Ephrin-A5 Is Required for Optimal Fertility and a Complete Ovulatory Response to Gonadotropins in the Female Mouse.

Follicle growth and ovulation involve the coordinated expression of many genes, driven by FSH and LH. Reports indicate that Eph receptors and ephrins are expressed in the ovary, suggesting roles in follicle growth and/or ovulation. We previously reported FSH-induced expression of ephrin-A5 (EFNA5) and 4 of its cognate Eph receptors in mouse granulosa cells. We now report that female mice lacking EFNA5 are subfertile, exhibit a compromised response to LH, and display abnormal ovarian histology after superovulation. Efna5(-/-) females litters were 40% smaller than controls, although no difference in litter frequency was detected. The ovarian response to superovulation was also compromised in Efna5(-/-) females, with 37% fewer oocytes ovulated than controls. These results corresponded with a reduction in ovarian mRNA levels of several LH-responsive genes, including Pgr, Ptgs2, Tnfaip6, Ereg, Btc, and Adamts4, suggesting that Efna5(-/-) ovaries exhibit a partially attenuated response to LH. Histopathological analysis indicated that superovulated Efna5(-/-) females exhibited numerous ovarian defects, including intraovarian release of cumulus oocyte complexes, increased incidence of oocytes trapped within luteinized follicles, granulosa cell and follicular fluid emboli, fibrin thrombi, and interstitial hemorrhage. In addition, adult Efna5(-/-) ovaries exhibited a 4-fold increase in multioocyte follicles compared with controls, although no difference was detected in 3-week-old mice, suggesting the possibility of follicle merging. Our observations indicate that loss of EFNA5 in female mice results in subfertility and imply that Eph-ephrin signaling may also play a previously unidentified role in the regulation of fertility in women.

Evaluating the effectiveness of cancer drug sensitization in vitro and in vivo.

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

BRCA2 inhibition enhances cisplatin-mediated alterations in tumor cell proliferation, metabolism, and metastasis.

Tumor cells have unstable genomes relative to non-tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti-cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2-targeting second-generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non-tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency in vivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti-cancer agent.

The ephrin signaling pathway regulates morphology and adhesion of mouse granulosa cells in vitro.

Follicle-stimulating hormone (FSH)-mediated changes in granulosa cell adhesion and morphology are essential for preovulatory follicle development, given the dramatic changes in follicle size and granulosa cell number that occur during this transition. Members of the Eph-ephrin family of cell-positioning and adhesion molecules, a family that consists of ephrin ligands and their Ephrin (Eph) receptors, regulate cell location, adhesion, and migration during embryonic development and tumor growth. However, very little is known about ephrin signaling during folliculogenesis. We have found that FSH increases the expression of several members of the Eph-ephrin family and that this signaling regulates granulosa cell morphology and adhesion. FSH induced increased mRNA levels of the ephrin ligand, ephrin-A5 (Efna5), and its receptors, Eph receptors A3, A5, and A8 (Epha3, Epha5, and Epha8, respectively), in granulosa cells. Immunofluorescence studies indicated that EFNA5 and EPHA5 are located in the membrane of granulosa cells of developing mouse follicles. Eph-ephrin signaling directly affected granulosa cell morphology and adhesion. Recombinant EFNA5 reduced cell spreading and increased cell rounding in mouse primary granulosa cells and in a rat granulosa cell line, whereas EPHA5 reduced granulosa cell adhesion in both model systems. Both FSH and forskolin also increased Efna5 and Epha5 mRNA levels in rat and human granulosa cell lines, indicating that FSH regulates these genes via the cAMP-dependent protein kinase A pathway and that this regulation is conserved across different species. The present study identifies Eph-ephrin signaling as a novel FSH-mediated pathway regulating granulosa cell morphology and adhesion.

Compromised fertility disrupts Peg1 but not Snrpn and Peg3 imprinted methylation acquisition in mouse oocytes.

Growth and maturation of healthy oocytes within follicles requires bidirectional signaling and intercellular gap junctional communication. Aberrant endocrine signaling and loss of gap junctional communication between the oocyte and granulosa cells leads to compromised folliculogenesis, oocyte maturation, and oocyte competency, consequently impairing fertility. Given that oocyte-specific DNA methylation establishment at imprinted genes occurs during this growth phase, we determined whether compromised endocrine signaling and gap junctional communication would disrupt de novo methylation acquisition using ERß and connexin37 genetic models. To compare mutant oocytes to control oocytes, DNA methylation acquisition was first examined in individual, 20-80 µm control oocytes at three imprinted genes, Snrpn, Peg3, and Peg1. We observed that each gene has its own size-dependent acquisition kinetics, similar to previous studies. To determine whether compromised endocrine signaling and gap junctional communication disrupted de novo methylation acquisition,individual oocytes from Esr2- and Gja4-deficient mice were also assessed for DNA methylation establishment. We observed no aberrant or delayed acquisition of DNA methylation at Snrpn, Peg3, or Peg1 in oocytes from Esr2-deficient females, and no perturbation in Snrpn or Peg3de novo methylation in oocytes from Gja4-null females. However, Gja4 deficiency resulted in a loss or delay in methylation acquisition at Peg1. One explanation for this difference between the three loci analyzed is the late establishment of DNA methylation at the Peg1 gene. These results indicate that compromised fertility though impaired intercellular communication can lead to imprinting acquisition errors. Further studies are required to determine the effects of subfertility/infertility originating from impaired signaling and intercellular communication during oogenesis on imprint maintenance during preimplantation development.

Expression of extracellular matrix components is disrupted in the immature and adult estrogen receptor ß-null mouse ovary.

Within the ovary, Estrogen Receptor ß (ERß) is localized to the granulosa cells of growing follicles. 17ß-estradiol (E2) acting via ERß augments the actions of follicle stimulating hormone in granulosa cells, leading to granulosa cell differentiation and formation of a preovulatory follicle. Adult ERß-null females are subfertile and possess ovaries with reduced numbers of growing follicles and corpora lutea. Because the majority of E2 production by granulosa cells occurs once puberty is reached, a role for ERß in the ovary prior to puberty has not been well examined. We now provide evidence that lack of ERß disrupts gene expression as early as post-natal day (PND) 13, and in particular, we identify a number of genes of the extracellular matrix (ECM) that are significantly higher in ERß-null follicles than in wildtype (WT) follicles. Considerable changes occur to the ECM occur during normal folliculogenesis to allow for the dramatic growth, cellular differentiation, and reorganization of the follicle from the primary to preovulatory stage. Using quantitative PCR and immunofluorescence, we now show that several ECM genes are aberrantly overexpressed in ERß-null follicles. We find that Collagen11a1, a protein highly expressed in cartilage, is significantly higher in ERß-null follicles than WT follicles as early as PND 13, and this heightened expression continues through PND 23-29 into adulthood. Similarly, Nidogen 2, a highly conserved basement membrane glycoprotein, is elevated in ERß-null follicles at PND 13 into adulthood, and is elevated specifically in the ERß-null focimatrix, a basal lamina-like matrix located between granulosa cells. Focimatrix laminin and Collagen IV expression were also higher in ERß-null ovaries than in WT ovaries at various ages. Our findings suggest two novel observations: a) that ERß regulates granulosa cell gene expression ovary prior to puberty, and b) that ERß regulates expression of ECM components in the mouse ovary.

Minireview: Estrogen receptor-beta: mechanistic insights from recent studies.

The discovery of estrogen receptor-beta (ERbeta) in 1996 stimulated great interest in the physiological roles and molecular mechanisms of ERbeta action. We now know that ERbeta plays a major role in mediating estrogen action in several tissues and organ systems, including the ovary, cardiovascular system, brain, and the immune system, and that ERbeta and ERalpha generally play distinct physiological roles in the body. Although significant progress has been made toward understanding the molecular mechanisms of ERbeta action, particularly in vitro, there remains a large gap in our understanding of the mechanisms by which ERbeta elicits its biological functions in a true physiological context.

Profile of estrogen-responsive genes in an estrogen-specific mammary gland outgrowth model.

Both ovarian and pituitary hormones are required for the pubertal development of the mouse mammary gland. Estradiol directs ductal elongation and branching, while progesterone leads to tertiary branching and alveolar development. The purpose of this investigation was to identify estrogen-responsive genes associated with pubertal ductal growth in the mouse mammary gland in the absence of other ovarian hormones and at different stages of development. We hypothesized that the estrogen-induced genes and their associated functions at early stages of ductal elongation would be distinct from those induced after significant ductal elongation had occurred. Therefore, ovariectomized prepubertal mice were exposed to 17beta-estradiol from two to 28 days, and mammary gland global gene expression analyzed by microarray analysis at various times during this period. We found that: (a) gene expression changes in our estrogen-only model mimic those changes that occur in normal pubertal development in intact mice, (b) both distinct and overlapping gene profiles were observed at varying extents of ductal elongation, and (c) cell proliferation, the immune response, and metabolism/catabolism were the most common functional categories associated with mammary ductal growth. Particularly striking was the novel observation that genes active during carbohydrate metabolism were rapidly and robustly decreased in response to estradiol. Lastly, we identified mammary estradiol-responsive genes that are also co-expressed with estrogen receptor alpha in human breast cancer. In conclusion, our genomic data support the physiological observation that estradiol is one of the primary hormonal signals driving ductal elongation during pubertal mammary development.

Estrogen receptor beta is required for optimal cAMP production in mouse granulosa cells.

Granulosa cells of preovulatory follicles differentiate in response to FSH, and this differentiation is augmented by estradiol. We have previously shown that FSH-mediated granulosa cell differentiation requires functional estrogen receptor-beta (ERbeta) by demonstrating that the granulosa cells of ERbeta(-/-) FSH-treated mice are unable to maximally induce expression of the LH receptor (an indicator of granulosa cell differentiation) compared with ERbeta(+/+) controls. As a result, FSH-primed ERbeta(-/-) granulosa cells exhibit a reduced response to a subsequent ovulatory dose of LH. In this study, we further characterized the attenuated response of ERbeta(-/-) granulosa cells to stimulation by LH and FSH using isolated mouse granulosa cells and primary granulosa cell cultures. We observed a 50% reduction in cAMP levels in cultured ERbeta(-/-) granulosa cells exposed to LH compared with ERbeta(+/+) controls. We also observed an attenuated genomic response in granulosa cells isolated from FSH-primed ERbeta(-/-) mice compared with ERbeta(+/+) controls. Our data indicate that this attenuated response may result from inadequate levels of cAMP, because cAMP levels in cultured ERbeta(-/-) granulosa cells exposed to forskolin were approximately 50% lower than in ERbeta(+/+) granulosa cells. Phosphorylation of cAMP regulatory element binding protein, an indicator of protein kinase A activity, was also reduced in FSH-treated ERbeta(-/-) granulosa cells compared with ERbeta(+/+) controls. These are the first data to indicate that ERbeta plays a role in the induction of the cAMP pathway in mouse granulosa cells and that disruption of proper ERbeta signaling associated with this pathway may cause negative effects on ovulation and fertility.

Estrogen receptors and human disease.

Estrogens influence many physiological processes in mammals, including but not limited to reproduction, cardiovascular health, bone integrity, cognition, and behavior. Given this widespread role for estrogen in human physiology, it is not surprising that estrogen is also implicated in the development or progression of numerous diseases, which include but are not limited to various types of cancer (breast, ovarian, colorectal, prostate, endometrial), osteoporosis, neurodegenerative diseases, cardiovascular disease, insulin resistance, lupus erythematosus, endometriosis, and obesity. In many of these diseases, estrogen mediates its effects through the estrogen receptor (ER), which serves as the basis for many therapeutic interventions. This Review will describe diseases in which estrogen, through the ER, plays a role in the development or severity of disease.

Estrogen receptor-beta is critical to granulosa cell differentiation and the ovulatory response to gonadotropins.

The process of granulosa cell differentiation that occurs in preovulatory follicles is dependent on FSH but requires augmentation by estradiol. To determine which estrogen receptor (ER) form mediates the effects of estradiol during gonadotropin-induced follicle growth, differentiation, and rupture, we characterized the response of ERalpha- and ERbeta-null mice to gonadotropin-induced ovulation. Immature mice were treated with an ovulatory regimen of exogenous gonadotropins and tissues were collected at distinct time points for morphological, biochemical, gene expression, and immunohistochemical analyses. Granulosa cells of ERbeta knockout (ERKO) preovulatory follicles exhibited an attenuated response to FSH-induced differentiation, as evident by reduced aromatase activity and estradiol synthesis, and insufficient expression of LH receptor. As a result, betaERKO ovaries were unable to fully respond to an ovulatory bolus of gonadotropin, leading to a reduced rate of follicle rupture; insufficient induction of prostaglandin-synthase 2 and progesterone receptor; an aberrant increase in aromatase activity and plasma estradiol; and incomplete expansion of the cumulus-oocyte complex. Parallel characterization of alphaERKO females indicated a minimal role for ERalpha in granulosa cell differentiation, ovulation, and the concomitant changes in gene expression, although some abnormalities were revealed. These studies demonstrate that ERbeta-mediated estradiol actions are vital to FSH-induced granulosa cell differentiation; and in the absence of ERbeta, preovulatory follicles are deficient in the necessary cellular organization (i.e. antrum and cumulus oocyte complex), enzymatic activity (i.e. capacity to convert androgen precursor to estradiol), and receptor signaling pathways (i.e. LH receptor) to respond to a gonadotropin surge and expel a healthy oocyte.

Estradiol regulates the thioredoxin antioxidant system in the mouse uterus.

The rodent uterus responds to acute estradiol (E2) treatment with a series of well characterized physiological responses. In a recent screen for genes involved in this response, we found that several genes in the thioredoxin (Txn) pathway were rapidly modified after E2 treatment in the mouse uterus. Txn is a 12-kDa protein with multiple roles in the cell, including protection against oxidative stress and apoptosis, regulation of transcription factor activity, and regulation of cellular proliferation. Txn in combination with Txn reductase (Txnrd) and Txn-interacting protein (Txnip) constitute the mammalian Txn pathway. This pathway exists in multiple locations in the cell, including the cytosol and mitochondria. To analyze the levels of Txn, Txnrd, and Txnip in the uterus, we treated ovariectomized adult mice with a time course of E2 and analyzed mRNA levels by real-time PCR. E2 rapidly decreased the expression of Txnip, but increased the levels of cytosolic Txn1 and Txnrd1 as well as mitochondrial Txn2. Using the ER antagonist, ICI 182,780, and mice lacking functional estrogen receptor alpha (ERalpha), we demonstrate that these E2-mediated changes require ERalpha, but not ERbeta. The repression of Txnip by E2 was also demonstrated in vitro in MCF-7 human breast cancer cells. This repression was blocked by treatment with the histone deacetylase inhibitor, trichostatin A, suggesting that repression by E2 may involve regulation of histone acetylation. We conclude that the rapid E2-mediated activation of the Txn pathway is an important step in the response of the mammalian uterus to estrogen.

Estrogen receptor-dependent genomic responses in the uterus mirror the biphasic physiological response to estrogen.

The physiological responses of the rodent uterus to acute estrogen (E) dosing can be divided into early and late events. Examples of early responses include increased RNA transcription, hyperemia, and water imbibition 2 and 6 h following E administration respectively, whereas later responses include cycles of DNA synthesis and mitosis of epithelial cells beginning 10 and 16 h after E. The development of estrogen receptor (ER) knockout (ERKO) mice, combined with microarray technology, has allowed us to design a genomic approach to study the acute response of the rodent reproductive tract to E. To determine whether early and late biological responses are correlated with altered regulation of a single set of genes or distinct sets of genes characteristic of early and late responses, uterine RNA was obtained from ovariectomized mice that were treated with vehicle or with estradiol for 2 h (early) or 24 h (late). Samples were also prepared from identically treated mice that lacked either ERalpha (alphaERKO) or ERbeta (betaERKO) to address the relative contributions of the ERs in the uterine responses. Microarray analysis of the relative expression of 8700 mouse cDNAs indicated distinct clusters of genes that were regulated both positively and negatively by E in the early or late phases as well as clusters of genes regulated at both times. Both early and late responses by the betaERKO samples were indistinguishable from those of WT samples, whereas the alphaERKO showed little change in gene expression in response to E, indicating the predominant role for ERalpha in the genomic response. Further studies indicated that the genomic responses in samples from intermediate time points (6 h, 12 h) fall within the early or late clusters, rather than showing unique clusters regulated in the intermediary period. The use of this genomic approach has illustrated how physiological responses are reflected in genomic patterns. Furthermore, the identification of functional gene families that are regulated by E in the uterus combined with the utilization of genetically altered experimental animal models can help to uncover and define novel mechanisms of E action.

Differential activation of the IkappaBalpha and mouse mammary tumor virus promoters by progesterone and glucocorticoid receptors.

The glucocorticoid and progesterone receptors (GR and PR) are structurally homologous and bind a common hormone response element (HRE). The mechanisms by which receptors activate specific promoters when multiple steroids are present in a cell is a critical question in endocrinology. To investigate how co-existing steroid receptors regulate gene transcription, we have compared two hormone-responsive promoters in T47D/A1-2 human breast cancer cells expressing both the GR and PR. The promoters chosen were those for the mouse mammary tumor virus (MMTV) and the gene for IkappaBalpha, the inhibitor of the ubiquitous transcription factor, nuclear factor kappa B (NFkappaB). Several differences between glucocorticoid and progestin activation of the IkappaBalpha and MMTV promoters were revealed. Both steroids activated the endogenous IkappaBalpha promoter, while only glucocorticoids activated a stably integrated MMTV promoter. In combination, progestins enhanced glucocorticoid activation of IkappaBalpha, but antagonized glucocorticoid activation of MMTV. These differences in steroid receptor competition were further demonstrated when levels of the PR were reduced by prolonged treatment with progestin. Under these conditions, the PR no longer competes effectively with the GR for activation of the MMTV promoter. However, on the IkappaBalpha promoter, the GR and PR still activate the promoter in a cooperative fashion. Another difference between the two promoters is their chromatin structure. In this cell line, the MMTV promoter chromatin is "closed" and insensitive to restriction enzyme cleavage, while the IkappaBalpha promoter is "open." Using PR antagonists, we demonstrate that at least one cofactor complex, the BRG-1 chromatin remodeling complex, differentially contributes to activation of both promoters.

Chromatin remodeling and tissue-selective responses of nuclear hormone receptors.

Chromatin structure of eukaryotic genes regulates gene expression by controlling the accessibility of regulatory factors. To overcome the inhibitory nature of chromatin, protein complexes that modify higher order chromatin organization and histone-DNA contacts are critical players in regulating transcription. For example, nuclear hormone receptors regulate transcription by interacting with ATP-dependent chromatin-remodeling complexes and coactivators, which include histone acetyltransferases and histone methylases that modify the basic residues of histones. A growing number of tissue-specific nuclear hormone receptor ligands, termed "selective modulators", owe their specificity, at least in part, to the differential recruitment of these chromatin-modifying coactivators. The molecular mechanisms by which these compounds modulate the functions of nuclear hormone receptors are discussed here.

Proteasomal inhibition enhances glucocorticoid receptor transactivation and alters its subnuclear trafficking.

The ubiquitin-proteasome pathway regulates the turnover of many transcription factors, including steroid hormone receptors such as the estrogen receptor and progesterone receptor. For these receptors, proteasome inhibition interferes with steroid-mediated transcription. We show here that proteasome inhibition with MG132 results in increased accumulation of the glucocorticoid receptor (GR), confirming that it is likewise a substrate for the ubiquitin-proteasome degradative pathway. Using the mouse mammary tumor virus (MMTV) promoter integrated into tissue culture cells, we found that proteasome inhibition synergistically increases GR-mediated transactivation. This increased activation was observed in a number of cell lines and on various MMTV templates, either as transiently transfected reporters or stably integrated into chromatin. These observations suggest that the increase in GR-mediated transcription due to proteasome inhibition may occur downstream of the initial chromatin remodeling step. In support of this concept, the increase in transcription did not correlate with an increase in chromatin remodeling, as measured by restriction enzyme hypersensitivity, or transcription factor loading, as exemplified by nuclear factor 1. To investigate the relationship between GR turnover, transcription, and subnuclear trafficking, we examined the effect of proteasome inhibition on the mobility of the GR within the nucleus and association of the GR with the nuclear matrix. Blocking GR turnover reduced the mobility of the GR within the nucleus, and this correlated with increased association of the receptor with the nuclear matrix. As a result of proteasome inhibition, GR mobility within the nucleus was reduced while its association with the nuclear matrix was increased. Thus, while altered nuclear mobility of steroid receptors may be a common feature of proteasome inhibition, GR is unique in its enhanced transactivation activity that results when proteasome function is compromised. Proteasomes may therefore impact steroid receptor action at multiple levels and exert distinct effects on individual receptor types.

Proteasome inhibitors reduce luciferase and beta-galactosidase activity in tissue culture cells.

Reporter enzymes are commonly used in cell biology to study transcriptional activity of genes. Recently, reporter enzymes in combination with compounds that inhibit proteasome function have been used to study the effect of blocking transcription factor degradation on gene activation. While investigating the effect of proteasome inhibition on steroid receptor activation of the mouse mammary tumor virus (MMTV) promoter, we found that treatment with proteasome inhibitors enhanced glucocorticoid activation of the promoter attached to a chloramphenicol acetyltransferase (CAT) reporter, but inhibited activation of MMTV attached to a firefly luciferase or beta-galactosidase reporter. MMTV RNA levels under these conditions correlated with the promoter activity observed using the CAT reporter, suggesting that proteasome inhibitor treatment interfered with luciferase or beta-galactosidase reporter assays. Washout experiments demonstrated that the majority of luciferase activity was lost if the proteasome inhibitor was added at the same time luciferase was produced, not once the functional protein was made, suggesting that proteasome inhibition interferes with production of luciferase protein. Indeed, we found that proteasome inhibitor treatment dramatically reduced the levels of luciferase and beta-galactosidase protein produced, as determined by Western blot. Thus, treatment with proteasome inhibitors interferes with luciferase and beta-galactosidase reporter assays, possibly by inhibiting production of a functional reporter protein.

The histone deacetylase inhibitor trichostatin A blocks progesterone receptor-mediated transactivation of the mouse mammary tumor virus promoter in vivo.

Post-translational modifications of histones play an important role in modulating gene transcription within chromatin. We used the mouse mammary tumor virus (MMTV) promoter, which adopts an ordered nucleosomal structure, to investigate the impact of a specific inhibitor of histone deacetylase, trichostatin A (TSA), on progesterone receptor-activated transcription. TSA induced global histone hyperacetylation, and this effect occurred independently of the presence of hormone. Interestingly, chromatin immunoprecipitation analysis revealed no significant change in the level of acetylated histones associated with the MMTV promoter following high TSA treatment. In human breast cancer cells, in which the MMTV promoter adopts a constitutively "open" chromatin structure, treatment with TSA converted the MMTV promoter into a closed structure. Addition of hormone did not overcome this TSA-induced closure of the promoter chromatin. Furthermore, TSA treatment resulted in the eviction of the transcription factor nuclear factor-1 from the promoter and reduced progesterone receptor-induced transcription. Kinetic experiments revealed that a loss of chromatin-remodeling proteins was coincident with the decrease in MMTV transcriptional activity and the imposition of repressed chromatin architecture at the promoter. These results demonstrate that deacetylase inhibitor treatment at levels that induce global histone acetylation may leave specific regulatory regions relatively unaffected and that this treatment may lead to transcriptional inhibition by mechanisms that modify chromatin-remodeling proteins rather than by influencing histone acetylation of the local promoter chromatin structure.