is responsible for antiglucocorticoid action. The efficiency of whole cell nuclear binding of covalent receptor-DM complexes was found to be approximately 50% of that seen with receptor-dex complexes. Analyses of long terminal repeat initiated v-rasH mRNA and MT-I mRNA inductions by dex in cells previously exposed to a subsaturating concentration of DM indicated that receptor-DM complexes do not inhibit by a competitive mechanism the transcriptional activation of these glucocorticoid responsive genes by receptor-dex complexes. These results do not rule out the possibility, however, that covalent receptor-DM complexes may still bind to the biologically active nuclear sites. The implications of this result concerning the mechanism of DM irreversible antiglucocorticoid action are discussed.

Overexpression of the human vitamin D3 receptor in mammalian cells using recombinant adenovirus vectors.

The human vitamin D3 receptor (hVDR) cDNA was cloned into the E1 region of the adenovirus genome to generate recombinant viruses which were used to infect 293 (adenovirus-transformed human fetal kidney) cells. High salt extracts from cells infected with the recombinant viruses were subjected to immunoblot analysis using a monoclonal antibody to chicken VDR and were shown to contain large quantities of a protein of approximately 50 kDa with a migration identical to that of the hVDR in T47D (human mammary adenocarcinoma) cells. Scatchard analysis showed that the infected cells express approximately 100-fold more receptor than T47D cells and that this receptor binds 1,25-dihydroxyvitamin D3 with high affinity. The overexpressed hVDR also binds to DNA-cellulose and is eluted with a KCl concentration similar to that determined for fully active endogenous VDR. Nuclear extracts from cells infected with the hVDR-expressing adenoviruses contain an activity that specifically binds an oligonucleotide with sequences from the rat osteocalcin vitamin D3 response element, as determined by gel mobility shift. This interaction can be inhibited by the presence of an anti-VDR antibody, but not by nonspecific immunoglobulins. We conclude, therefore, that the overexpressed receptor has the ligand- and DNA-binding characteristics defined for endogenous VDR and that adenoviruses can be used to efficiently express large quantities of functional hVDR in a human cell line. Finally, a second binding activity, specific for the vitamin D response element, but distinct from the VDR, has been identified in extracts from uninfected cells.

Proopiomelanocortin gene promoter elements required for constitutive and glucocorticoid-repressed transcription.

The POMC gene is expressed predominantly in the anterior pituitary. The high level of POMC transcription in this tissue is modulated by peptide hormones and repressed by glucocorticoids. In this present study we have investigated promoter elements required for the high basal transcription and glucocorticoid repression using transient transfection and in vitro transcription assays. We first determined that the region between -77 to -51 of the promoter, which has previously been shown to harbor a glucocorticoid receptor-binding site, is required for high basal expression both in vivo and in vitro. This promoter domain is also required for glucocorticoid repression of transcription in vivo. Two site-directed mutants within this area both decreased basal transcription, but were fully repressed by glucocorticoids, implying that the -77 to -51 region is a complex regulatory region harboring separable basal and glucocorticoid-repressible elements. Electrophoretic mobility shift and exonuclease III footprinting analysis revealed the existence of two factors that bind in this region. We also examined the effect of broad promoter deletions on basal expression and glucocorticoid repression. These experiments revealed that the region between -480 and -320 is also required for glucocorticoid repression. Taken together, the data suggest a model in which high basal transcription is generated by direct interaction of factors binding between -480 to -320 and -77 to -51. Glucocorticoid repression could occur by direct receptor disruption of these interactions.