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Chromosome-Wide Mapping of Estrogen Receptor Binding Reveals Long-Range Regulation Requiring the Forkhead Protein FoxA1
Abstract
Estrogen plays an essential physiologic role in reproduction and a pathologic one in breast cancer. The completion of the human genome has allowed the identification of the expressed regions of protein-coding genes; however, little is known concerning the organization of their cis-regulatory elements. We have mapped the association of the estrogen receptor (ER) with the complete nonrepetitive sequence of human chromosomes 21 and 22 by combining chromatin immunoprecipitation (ChIP) with tiled microarrays. ER binds selectively to a limited number of sites, the majority of which are distant from the transcription start sites of regulated genes. The unbiased sequence interrogation of the genuine chromatin binding sites suggests that direct ER binding requires the presence of Forkhead factor binding in close proximity. Furthermore, knockdown of FoxA1 expression blocks the association of ER with chromatin and estrogen-induced gene expression demonstrating the necessity of FoxA1 in mediating an estrogen response in breast cancer cells.
... In other words, genes can be regulated by enhancers located virtually anywhere on the genome. ERα is known to mainly regulate expression of genes through binding to distant enhancers, as only about 5% of its binding sites are located within a distance of 5 kb from Transcription Start Site (TSS) of any gene 5 . ...
... (Recall that both classes comprise segments with ERα ChIP peaks and H3K27Ac marks, with the only difference between classes being that the positive class is transcribed as eRNAs.) FOXA1 is perhaps the most wellknown co-regulator of ERα 5,30 . As shown in Fig. 4e and consistent with previous findings, our models predict that FOXA1 activates a large fraction (nearly 50%) of examined enhancers, in both classes. ...
Estrogen Receptor α (ERα) is a major lineage determining transcription factor (TF) in mammary gland development. Dysregulation of ERα-mediated transcriptional program results in cancer. Transcriptomic and epigenomic profiling of breast cancer cell lines has revealed large numbers of enhancers involved in this regulatory program, but how these enhancers encode function in their sequence remains poorly understood. A subset of ERα-bound enhancers are transcribed into short bidirectional RNA (enhancer RNA or eRNA), and this property is believed to be a reliable marker of active enhancers. We therefore analyze thousands of ERα-bound enhancers and build quantitative, mechanism-aware models to discriminate eRNAs from non-transcribing enhancers based on their sequence. Our thermodynamics-based models provide insights into the roles of specific TFs in ERα-mediated transcriptional program, many of which are supported by the literature. We use in silico perturbations to predict TF-enhancer regulatory relationships and integrate these findings with experimentally determined enhancer-promoter interactions to construct a gene regulatory network. We also demonstrate that the model can prioritize breast cancer-related sequence variants while providing mechanistic explanations for their function. Finally, we experimentally validate the model-proposed mechanisms underlying three such variants.
... Furthermore, we observed a significant correlation between these epigenetic groups and the motif enrichment of two transcription factors (TFs): forkhead box A1 (FOXA1) and grainyhead-like 2 (GRHL2). FOXA1 functions as a pioneer factor for ER, promoting luminal-lineage proliferation [12,13], whereas GRHL2 is known to be involved in reprogramming ER signaling in breast cancer development [14]. Our knockdown experiments demonstrated that these TFs maintain accessibility to region-specific accessible regions. ...
... FOXA1 is a well-established regulator, that acts as a pioneer factor for ER and drives estrogen-dependent proliferation in ERpositive breast cancer [12,13]. Overexpression or activating mutations of FOXA1 promote breast cancer aggressiveness and are associated with poorer outcomes [26,27]. ...
Breast cancer is a heterogeneous disease, and breast cancer cell lines are invaluable for studying this heterogeneity. However, the epigenetic diversity across these cell lines remains poorly understood. In this study, we performed genome-wide chromatin accessibility analysis on 23 breast cancer cell lines, including 2 estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative (ER+/HER2−), 3 ER+/HER2+, 3 HER2+, and 15 triple-negative breast cancer (TNBC) lines. These cell lines were classified into three groups based on their chromatin accessibility: the receptor-positive group (Group-P), TNBC basal group (Group-B), and TNBC mesenchymal group (Group-M). Motif enrichment analysis revealed that only Group-P exhibited coenrichment of forkhead box A1 (FOXA1) and grainyhead-like 2 (GRHL2) motifs, whereas Group-B was characterized by the presence of the GRHL2 motif without FOXA1. Notably, Group-M did not show enrichment of either FOXA1 or GRHL2 motifs. Furthermore, gene ontology analysis suggested that group-specific accessible regions were associated with their unique lineage characteristics. To investigate the epigenetic landscape regulatory roles of FOXA1 and GRHL2, we performed knockdown experiments targeting FOXA1 and GRHL2, followed by assay for transposase-accessible chromatin sequencing analysis. The findings revealed that FOXA1 maintains Group-P–specific regions while suppressing Group-B–specific regions in Group-P cells. In contrast, GRHL2 preserves commonly accessible regions shared between Group-P and Group-B in Group-B cells, suggesting that FOXA1 and GRHL2 play a pivotal role in preserving distinct chromatin accessibility patterns for each group. Specifically, FOXA1 distinguishes between receptor-positive and TNBC cell lines, whereas GRHL2 distinguishes between basal-like and mesenchymal subtypes in TNBC lines.
... There are also protein-centric methods targeting genome-wide chromatin interactions mediated by a specific protein of interest. One of the earliest such methods is ChIP-3C [10], also known as ChIP-loop, which combines chromatin immunoprecipitation (ChIP) with 3C to facilitate detection of protein-specific chromatin interactions. The protocol of ChIP-3C for preparing proximal ligation is similar to 3C, including crosslinking and restriction enzyme digestion, but the crosslinked chromatin is first ChIP-enriched before ligation. ...
... Current experimental results from ChIA-PET, HiChIP, and PLAC-seq are indispensable resources for understanding micro-scale mechanisms of long-range and spatial gene regulations. It has been found that particular protein signaling and genome structural variation may alter the long-range regulatory mechanism, hence causing certain diseases [16,17], especially cancer [10,[18][19][20]. Output from these experimental procedures is a list of long-range interaction frequencies (counts) between pairs of anchors (genomic segments), which may or may not be on the same chromosome (Table 1 provides a partial list from a biological experiment as an example). ...
The ability to simulate high-throughput data with high fidelity to real experimental data is fundamental for benchmarking methods used to detect true long-range chromatin interactions mediated by a specific protein. Yet, such tools are not currently available. To fill this gap, we develop an in silico experimental procedure, ChIA-Sim, which imitates the experimental procedures that produce real ChIA-PET, Hi-ChIP, or PLAC-seq data. We show the fidelity of ChIA-Sim to real data by using guiding characteristics of several real datasets to generate data using the simulation procedure. We also used ChIA-Sim data to demonstrate the use of our in silico procedure in benchmarking methods for significant interactions analysis by evaluating four methods for significant interaction calling (SIC). In particular, we assessed each method’s performance in terms of correct identification of long-range interactions. We further analyzed four experimental datasets from publicly available databases and shew that the trend of the results are consistent with those seen in data generated from ChIA-Sim. This serves as additional evidence that ChIA-Sim closely resembles data produced from the experimental protocols it models after.
... Studies in C. elegans have found that first introns are more conserved in length, are bound by more transcription factors, and that the transcription factors that bind first introns are largely different from those binding promoters [68]. Studies of the ERβ homolog, ERα, in human breast cancer cells have also found that ERα does not primarily bind promoter regions (approximately 3% of sites are in the proximal promoter region [69,70]). From this, we propose that intron 1 binding, rather than promoter binding, may be a main mechanism for transcriptional activation of endogenous ERβ target genes. ...
... However, it should be noted that a complete overlap between identified ChIP-binding sites and regulated genes is not expected. The identified overlap here is similar to what has been previously demonstrated for ERα in homogenous cell lines [43,69,73]. In addition, our study does not separate the binding and activity of the two ERβ splice variants present in the mouse ovary. ...
Background
Estrogen receptor beta (ERβ, Esr2) plays a pivotal role in folliculogenesis and ovulation, yet its exact mechanism of action is mainly uncharacterized.
Results
We here performed ERβ ChIP-sequencing of mouse ovaries followed by complementary RNA-sequencing of wild-type and ERβ knockout ovaries. By integrating the ERβ cistrome and transcriptome, we identified its direct target genes and enriched biological functions in the ovary. This demonstrated its strong impact on genes regulating organism development, cell migration, lipid metabolism, response to hypoxia, and response to estrogen. Cell-type deconvolution analysis of the bulk RNA-seq data revealed a decrease in luteal cells and an increased proportion of theca cells and a specific type of cumulus cells upon ERβ loss. Moreover, we identified a significant overlap with the gene regulatory network of liver receptor homolog 1 (LRH-1, Nr5a2) and showed that ERβ and LRH-1 extensively bound to the same chromatin locations in granulosa cells. Using ChIP-reChIP, we corroborated simultaneous ERβ and LRH-1 co-binding at the ERβ-repressed gene Greb1 but not at the ERβ-upregulated genes Cyp11a1 and Fkbp5. Transactivation assay experimentation further showed that ERβ and LRH-1 can inhibit their respective transcriptional activity at classical response elements.
Conclusions
By characterizing the genome-wide endogenous ERβ chromatin binding, gene regulations, and extensive crosstalk between ERβ and LRH-1, along with experimental corroborations, our data offer genome-wide mechanistic underpinnings of ovarian physiology and fertility.
... Traditionally, ERs have been considered nuclear receptors, but it has been welldocumented that these receptors are also present on the membrane and in the cytoplasm. Nuclear ERα stimulates gene expression changes that promote cell cycle progression [6,7,8]. Moreover, the binding of ligands to plasma membrane ERα triggers rapid cellular changes through second messenger pathways, which also contribute to transcriptional effects of estrogen by regulating processes such as proliferation, cell migration, and development [9,10]. ...
... On the contrary, no significant uptake of nanoMIPs in the ERa negative cell line MDA-MB-231 was observed, as shown in Figure 6 c and d. It has been reported that ERa binds with Caveolin-1, which is one of the structural proteins of caveolae, and plays a major role in trafficking of ERα to and from the cell surface and maintaining an environment for cell signalling and endocytosis of ERα [46].Thus, the internalization of nanoMIPs into MCF-7 cells can likely be attributed to the ERa mediated caveolae dependent endocytosis [8,47]. Furthermore, the difference in MFI between MCF-7 cells and MDA-MB-231 cells subjected to 10 μg/mL of FLU-nanoMIPs (P ≤ 0.01) and FLU-DOX-nanoMIPs (P ≤ 0.001) was statistically significant (Figure 6 ...
Nanoparticles functionalized with specific receptors (e.g., antibodies, peptides) are used for targeted drug delivery of anti-cancer agents but their side effects include hypersensitivity reactions, toxicity, inflammation, and life-threatening allergic reactions (Anaphylaxis) [1,2]. Consequently, double imprinted molecularly imprinted nanoparticles (nanoMIPs) against a linear epitope of breast cancer cell receptor estrogen alpha (ER alpha) and loaded with an anti-cancer agent (doxorubicin, DOX) are synthesized via a solid-phase approach. Surface plasmon resonance (SPR) measurements reveal that the produced nanoMIPs exhibit KD values of 19 nM (against the epitope used for imprinting) and 10 nM (ER alpha receptor), and thus rival the affinity of nanoparticles decorated with natural affinity reagents (e.g., antibodies, peptides), whilst offering the advantages of low-cost and enhanced cellular uptake due to the receptor mediated endocytosis. We present the results of in vitro flow cytometry that DOX loaded nanoMIPs can preferentially bind to MCF-7 (ER alpha positive) breast cancer (BC) cells vs MDA-MB-231 (ER alpha negative) BC cells. Confocal imaging witnessed the above results and showed the sequential movement of the DOX loaded nanoMIPs from membrane to the nucleus of MCF-7 BC cells and achieve delivery of DOX once internalised in the cells (directly to the nucleus). As a result, enhanced cell toxicity in MCF-7 cells (~80%) as compared to MDA-MB-231 cells (~15%) is observed via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) cytotoxicity assay in a time dependent manner. Overall, this study provides a promising approach for the targeted drug delivery of chemotherapeutic drugs to breast cancer cells, which has the potential to significantly improve patient outcome whilst also reducing debilitating side effects of current treatment.
... Extranuclear, non-genomic signalling upon estrogen stimulation activates the anticipatory UPR by causing a release of calcium from EnR to the cytoplasm [21]. Upon estrogen stimulation, ER increases the expression of XBP1 by binding to the enhancer region of XBP1 gene [22]. MYC, an estrogen-responsive gene upregulates the expression of IRE1 during estrogen stimulation [23]. ...
... Thus, estrogen signalling increases the expression of both IRE1 and XBP1 leading to sustained activation of IRE1-XBP1 axis, which augments the production of XBP1s. Indeed, expression of the XBP1s protein is elevated by estrogen stimulation in ER-positive breast cancer cells [22,24,25]. Further, XBP1s protein physically interacts with ER and enhances the estrogenindependent transactivational function of ER [12,26] which generates a positive feedback loop consisting of XBP1s and ER. ...
Background
Endocrine-resistant breast cancers have elevated expression of XBP1, where it drives endocrine resistance by controlling the expression of its target genes. Despite the in-depth understanding of the biological functions of XBP1 in ER-positive breast cancer, effectors of endocrine resistance downstream of XBP1 are poorly understood. The aim of this study was to identify the XBP1-regulated genes contributing to endocrine resistance in breast cancer.
Methods
XBP1 deficient sub-clones in MCF7 cells were generated using the CRISPR-Cas9 gene knockout strategy and were validated using western blot and RT-PCR. Cell viability and cell proliferation were evaluated using the MTS assay and colony formation assay, respectively. Cell death and cell cycle analysis were determined using flow cytometry. Transcriptomic data was analysed to identify XBP1-regulated targets and differential expression of target genes was evaluated using western blot and qRT-PCR. Lentivirus and retrovirus transfection were used to generate RRM2 and CDC6 overexpressing clones, respectively. The prognostic value of the XBP1-gene signature was analysed using Kaplan–Meier survival analysis.
Results
Deletion of XBP1 compromised the upregulation of UPR-target genes during conditions of endoplasmic reticulum (EnR) stress and sensitized cells to EnR stress-induced cell death. Loss of XBP1 in MCF7 cells decreased cell growth, attenuated the induction of estrogen-responsive genes and sensitized them to anti-estrogen agents. The expression of cell cycle associated genes RRM2, CDC6, and TOP2A was significantly reduced upon XBP1 deletion/inhibition in several ER-positive breast cancer cells. Expression of RRM2, CDC6, and TOP2A was increased upon estrogen stimulation and in cells harbouring point-mutants (Y537S, D538G) of ESR1 in steroid free conditions. Ectopic expression of RRM2 and CDC6 increased cell growth and reversed the hypersensitivity of XBP1 KO cells towards tamoxifen conferring endocrine resistance. Importantly, increased expression of XBP1-gene signature was associated with poor outcome and reduced efficacy of tamoxifen treatment in ER-positive breast cancer.
Conclusions
Our results suggest that RRM2 and CDC6 downstream of XBP1 contribute to endocrine resistance in ER-positive breast cancer. XBP1-gene signature is associated with poor outcome and response to tamoxifen in ER-positive breast cancer.
... Forkhead protein FOXA1 is a key pioneer factor mediating ERchromatin interactions, and its impact on ER global binding, endocrine response, and resistance has been broadly reported in ER þ breast cancer (31)(32)(33), but limited studies have examined a role in ESR1mutant disease. Jeselsohn and colleagues revealed weaker FOXA1 binding in the Y537S-mutant ER cistrome (24), while Arnesen and colleagues found FOXA1 bound to a significant fraction of accessible chromatin regions in both Y537S-and D538G-mutant cells (15). ...
... Given that FOXA1 is an important epigenetic regulator that determines ER-chromatin interactions (31)(32)(33), we sought to compare the global FOXA1-ER genomic occupancy in ESR1 WT and mutant cells. We previously performed FOXA1 ChIP-seq (34) and ER ChIP-seq analysis (46) in T47D Y537S and D538G genome-edited cells (Supplementary Table. ...
Estrogen receptor alpha (ER/ESR1) mutations occur in 30% to 40% of endocrine resistant ER-positive (ER+) breast cancer. Forkhead box A1 (FOXA1) is a key pioneer factor mediating ER–chromatin interactions and endocrine response in ER+ breast cancer, but its role in ESR1-mutant breast cancer remains unclear. Our previous FOXA1 chromatin immunoprecipitation sequencing (ChIP-seq) identified a large portion of redistributed binding sites in T47D genome-edited Y537S and D538G ESR1–mutant cells. Here, we further integrated FOXA1 genomic binding profile with the isogenic ER cistrome, accessible genome, and transcriptome data of T47D cell model. FOXA1 redistribution was significantly associated with transcriptomic alterations caused by ESR1 mutations. Furthermore, in ESR1-mutant cells, FOXA1-binding sites less frequently overlapped with ER, and differential gene expression was less associated with the canonical FOXA1–ER axis. Motif analysis revealed a unique enrichment of retinoid X receptor (RXR) motifs in FOXA1-binding sites of ESR1-mutant cells. Consistently, ESR1-mutant cells were more sensitive to growth stimulation with the RXR agonist LG268. The mutant-specific response was dependent on two RXR isoforms, RXR-α and RXR-β, with a stronger dependency on the latter. In addition, T3, the agonist of thyroid receptor (TR) also showed a similar growth-promoting effect in ESR1-mutant cells. Importantly, RXR antagonist HX531 blocked growth of ESR1-mutant cells and a patient-derived xenograft (PDX)-derived organoid with an ESR1 D538G mutation. Collectively, our data support the evidence for a stronger RXR response associated with FOXA1 reprograming in ESR1-mutant cells, suggesting development of therapeutic strategies targeting RXR pathways in breast tumors with ESR1 mutation.
Implications
It provides comprehensive characterization of the role of FOXA1 in ESR1-mutant breast cancer and potential therapeutic strategy through blocking RXR activation.
... Higher CCND1 expression has been linked to an increased risk of death in ER+ breast cancer 53 . Drugs designed to inhibit the action of CDK4 have recently been shown to improve prognosis in hormone dependent cancers, beyond the use of endocrine therapy alone 54,55 . GATA3 and FOXA1 showed very high synthetic-lethal dependency relations with DLV 1 in this cell line data. ...
Cancers are commonly characterised by a complex pathology encompassing genetic, microscopic and macroscopic features, which can be probed individually using imaging and omics technologies. Integrating this data to obtain a full understanding of pathology remains challenging. We introduce a new method called Deep Latent Variable Path Modelling (DLVPM), which combines the representational power of deep learning with the capacity of path modelling to identify relationships between interacting elements in a complex system. To evaluate the capabilities of DLVPM, we initially trained a foundational model to map dependencies between SNV, Methylation, miRNA-Seq, RNA-Seq and Histological data using Breast Cancer data from The Cancer Genome Atlas (TCGA). This method exhibited superior performance in mapping associations between data types compared to classical path modelling. We additionally performed successful applications of the model to: stratify single-cell data, identify synthetic lethal interactions using CRISPR-Cas9 screens derived from cell-lines, and detect histologic-transcriptional associations using spatial transcriptomic data. Results from each of these data types can then be understood with reference to the same holistic model of illness.
... AR-V7 can bind to compact chromatin regions. FOXA1, a pioneer transcription factor, functions to decompact chromatin structure through its winged-helix forkhead DNA binding domain, thereby facilitating the binding of AR and the estrogen receptor (54,55). To examine the levels of FOXA1 binding at AR-V7 unique sites, we performed ChIP-seq of FOXA1 in LN-tet-ARV7 cells treated with or without doxycycline. ...
One critical mechanism through which prostate cancer (PCa) adapts to treatments targeting androgen receptor (AR) signaling is the emergence of ligand-binding domain-truncated and constitutively active AR splice variants, particularly AR-V7. While AR-V7 has been intensively studied, its ability to activate distinct biological functions compared to the full-length AR (AR-FL), and its role in regulating the metastatic progression of castration-resistant PCa (CRPC), remains unclear. Our study found that, under castrated conditions, AR-V7 strongly induced osteoblastic bone lesions, a response not observed with AR-FL overexpression. Through combined ChIP-seq, ATAC-seq, and RNA-seq analyses, we demonstrated that AR-V7 uniquely accesses the androgen-responsive elements in compact chromatin regions, activating a distinct transcription program. This program was highly enriched for genes involved in epithelial-mesenchymal transition and metastasis. Notably, we discovered that SOX9, a critical metastasis driver gene, was a direct target and downstream effector of AR-V7. Its protein expression was dramatically upregulated in AR-V7-induced bone lesions. Moreover, we found that Ser81 phosphorylation enhanced AR-V7's pro-metastasis function by selectively altering its specific transcription program. Blocking this phosphorylation with CDK9 inhibitors impaired the AR-V7-mediated metastasis program. Overall, our study has provided molecular insights into the role of AR splice variants in driving the metastatic progression of CRPC.
... We first defined direct ER target genes using our previously-generated PRO-seq data from parental T47D cells that were hormone starved for three days and then acutely stimulated with estrogen or a DMSO vehicle control for 90 minutes (S8 Fig) [39]. We exclusively focused on estrogen-activated genes because ER directly activates these genes [8,42,43]. 65 ER target genes were activated, and 58 were repressed by acute TRPS1 depletion (Fig 5A). To test the robustness of this change in ER target gene transcription, we additionally performed PRO-seq in each of the three independent TRPS1-dTAG clones generated from the parental T47D cells. ...
Common genetic variants in the repressive GATA-family transcription factor (TF) TRPS1 locus are associated with breast cancer risk, and luminal breast cancer cell lines are particularly sensitive to TRPS1 knockout. We introduced an inducible degron tag into the native TRPS1 locus within a luminal breast cancer cell line to identify the direct targets of TRPS1 and determine how TRPS1 mechanistically regulates gene expression. We acutely deplete over 80 percent of TRPS1 from chromatin within 30 minutes of inducing degradation. We find that TRPS1 regulates transcription of hundreds of genes, including those related to estrogen signaling. TRPS1 directly regulates chromatin structure, which causes estrogen receptor alpha (ER) to redistribute in the genome. ER redistribution leads to both repression and activation of dozens of ER target genes. Downstream from these primary effects, TRPS1 depletion represses cell cycle-related gene sets and reduces cell doubling rate. Finally, we show that high TRPS1 activity, calculated using a gene expression signature defined by primary TRPS1-regulated genes, is associated with worse breast cancer patient prognosis. Taken together, these data suggest a model in which TRPS1 modulates the genomic distribution of ER, both activating and repressing transcription of genes related to cancer cell fitness.
... Inhibition of the IRE1-XBP1 axis abrogated the activation of the estrogen pathway by HyT-induced destabilisation of ERHT [13]. The expression of XBP1 is upregulated upon E2-stimulation, which, in turn, potentiates estrogen receptor (ER)-dependent transcriptional activity [14,15]. Ectopic expression of spliced XBP1 can bring about estrogen-independent growth and provide resistance to endocrine therapy [16]. ...
X-box binding protein 1 (XBP1) is a transcription factor that plays a crucial role in the unfolded protein response (UPR), a cellular stress response pathway involved in maintaining protein homeostasis in the endoplasmic reticulum (EnR). While the role of XBP1 in UPR is well-characterised, emerging evidence suggests its involvement in endocrine resistance in breast cancer. The transcriptional activity of spliced XBP1 (XBP1s) is a major component of its biological effects, but the targets of XBP1s in estrogen receptor (ER)-positive breast cancer are not well understood. Here, we show that the expression of miR-378 and PPARGC1B (host gene of miR-378) is downregulated during UPR. Using chemical and genetic methods, we show that XBP1s is necessary and sufficient for the downregulation of miR-378 and PPARGC1B. Our results show that overexpression of miR-378 significantly suppressed cell growth, colony formation, and migration of ER-positive breast cancer cells. Further, we found that expression of miR-378 sensitised the cells to UPR-induced cell death and anti-estrogens. The expression of miR-378 and PPARGC1B was downregulated in breast cancer, and higher expression of miR-378 is associated with better outcomes in ER-positive breast cancer. We found that miR-378 upregulates the expression of several genes that regulate type I interferon signalling. Analysis of separate cohorts of breast cancer patients showed that a gene signature derived from miR-378 upregulated genes showed a strong association with improved overall and recurrence-free survival in breast cancer. Our results suggest a growth-suppressive role for miR-378 in ER-positive breast cancer where downregulation of miR-378 by XBP1 contributes to endocrine resistance in ER-positive breast cancer.
... LncRNA RP11-10E18.7 was found to be associated with miRNA hsa-miR-181a-5p that regulated gene SRPK2 in blood (34). Forkhead box A1 (FOXA1) is a forkhead box transcription factor expressed in mammary luminal epithelial cells (LECs) (35). FOXA1 mutations are a hallmark of ER + BC and have been widely regarded as a determinant of breast tumor response to endocrine therapy as well as a marker for favorable patient prognosis (36). ...
Background
As either oncogenes or tumor suppressor genes, long non-coding RNAs (lncRNAs) have a major role in both tumorigenesis and progression of human cancers, including breast cancer (BC). However, the statistical correlation between the lncRNA-lncRNA interaction and prognosis of BC remains unclear.
Methods
We analyzed the fragments per kilobase per million (FPKM) lncRNA expression data in tumor tissue samples from 890 female patients with BC in The Cancer Genome Atlas (TCGA) between May 2021 and October 2022. The Cox proportional hazards model adjusted for age, race, clinical stage, neoadjuvant therapy, estrogen receptor (ER), and progesterone receptor (PR) was adopted to evaluate the lncRNA-lncRNA interaction regarding overall survival (OS) of BC. The multiple comparison was corrected by Bonferroni method.
Results
RP11-10E18.7×RP11-481C4.2 was significantly associated with OS of BC patients [hazard ratio (HR)interaction =1.04, 95% confidence interval (CI): 1.03–1.06, P=3.35×10⁻⁹]. Then, gene-gene interaction analysis was performed for genes co-expressed with lncRNAs. FOXA1×U2SURP (HRinteraction =1.49, 95% CI: 1.28–1.73, P=2.16×10⁻⁷) was found to have a similar interactive pattern to RP11-10E18.7×RP11-481C4.2. after classifying the patients by intersection (3.47), we observed that the effect of FOXA1 opposite in patients with different U2SURP expression level (HRhigh vs. low =0.58, 95% CI: 0.34–0.99, P=0.046 in low expression of U2SURP; HRhigh vs. low =1.56, 95% CI: 1.18–2.87, P=0.029 in high expression of U2SURP).
Conclusions
Our comprehensive study identified RP11-10E18.7×RP11-481C4.2 as a potential biomarker of BC prognosis. The results play an essential role in the impact of lncRNA-lncRNA interaction on BC survival. Our findings elucidated potential molecular mechanisms of BC progression under complex association patterns and provided potential dynamic and reversible therapeutic targets for BC patients.
... In this case, we search 25 kb upstream of the transcription start site, 10 kb downstream of the transcription end site, and the entirety of the gene (both introns and exons). We chose this distance because, while cis-regulatory elements can be found far away from the transcription start site (Carroll et al., 2005), the majority are within 25 kb and can also be found in intragenic regions (Welboren et al., 2009;Zheng et al., 2016). To see if the male-biased gene set had more AREs than expected, we randomly sampled an equal number of genes (n = 1,043) from all genes in the human genome 1,000 times to generate two distributions. ...
In males of many vertebrate species, sexual selection has led to the evolution of sexually dimorphic traits, which are often developmentally controlled by androgen signaling involving androgen response elements (AREs). Evolutionary changes in the number and genomic locations of AREs can modify patterns of receptor regulation and potentially alter gene expression. Here, we use recently sequenced primate genomes to evaluate the hypothesis that the strength of sexual selection is related to the genome-wide number of AREs in a diversifying lineage. In humans, we find a higher incidence of AREs near male-biased genes and androgen-responsive genes when compared with randomly selected genes from the genome. In a set of primates, we find that gains or losses of AREs proximal to genes are correlated with changes in male expression levels and the degree of sex-biased expression of those genes. In a larger set of primates, we find that an increase in one indicator of sexual selection, canine size sexual dimorphism, is correlated with genome-wide ARE counts. Our results suggest that the responsiveness of the genome to androgens in humans and their close relatives has been shaped by sexual selection that arises from competition among males for mating access to females.
... Since some of the various signatures derived the BC360 panel have been trained for use in BCa, it was not unexpected that there were no clear correlations between these PCas ( Figure 1A). Across the FMRP cohort, there was a strong expression of FOXA1, an AR and ER transcription factor required to facilitate chromatin binding (72,73). Interestingly there was strong activation of ER signaling pathways as shown by the high ER Signaling scores which capture the impact of ER pathways on downstream signaling (Table S3). ...
The diverse clinical outcomes of prostate cancer have led to the development of gene signature assays predicting disease progression. Improved prostate cancer progression biomarkers are needed as current RNA biomarker tests have varying success for intermediate prostate cancer. Interest grows in universal gene signatures for invasive carcinoma progression. Early breast and prostate cancers share characteristics, including hormone dependence and BRCA1/2 mutations. Given the similarities in the pathobiology of breast and prostate cancer, we utilized the NanoString BC360 panel, comprising the validated PAM50 classifier and pathway-specific signatures associated with general tumor progression as well as breast cancer-specific classifiers. This retrospective cohort of primary prostate cancers (n=53) was stratified according to biochemical recurrence (BCR) status and the CAPRA-S to identify genes related to high-risk disease. Two public cohorts (TCGA-PRAD and GSE54460) were used to validate the results. Expression profiling of our cohort uncovered associations between PIP and INHBA with BCR and high CAPRA-S score, as well as associations between VCAN, SFRP2, and THBS4 and BCR. Despite low levels of the ESR1 gene compared to AR, we found strong expression of the ER signaling signature, suggesting that BCR may be driven by ER-mediated pathways. Kaplan-Meier and univariate Cox proportional hazards regression analysis indicated the expression of ESR1, PGR, VCAN, and SFRP2 could predict the occurrence of relapse events. This is in keeping with the pathways represented by these genes which contribute to angiogenesis and the epithelial-mesenchymal transition. It is likely that VCAN works by activating the stroma and remodeling the tumor microenvironment. Additionally, SFRP2 overexpression has been associated with increased tumor size and reduced survival rates in breast cancer and among prostate cancer patients who experienced BCR. ESR1 influences disease progression by activating stroma, stimulating stem/progenitor prostate cancer, and inducing TGF-b. Estrogen signaling may therefore serve as a surrogate to AR signaling during progression Frontiers in Oncology
... The minimal ERE is a palindromic DNA motif with the consensus sequence 5′-GGTCAnnnTGACC-3′. 6 Activity of the ER depends on co-activators such FOXA1 that change the chromatin state. [7][8][9] It can also act as a co-activator itself together with the AP1 or SP1 transcription factors, or by ligandindependent transcriptional activation upon phosphorylation of critical residues including serine 118. [10][11][12] Additional roles for the ER include chromatin remodelling through interaction with the histone H3 lysine 9 demethylase KDM4B 13,14 and rapid, so-called non-genomic effects that have been described for a subpopulation of ER located at the plasma membrane. ...
Background
Oestrogen receptor alpha (ER) is involved in cell growth and proliferation and functions as a transcription factor, a transcriptional coregulator, and in cytoplasmic signalling. It affects, for example, bone, endometrium, ovaries and mammary epithelium. It is a key biomarker in clinical management of breast cancer, where it is used as a prognostic and treatment‐predictive factor, and a therapeutical target. Several ER isoforms have been described, but transcript annotation in public databases is incomplete and inconsistent, and functional differences are not well understood.
Methods
We have analysed short‐ and long‐read RNA sequencing data from breast tumours, breast cancer cell lines, and normal tissues to create a comprehensive annotation of ER transcripts and combined it with experimental studies of full‐length protein and six alternative isoforms.
Results
The isoforms have varying transcription factor activity, subcellular localisation, and response to the ER‐targeting drugs tamoxifen and fulvestrant. Antibodies differ in ability to detect alternative isoforms, which raises concerns for the interpretation of ER‐status in routine pathology.
Conclusions
Future work should investigate the effects of alternative isoforms on patient survival and therapy response. An accurate annotation of ER isoforms will aid in interpretation of clinical data and inform functional studies to improve our understanding of the ER in health and disease.
... Using this approach, we identi ed 23793 ERa bound sites after DMSO treatment. These ndings support previous studies that ERa exhibits signi cant constitutive chromatin binding in the absence of external ligand activation (39). To determine the ERa binding pro les after ligand activation, we compared MCF-7 cells treated for 1 h with 10 nM E2, 10 mM RES or 10 mM DIM to DMSO treated samples. ...
Background
Inhibitory crosstalk between estrogen receptor alpha (ERα) and aryl hydrocarbon receptor (AHR) regulates 17β-estradiol (E2)-dependent breast cancer cell signalling. ERα and AHR are ligand activated transcription factors that mediate the actions of E2 and pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively. Like ERα, AHR binds many dietary compounds, including resveratrol (RES) and 3,3´diindolylmethane (DIM) often found in berries and cruciferous vegetables, respectively. RES activates ERα but inhibits AHR, whereas DIM activates ERα and AHR. Thus, dietary intake of RES and DIM will modulate both receptor activities. Here we used chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) and transcriptomics (RNA-seq) to study ERα and AHR crosstalk after treatment of MCF-7 human breast cancer cells with DIM, RES, E2 or TCDD alone or in combination.
Methods and Results
1 h or 6 h ligand treated MCF-7 cells were profiled for ERα and AHR binding sites using ChIP-seq or gene expression changes using RNA-seq, respectively. E2 resulted in 866 differentially expressed genes (DEGs), 532 of which were also bound by ERα. DIM and RES resulted in 577 and 446 DEGs, respectively, with 278 of the RES and 393 of the DIM DEGs also bound by ERα. An estrogen response element (ERE) was enriched after treatments with all three ligands, while an AHR response element (AHRE) was also enriched in the DIM-ERα but not after E2-ERα bound sites. An ERE was enriched in E2 + TCDD and DIM AHR bound sites but not for the TCDD-AHR bound sites. We found that 90% of the DEGs closest to an AHR binding site after DIM or E2 + TCDD also had an ERE and 60% of coregulated genes were common to both treatments. Collectively, our data show that DIM activates ERα and AHR like that of E2 + TCDD, while RES activates ERα but inhibits AHR.
Conclusion
The dietary compounds DIM and RES differentially regulate ERα and AHR action and likely other signalling pathways, which need to be considered to properly interpret their cellular and biological responses. These data further highlight the complex crosstalk between ERa and AHR and suggest that when both receptors are activated ERa dominants causing the preferential recruitment of AHR to ERa target genes.
... In addition, the majority of ER binding sites are adjacent to genes that are unresponsive to estrogens. These observations suggest that the canonical mode of ER function is involved in longrange control of gene regulation (Carroll et al., 2005;Carroll et al., 2006;M. Ding, Wang, Chen, Shen, & Xu, 2012;Hah et al., 2011;Ikeda, Horie-Inoue, & Inoue, 2015;Kittler et al., 2013;C. ...
The nuclear receptor (NR) superfamily comprises 48 transcription factors in humans that control a plethora of gene network programs involved in a wide range of physiological processes. In this review, we will summarize and discuss recent progress in NR biology and drug development derived from the integration of various approaches, including biophysical techniques, structural studies, and translational investigations. We also highlight how defective NR signaling results in various diseases and disorders and how NRs can be targeted for therapeutic intervention via modulation via binding to synthetic lipophilic ligands. Furthermore, we also review recent studies that improved our understanding of NR structure and signaling. Significance Statement Nuclear receptors are ligand regulated transcription factors that function as key regulators of a wide range of physiological functions. NRs also serve as receptors for myriad drugs and in this review we provide an update of recent research into the function of this family of drug targets.
... Moreover, we found that patients with the combined low expression levels of AGR2 and FOXA1 had the best PFS than the rest in ER-positive breast cancer. Consistent with our study, many previous studies have also found that a higher expression of either AGR2 or FOXA1 was related to ER/PR-positive tumors [8,17,19,24,28], which was supported by the findings that FOXA1 was required for the expression of ER-regulated genes [4,29,30], and AGR2 was the target of ER [31]. The positive association of AGR2 expression with HER2 status has also been reported in a previous study [18], which may be explained to some extent by that activation of HER2 leads to the activation of extracellular signal-regulated kinases 1,2 (Erk1,2) and Akt, and these kinases were involved in the up-regulation of AGR2 expression [32,33]. ...
Background
The prognostic role of either forkhead box A1 (FOXA1) or anterior gradient 2 (AGR2) in breast cancer has been found separately. Considering that there were interplays between them depending on ER status, we aimed to assess the statistical interaction between AGR2 and FOXA1 on breast cancer prognosis and examine the prognostic role of the combination of them by ER status.
Methods
AGR2 and FOXA1 expression in tumor tissues were evaluated with tissue microarrays by immunohistochemistry in 915 breast cancer patients with follow up data. The expression levels of these two markers were treated as binary variables, and many different cutoff values were tried for each marker. Survival and Cox proportional hazard analyses were used to evaluate the relationship between AGR2, FOXA1 and prognosis, and the statistical interaction between them on the prognosis was assessed on multiplicative scale.
Results
Statistical interaction between AGR2 and FOXA1 on the PFS was significant with all the cutoff points in ER-positive breast cancer patients but not ER-negative ones. Among ER-positive patients, the poor prognostic role of the high level of FOXA1 was significant only in patients with the low level of AGR2, and vice versa. When AGR2 and FOXA1 were considered together, patients with low levels of both markers had significantly longer PFS compared with all other groups.
Conclusions
There was a statistical interaction between AGR2 and FOXA1 on the prognosis of ER-positive breast cancer. The combination of AGR2 and FOXA1 was a more useful marker for the prognosis of ER-positive breast cancer patients.
... Notably, chromatin modifiers like EZH2 and LSD1 gain histone-independent activities, modifying critical transcription factors/cofactors and further altering transcription networks during cancer progression (6,7). In a recent study, we identified that lysine 270 of FOXA1, a critical linage-specific pioneer factor of AR and ER (estrogen receptor) (8)(9)(10), is a nonhistone substrate of LSD1. The LSD1-mediated demethylation of FOXA1 stabilizes its chromatin binding, promoting PCa progression (11,12). ...
Dysregulation of histone lysine methyltransferases and demethylases is one of the major mechanisms driving the epigenetic reprogramming of transcriptional networks in castration-resistant prostate cancer (CRPC). In addition to their canonical histone targets, some of these factors can modify critical transcription factors, further impacting oncogenic transcription programs. Our recent report demonstrated that LSD1 can demethylate the lysine 270 of FOXA1 in prostate cancer (PCa) cells, leading to the stabilization of FOXA1 chromatin binding. This process enhances the activities of the androgen receptor and other transcription factors that rely on FOXA1 as a pioneer factor. However, the identity of the methyltransferase responsible for FOXA1 methylation and negative regulation of the FOXA1-LSD1 oncogenic axis remains unknown. SETD7 was initially identified as a transcriptional activator through its methylation of histone 3 lysine 4, but its function as a methyltransferase on nonhistone substrates remains poorly understood, particularly in the context of PCa progression. In this study, we reveal that SETD7 primarily acts as a transcriptional repressor in CRPC cells by functioning as the major methyltransferase targeting FOXA1-K270. This methylation disrupts FOXA1-mediated transcription. Consistent with its molecular function, we found that SETD7 confers tumor suppressor activity in PCa cells. Moreover, loss of SETD7 expression is significantly associated with PCa progression and tumor aggressiveness. Overall, our study provides mechanistic insights into the tumor-suppressive and transcriptional repression activities of SETD7 in mediating PCa progression and therapy resistance.
... For example, the chromosomal loops on chr11 are complemented by the binding of CTCF, FOXA1, and ESRRA, molecules with potential roles in breast cancer risk. Regarding ESRRA, it is known that the closely related estrogen receptor (ER) binds throughout the genome at enhancers distant from the start sites of genes it regulates (Carroll et al, 2005;Eeckhoute et al, 2006), including CCND1. Several proteins are involved in ER recruitment, including the pioneer factor FOXA1 (Eeckhoute et al, 2006;Hurtado et al, 2011) and CTCF, which acts upstream of FOXA1 to drive ERmediated transcription via chromosome loops and partitions the genome into ER-responsive blocks (Chan & Song, 2008). ...
Hundreds of common variants have been found to confer small but significant differences in breast cancer risk, supporting the widely accepted polygenic model of inherited predisposition. Using a novel closed-pattern mining algorithm, we provide evidence that rare haplotypes may refine the association of breast cancer risk with common germline alleles. Our method, called Chromosome Overlap, consists in iteratively pairing chromosomes from affected individuals and looking for noncontiguous patterns of shared alleles. We applied Chromosome Overlap to haplotypes of genotyped SNPs from female breast cancer cases from the UK Biobank at four loci containing common breast cancer-risk SNPs. We found two rare (frequency <0.1%) haplotypes bearing a GWAS hit at 11q13 (hazard ratio = 4.21 and 16.7) which replicated in an independent, European ancestry population at P < 0.05, and another at 22q12 (frequency <0.2%, hazard ratio = 2.58) which expanded the risk pool to noncarriers of a GWAS hit. These results suggest that rare haplotypes (or mutations) may underlie the "synthetic association" of breast cancer risk with at least some common variants.
... Epithelial-mesenchymal metamorphosis and cancerous stem cells in pancreatic cancer are caused by FOXM1 overexpression [111]. The pulmonary vasculature's embryonic development depends on FOXM1, which is also involved in colon CSC proliferation and self-renewal. ...
Breast cancer has been acknowledged as one of the most notorious cancers, responsible for millions of deaths around the globe. Understanding the various factors, genetic mutations, comprehensive pathways, etc., that are involved in the development of breast cancer and how these affect the development of the disease is very important for improving and revitalizing the treatment of this global health issue. The forkhead-box gene family, comprising 19 subfamilies, is known to have a significant impact on the growth and progression of this cancer. The article looks into the various forkhead genes and how they play a role in different types of cancer. It also covers their impact on cancer drug resistance, interaction with microRNAs, explores their potential as targets for drug therapies, and their association with stem cells.
... We first defined direct ER target genes using our previously-generated PRO-seq data from parental T47D cells that were hormone starved for three days and then acutely stimulated with estrogen or a DMSO vehicle control for 90 minutes ( Figure S2) (Scott et al. 2022). We exclusively focused on estrogen-activated genes because ER directly activates these genes (Carroll et al. 2005;Guertin, MJ et al. 2014;Hah et al. 2011). Dozens of ER target genes were activated and repressed by acute TRPS1 depletion ( Figure 5A). ...
Breast cancer is the most frequently diagnosed cancer in women. The most common subtype is luminal breast cancer, which is typically driven by the estrogen receptor alpha (ER), a transcription factor (TF) that activates many genes required for proliferation. Multiple effective therapies target this pathway, but individuals often develop resistance. Thus, there is a need to identify additional targets that regulate ER activity and contribute to breast tumor progression. TRPS1 is a repressive GATA-family TF that is overexpressed in breast tumors. Common genetic variants in the TRPS1 locus are associated with breast cancer risk, and luminal breast cancer cell lines are particularly sensitive to TRPS1 knockout. However, we do not know how TRPS1 regulates target genes to mediate these breast cancer patient and cellular outcomes. We introduced an inducible degron tag into the native TRPS1 locus within a luminal breast cancer cell line to identify the direct targets of TRPS1 and determine how TRPS1 mechanistically regulates gene expression. We acutely deplete over eighty percent of TRPS1 from chromatin within 30 minutes of inducing degradation. We find that TRPS1 regulates transcription of hundreds of genes, including those related to estrogen signaling. TRPS1 directly regulates chromatin structure, which causes ER to redistribute in the genome. ER redistribution leads to both repression and activation of dozens of ER target genes. Downstream from these primary effects, TRPS1 depletion represses cell cycle-related gene sets and reduces cell doubling rate. Finally, we show that high TRPS1 activity, calculated using a gene expression signature defined by primary TRPS1-regulated genes, is associated with worse breast cancer patient prognosis. Taken together, these data suggest a model in which TRPS1 modulates the activity of other TFs, both activating and repressing transcription of genes related to cancer cell fitness.
... Despite the major oncogenic role that ER plays in endometrial cancer, ER coregulatory proteins in this disease remain relatively unknown. In breast cancer, several regulatory proteins that help ER regulate gene expression have been found, including Forkhead box A1 (FOXA1), GATA Binding Protein 3 (GATA3), Growth Regulating Estrogen Receptor Binding 1 (GREB1), and Nuclear Receptor Coactivator 3 (NCOA3/SRC-3) [30][31][32][33]. To gain insight into endometrial cancer ER associated cofactors, we performed RIME in Ishikawa parental cells and compared the data to published breast cancer ER RIME datasets. ...
Activating estrogen receptor alpha (ER; also known as ESR1) mutations are present in primary endometrial and metastatic breast cancers, promoting estrogen-independent activation of the receptor. Functional characterizations in breast cancer have established unique molecular and phenotypic consequences of the receptor, yet the impact of ER mutations in endometrial cancer has not been fully explored. In this study, we used CRISPR-Cas9 to model the clinically prevalent ER-Y537S mutation and compared results with ER-D538G to discover allele-specific differences between ER mutations in endometrial cancer. We found that constitutive activity of mutant ER resulted in changes in the expression of thousands of genes, stemming from combined alterations to ER binding and chromatin accessibility. The unique gene expression programs resulted in ER-mutant cells developing increased cancer-associated phenotypes, including migration, invasion, anchorage-independent growth, and growth in vivo. To uncover potential treatment strategies, we identified ER-associated proteins via Rapid Immunoprecipitation and Mass Spectrometry of Endogenous Proteins and interrogated two candidates, CDK9 and NCOA3. Inhibition of these regulatory proteins resulted in decreased growth and migration, representing potential novel treatment strategies for ER-mutant endometrial cancer.
Implications
This study provides insight into mutant ER activity in endometrial cancer and identifies potential therapies for women with ER-mutant endometrial cancer.
... We also previously reported that IGFBP-2 induces PCa cell growth and contributes to chemoresistance [22,23]. More recently, we reported a novel correlation between IGFBP-2 and a pioneer transcription factor, FOXA1 in prostate cancer cells [24], that opens the chromatin for binding of additional transcription factors, such as the AR and its variants [25][26][27]. ...
Prostate cancer (PCa) is one of the leading causes of cancer-related deaths in men. Localised PCa can be treated effectively, but most patients relapse/progress to more aggressive disease. One possible mechanism underlying this progression is alternative splicing of the androgen receptor, with AR variant 7(ARV7) considered to play a major role. Using viability assays, we confirmed that ARV7-positive PCa cells were less sensitive to treatment with cabazitaxel and an anti-androgen-enzalutamide. Also, using live-holographic imaging, we showed that PCa cells with ARV7 exhibited an increased rate of cell division, proliferation, and motility, which could potentially contribute to a more aggressive phenotype. Furthermore, protein analysis demonstrated that ARV7 knock-down was associated with a decrease in insulin-like growth factor-2 (IGFBP-2) and forkhead box protein A1(FOXA1). This correlation was confirmed in-vivo using PCa tissue samples. Spearman rank correlation analysis showed significant positive associations between ARV7 and IGFBP-2 or FOXA1 in tissue from patients with PCa. This association was not present with the AR. These data suggest an interplay of FOXA1 and IGFBP-2 with ARV7-mediated acquisition of an aggressive prostate cancer phenotype.
... 70 These tumors were also enriched for AR and HER2 expression, as well as expression of FOXA1-a steroid nuclear receptor pioneering factor implicated in genomic binding of ER and AR. 70,[72][73][74] As with the Farmer et al. study, 70% of cases demonstrated apocrine features on histopathologic review. Furthermore, the TNBC cell line model MDA-MB-453 was found to recapitulate this distinct luminal molecular signature and proliferate in the presence of androgens-which was abrogated by treatment with an AR antagonist-providing important in vitro functional validation of correlative gene expression data that AR may be an oncogenic driver. ...
Aberrant estrogen receptor (ER) signaling is central to the pathogenesis of many breast cancers. Like ER, the androgen receptor (AR) is a steroid nuclear receptor that is frequently expressed in breast cancer and has long been considered an attractive therapeutic target. Although androgens were historically employed in the treatment of breast cancer, this strategy has largely fallen out of favor with the advent of modern anti--estrogens, due to virilizing effects from androgens, as well as concerns that androgens could be converted to estrogens to fuel tumor growth. Recent molecular advances, however, including the development of selective androgen receptor modulators, have renewed interest in targeting the AR. Yet androgen signaling in breast cancer remains incompletely understood, and preclinical studies have yielded conflicting and sometimes contradictory evidence regarding the role of AR, resulting in clinical investigations into both AR agonists and antagonists. It is increasingly recognized that AR may very well be context-specific, with divergent actions in ER-positive versus ER-negative disease. Here, we will summarize our current understanding of AR biology and insights from recent investigations into AR-directed therapies in breast cancer.
... Deoxyribonucleic acid methylation at the promoter region is a widely accepted epigenetic mechanism afecting gene expression. However, the binding sites for transcription International Journal of Clinical Practice factors might be a great distance from the transcriptional start sites [34], and those located in introns or those that are distal to genes might interact with promoters via looping chromatin [35,36]. Xue et al. reported that the hypermethylation of CpG islands spanning exon II to intron III activated SF-1 gene mRNA expression in endometriosis [17]. ...
Objective:
Endometriosis, which is a common disease affecting approximately 10% of women of reproductive age, usually causes dysmenorrhea and infertility, thus seriously harming the patients' physical and mental health. However, there is a mean delay of 6.7 years between the onset of the symptoms and the surgical diagnosis of endometriosis. There is an increasing amount of evidence that suggests that epigenetic aberrations, including deoxyribonucleic acid (DNA) methylation, play a definite role in the pathogenesis of endometriosis. This study aimed to explore the noninvasive or minimally invasive biomarkers of this disease.
Materials and methods:
Six patients with surgically confirmed ovarian endometriosis and six patients who received IUD implantation for contraception without endometriosis were recruited in the East Hospital of Tongji University in 2018. The genome methylation profiling of the eutopic and ectopic endometrium of ovarian endometriosis patients and normal endometrial specimens from healthy women was determined using a methylation microarray test. The test screened methylation-differentiated 5'-C-phosphate-G-3' (CpG) sites and then located the target genes affected by these sites following sequence alignment. Then, an additional 22 patients and 26 healthy controls were enrolled to further verify the difference in the selected genes between endometriosis patients and healthy women. The differential DNA methylation of the selected genes was validated via direct bisulfite sequencing and analysis of their messenger ribonucleic acid (mRNA) levels using quantitative reverse transcription polymerase chain reaction (qRT-PCR).
Results:
Fifteen differentially methylated CpG sites were found among the patients and healthy women, and five CpG sites were mapped to the introns of the human leukocyte antigen-C (HLA-C) gene; these were highly polymorphic between different HLA-C alleles and were HLA-C∗07 specific. The results indicated that the HLA-C∗07 carrier patients exhibited significantly higher DNA methylation levels at the intron VII of HLA-C compared to the HLA-C∗07 carrier healthy controls. High HLA-C∗07 mRNA levels were also observed using qRT-PCR with HLA-C∗07-specific primers, which indicated that the hypermethylation of CpG in intron VII might suppress a silencer that regulates HLA-C∗07 expressions.
Conclusion:
Deoxyribonucleic acid hypermethylation in the intron VII of the HLA-C∗07 gene appears to regulate the expression of HLA-C∗07. The aberrant DNA methylation in this region was positively correlated with the occurrence of endometriosis.
... The RNA-Seq data are not in agreement with the MR and GR ChIP-Seq results discussed above if we consider overall ChIP signal (binding intensity and number of peaks) to correlate with gene response (40). Among the four experimental conditions (two cell lines and two hormones), the parental line treated with Aldo generates the fewest MR and GR peaks but exhibited the second strongest transcriptional response. ...
The glucocorticoid and mineralocorticoid receptors (GR and MR, respectively) have distinct, yet overlapping physiological and pathophysiological functions. There is strong indication that both receptors interact both functionally and physically, but the precise role of this interdependence is poorly understood. Here, we analyzed the impact of GR co-expression on MR genome-wide chromatin binding and transcriptional responses to aldosterone and glucocorticoids, both physiological ligands of this receptor. Our data show that GR co-expression alters MR genome-wide binding in a locus- and ligand-specific way. MR binding to consensus DNA sequences is affected by GR. Transcriptional responses of MR in the absence of GR are weak and show poor correlation with chromatin binding. In contrast, co-expression of GR potentiated MR-mediated transcription, particularly in response to aldosterone. Finally, single-molecule tracking of MR suggests that the presence of GR contributes to productive binding to chromatin. Together, our data indicate that co-expression of GR potentiates aldosterone-mediated MR transcriptional activity, even in the absence of glucocorticoids.
... ERs are expressed in most of BC. Two-thirds of all BCs are luminal subtypes, and ER activity is essential for their growth (Carroll et al., 2005). Because estrogen signaling is necessary for the progression of ER-positive BC, ER inhibitors can be used to treat the condition (Ali and Coombes, 2002). ...
Forkhead box protein A1 (FOXA1) is pioneer factor has a dual role either as growth stimulator or a repressor and the significance role of this protein as a therapeutic biomarker still controversial, so the aim of this study was to identify the precise role of FOXA1 protein expression in treatment of BC patients by immunohistochemical (IHC) staning method. In this study, seventy three tissues samples embedded in wax block were taken from BC patients with (52.71+11.55) years who had surgery between 2014-2020 and thirty eight of normal (non tumoral) breast tissues samples with (54.17+10.84) years as a control group. There was a significant association between positive FOXA1 protein expression and Luminal A and Luminal B of BC (P=0.011). Moreover, FOXA1 positivity was elevated significantly in patients with positive Estrogen receptors (ER) expression (P=0.0001), positive Progesterone receptor (PR) expression (P=0.016). Furthermore, there was no significant correlation between FOXA1 expression in BC patients and Her2 expression (P=0.764). FOXA1 positive expression can be employed as a therapeutic marker to predict hormone responsiveness.
Chromatin is densely packed with nucleosomes, which limits the accessibility of many chromatin-associated proteins. Pioneer factors (PFs) are usually viewed as a special group of sequence-specific transcription factors (TFs) that can recognize nucleosome-embedded motifs, invade compact chromatin, and generate open chromatin regions. Through this process, PFs initiate a cascade of events that play key roles in gene regulation and cell differentiation. A current debate in the field is if PFs belong to a unique subset of TFs with intrinsic "pioneering activity", or if all TFs have the potential to function as PFs within certain cellular contexts. There are also different views regarding the key feature(s) that define pioneering activity. In this review, we present evidence from the literature related to these alternative views and discuss how to potentially reconcile them. It is possible that both intrinsic properties, like tight nucleosome binding and structural compatibility, and cellular conditions, like concentration and co-factor availability, are important for PF function.
Breast cancer bone metastases (BMET) are incurable, primarily osteolytic, and occur most commonly in estrogen receptor-α positive (ER+) breast cancer. ER+ human breast cancer BMET modeling in mice has demonstrated an estrogen (E2)-dependent increase in tumor-associated osteolysis and bone-resorbing osteoclasts, independent of estrogenic effects on tumor proliferation or bone turnover, suggesting a possible mechanistic link between tumoral ERα-driven osteolysis and ER+ bone progression. To explore this question, inducible secretion of the osteolytic factor, parathyroid hormone–related protein (PTHrP), was utilized as an in vitro screening bioassay to query the osteolytic potential of estrogen receptor- and signaling pathway–specific ligands in BMET-forming ER+ human breast cancer cells expressing ERα, ERß, and G protein–coupled ER. After identifying genomic ERα signaling, also responsibility for estrogen's proliferative effects, as necessary and sufficient for osteolytic PTHrP secretion, in vivo effects of a genomic-only ER agonist, estetrol (E4), on osteolytic ER+ BMET progression were examined. Surprisingly, while pharmacologic effects of E4 on estrogen-dependent tissues, including bone, were evident, E4 did not support osteolytic BMET progression (vs robust E2 effects), suggesting an important role for nongenomic ER signaling in ER+ metastatic progression at this site. Because bone effects of E4 did not completely recapitulate those of E2, the relative importance of nongenomic ER signaling in tumor vs bone cannot be ascertained here. Nonetheless, these intriguing findings suggest that targeted manipulation of estrogen signaling to mitigate ER+ metastatic progression in bone may require a nuanced approach, considering genomic and nongenomic effects of ER signaling on both sides of the tumor/bone interface.
Estrogen Receptor 1 (ESR1; also known as ERα, encoded by ESR1 gene) is the main driver and prime drug target in luminal breast cancer. ESR1 chromatin binding is extensively studied in cell lines and a limited number of human tumors, using consensi of peaks shared among samples. However, little is known about inter-tumor heterogeneity of ESR1 chromatin action, along with its biological implications. Here, we use a large set of ESR1 ChIP-seq data from 70 ESR1 ⁺ breast cancers to explore inter-patient heterogeneity in ESR1 DNA binding to reveal a striking inter-tumor heterogeneity of ESR1 action. Of note, commonly shared ESR1 sites show the highest estrogen-driven enhancer activity and are most engaged in long-range chromatin interactions. In addition, the most commonly shared ESR1-occupied enhancers are enriched for breast cancer risk SNP loci. We experimentally confirm SNVs to impact chromatin binding potential for ESR1 and its pioneer factor FOXA1. Finally, in the TCGA breast cancer cohort, we can confirm these variations to associate with differences in expression for the target gene. Cumulatively, we reveal a natural hierarchy of ESR1–chromatin interactions in breast cancers within a highly heterogeneous inter-tumor ESR1 landscape, with the most common shared regions being most active and affected by germline functional risk SNPs for breast cancer development.
Forkhead box protein A1 (FOXA1), a pioneering transcriptional factor known for its critical roles in prostate and ERα−positive breast cancer, is also expressed in human epidermal growth factor receptor-2 (HER2/ErbB2)-positive breast cancers. However, its role in HER2-pos tumors is less well understood. Here we investigate the function of FOXA1 in HER2/ErbB2- positive breast cancers. The loss of FOXA1 was associated with a marked decrease in the viability of HER2-positive and HER2 amplified cell lines, suggesting a pivotal involvement of FOXA1 in these breast cancers. Employing patient-derived single-cell RNA sequencing and spatial transcriptomics, we demonstrate that FOXA1 is co-expressed with ErbB2 in HER2- positive breast cancers. Suppression of FOXA1 expression led to the reduction of HER2 expression and signaling. Chromatin Immunoprecipitation Sequencing (ChIP-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) identified FOXA1 binding motifs in the ErbB2 promoter and regulatory element regions, which controlled ErbB2 gene expression. Notably, FOXA1 knockdown was observed to enhance Epithelial-Mesenchymal Transition (EMT) signaling and impede luminal tumor differentiation. Furthermore, we find that FOXA1 and TRPS1 combine to regulate TEAD/YAP-TAZ activity. Taken together, these findings highlight the essential role of FOXA1 in maintaining HER2 expression and a luminal cell phenotype in HER2-positive breast cancers.
Inheritance of a BRCA2 pathogenic variant conveys a substantial life-time risk of breast cancer. Identification of the cell(s)-of-origin of BRCA2-mutant breast cancer and targetable perturbations that contribute to transformation remains an unmet need for these individuals who frequently undergo prophylactic mastectomy. Using preneoplastic specimens from age-matched, premenopausal females, here we show broad dysregulation across the luminal compartment in BRCA2mut/+ tissue, including expansion of aberrant ERBB3lo luminal progenitor and mature cells, and the presence of atypical oestrogen receptor (ER)-positive lesions. Transcriptional profiling and functional assays revealed perturbed proteostasis and translation in ERBB3lo progenitors in BRCA2mut/+ breast tissue, independent of ageing. Similar molecular perturbations marked tumours bearing BRCA2-truncating mutations. ERBB3lo progenitors could generate both ER⁺ and ER⁻ cells, potentially serving as cells-of-origin for ER-positive or triple-negative cancers. Short-term treatment with an mTORC1 inhibitor substantially curtailed tumorigenesis in a preclinical model of BRCA2-deficient breast cancer, thus uncovering a potential prevention strategy for BRCA2 mutation carriers.
Estrogen Receptor alpha (ERα) is the main driver and prime drug target in luminal breast. ERα chromatin binding is extensively studied in cell lines and a limited number of human tumors, using consensi of peaks shared among samples. However, little is known about inter-tumor heterogeneity of ERα chromatin action, along with its biological implications.
Here, we use a large set of ERα ChIP-seq data from 70 ERα+ breast cancers to explore inter-patient heterogeneity in ERα DNA binding, to reveal a striking inter-tumor heterogeneity of ERα action. Interestingly, commonly-shared ERα sites showed the highest estrogen-driven enhancer activity and were most-engaged in long-range chromatin interactions. In addition, the most-commonly shared ERα-occupied enhancers were enriched for breast cancer risk SNP loci. We experimentally confirm SNVs to impact chromatin binding potential for ERα and its pioneer factor FOXA1. Finally, in the TCGA breast cancer cohort, we could confirm these variations to associate with differences in expression for the target gene. Cumulatively, we reveal a natural hierarchy of ERα-chromatin interactions in breast cancers within a highly heterogeneous inter-tumor ERα landscape, with the most-common shared regions being most active and affected by germline functional risk SNPs for breast cancer development.
Inhibitory crosstalk between estrogen receptor alpha (ERα) and aryl hydrocarbon receptor (AHR) regulates 17β-estradiol (E2)-dependent breast cancer cell signaling. ERα and AHR are transcription factors activated by E2 and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively. Dietary ligands resveratrol (RES) and 3,3′diindolylmethane (DIM) also activate ERα while only DIM activates AHR and RES represses it. DIM and RES are reported to have anti-cancer and anti-inflammatory properties. Studies with genome-wide targets and AHR- and ERα-regulated genes after DIM and RES are unknown. We used chromatin immunoprecipitation with high-throughput sequencing and transcriptomics to study ERα as well as AHR coregulation in MCF-7 human breast cancer cells treated with DIM, RES, E2, or TCDD alone or E2+TCDD for 1 and 6 h, respectively. ERα bound sites after being DIM enriched for the AHR motif but not after E2 or RES while AHR bound sites after being DIM and E2+TCDD enriched for the ERE motif but not after TCDD. More than 90% of the differentially expressed genes closest to an AHR binding site after DIM or E2+TCDD also had an ERα site, and 60% of the coregulated genes between DIM and E2+TCDD were common. Collectively, our data show that RES and DIM differentially regulate multiple transcriptomic targets via ERα and ERα/AHR coactivity, respectively, which need to be considered to properly interpret their cellular and biological responses. These novel data also suggest that, when both receptors are activated, ERα dominates with preferential recruitment of AHR to ERα target genes.
The FOXA1 pioneer factor is an essential mediator of steroid receptor function in multiple hormone-dependent cancers, including breast and prostate cancers, enabling nuclear receptors such as estrogen receptor (ER) and androgen receptor (AR) to activate lineage-specific growth programs. FOXA1 is also highly expressed in non–small cell lung cancer (NSCLC), but whether and how it regulates tumor growth in this context is not known. Analyzing data from loss-of-function screens, we identified a subset of NSCLC tumor lines where proliferation is FOXA1 dependent. Using rapid immunoprecipitation and mass spectrometry of endogenous protein, we identified chromatin-localized interactions between FOXA1 and glucocorticoid receptor (GR) in these tumor cells. Knockdown of GR inhibited proliferation of FOXA1-dependent, but not FOXA1-independent NSCLC cells. In these FOXA1-dependent models, FOXA1 and GR cooperate to regulate gene targets involved in EGF signaling and G1–S cell-cycle progression. To investigate the therapeutic potential for targeting this complex, we examined the effects of highly selective inhibitors of the GR ligand-binding pocket and found that GR antagonism with ORIC-101 suppressed FOXA1/GR target expression, activation of EGF signaling, entry into the S-phase, and attendant proliferation in vitro and in vivo. Taken together, our findings point to a subset of NSCLCs harboring a dependence on the FOXA1/GR growth program and provide rationale for its therapeutic targeting.
Significance
NSCLC is the leading cause of cancer deaths worldwide. There is a need to identify novel druggable dependencies. We identify a subset of NSCLCs dependent on FOXA1-GR and sensitive to GR antagonism.
Aberrant activation of the forkhead protein FOXA1 is observed in advanced hormone-related cancers. However, the key mediators of high FOXA1 signaling remain elusive. We demonstrate that ectopic high FOXA1 (H-FOXA1) expression promotes estrogen receptor-positive (ER+) breast cancer (BC) metastasis in a xenograft mouse model. Mechanistically, H-FOXA1 reprograms ER-chromatin binding to elicit a core gene signature (CGS) enriched in ER+ endocrine-resistant (EndoR) cells. We identify Secretome14, a CGS subset encoding ER-dependent cancer secretory proteins, as a strong predictor for poor outcomes of ER+ BC. It is elevated in ER+ metastases vs. primary tumors, irrespective of ESR1 mutations. Genomic ER binding near Secretome14 genes is also increased in mutant ER-expressing or mitogen-treated ER+ BC cells and in ER+ metastatic vs. primary tumors, suggesting a convergent pathway including high growth factor receptor signaling in activating pro-metastatic secretome genes. Our findings uncover H-FOXA1-induced ER reprogramming that drives EndoR and metastasis partly via an H-FOXA1/ER-dependent secretome.
Aims:
To examine associations between the transcription factors CCCTC-binding factor (CTCF) and forkhead box protein A1 (FOXA1) and the androgen receptor (AR) and their association with components of the insulin-like growth factor (IGF)-pathway in a cohort of men with localized prostate cancer.
Methods:
Using prostate tissue samples collected during the Prostate cancer: Evidence of Exercise and Nutrition Trial (PrEvENT) trial (N = 70 to 92, depending on section availability), we assessed the abundance of CTCF, FOXA1, AR, IGFIR, p-mTOR, PTEN and IGFBP-2 proteins using a modified version of the Allred scoring system. Validation studies were performed using large, publicly available datasets (TCGA) (N = 489).
Results:
We identified a strong correlation between CTCF and AR staining with benign prostate tissue. CTCF also strongly associated with the IGFIR, with PTEN and with phospho-mTOR. FOXA1 was also correlated with staining for the IGF-IR, with IGFBP-2 and with staining for activated phosphor-mTOR. The staining for the IGF-IR was strongly correlated with the AR.
Conclusion:
Our findings emphasise the close and complex links between the endocrine controls, well known to play an important role in prostate cancer, and the transcription factors implicated by the recent genetic evidence.
Activation of the tyrosine kinase c-Src promotes breast cancer progression and poor outcomes, yet the underlying mechanisms are incompletely understood. Here, we show that deleting c-Src abrogates the activity of Forkhead Box M1 (FOXM1), a master transcriptional regulator of the cell cycle, in a genetically engineered model mimicking the Luminal B molecular subtype of breast cancer. By phosphorylating it on two tyrosine residues, c-Src stimulates the nuclear localization of FOXM1 and the expression of its target genes, including key regulators of G2-M cell cycle progression as well as c-Src itself. This positive feedback loop drives proliferation in genetically engineered and patient-derived models of Luminal B-like breast cancer. Targeting this mechanism, including through novel compounds that destabilize the FOXM1 protein, induces G2-M cell cycle arrest and apoptosis, blocking tumor progression and impairing metastasis. We identify a positive correlation between FOXM1 and c-Src expression in human breast cancer and show that the expression of FOXM1 target genes predicts poor outcomes and associates with the Luminal B subtype, which responds poorly to approved therapies. These findings indicate that a regulatory network centered on c-Src and FOXM1 is a targetable vulnerability in aggressive luminal breast cancers.
Transcription factors (TFs) represent the most commonly deregulated DNA-binding class of proteins associated with multiple human cancers. They can act as transcriptional activators or repressors that rewire the cistrome, resulting in cellular reprogramming during cancer progression. Deregulation of TFs is associated with the onset and maintenance of various cancer types including prostate cancer. An emerging subset of TFs has been implicated in the regulation of multiple cancer hallmarks during tumorigenesis. Here, we discuss the role of key TFs which modulate transcriptional cicuitries involved in the development and progression of prostate cancer. We further highlight the role of TFs associated with key cancer hallmarks, including, chromatin remodelling, genome instability, DNA repair, invasion, and metastasis. We also discuss the pluripotent function of TFs in conferring lineage plasticity, that aids in disease progression to neuroendocrine prostate cancer. At the end, we summarize the current understanding and approaches employed for the therapeutic targeting of TFs and their cofactors in the clinical setups to prevent disease progression.
Background
Adenomyosis is characterized by overgrowth of endometrial glands and stroma in the myometrium and is associated with reduced apoptosis. One of the key participants in one of the pathways of apoptosis is cytochrome c, whose expression can be regulated by actin-binding proteins involved in the formation of structures that provide cell motility. The aim of the study was to determine the content of actin-binding proteins, cytochrome c, and terminal members of the mitochondrial respiratory chain in endometrial biopsies of patients with adenomyosis and the control group.Methods and resultsThe content of all studied proteins was determined by Western blotting, and the mRNA content of the corresponding genes was determined by quantitative RT-PCR. The relative content of alpha-actinin1 and mRNA of the gene encoding it in biopsy specimens from patients with adenomyosis was higher than in controls by 86 and 84% (p < 0.05), respectively. The relative content of alpha-actinin4 did not change, as did cytochrome c. The content of cytochrome-c-oxidase and ATPsynthase in the group with adenomyosis exceeded the control level by 270 and 121% (p < 0.05), respectively, but the relative content of mRNA of these genes did not change, which may indicate a change in regulation at the level of protein synthesis.Conclusion
The results may indicate a local increase in the synthesis of ATP in pathological endometrial cells, which indicates the possible effectiveness of local application of H+-ATP synthase inhibitors (for example, macrolide antibiotic) to reduce the severity of clinical symptoms of adenomyosis.
Background
Breast cancer (BC) poses a serious threat to women worldwide. This research was designed to explore the association between the rs4784227 polymorphism of cancer susceptibility candidate gene 16 ( CASC16 ) and BC susceptibility and prognosis, aiming to provide further information for the early detection of BC and to accelerate comprehensive cancer management.
Methods
A total of 1,733 subjects were recruited for this case-control study, of which 828 are BC patients and 905 are healthy individuals. The relevance between SNP rs4784227 and BC risk in diverse genetic models was analyzed by using the SNPStats analysis program and was assessed by odds ratios (ORs) and 95% confidence intervals (CIs) using the binary logistic regression model. Pearson’s χ ² test was used to determine the correlation between the polymorphism and clinical characteristics of BC patients. Additionally, univariate survival analysis was performed by the Kaplan-Meier method and log-rank test, and multivariate survival analysis was performed by Cox regression.
Results
SNP rs4784227 was significantly associated with susceptibility to BC in the dominant model (CT/TT versus CC, OR = 1.237, 95% CI = 1.012–1.513, P = 0.038). The minor allele of SNP rs4784227 was significantly linked to an increased risk of BC (OR = 1.197, 95% CI = 1.022–1.401, P = 0.026). In addition, the rs4784227 polymorphism of CASC16 was associated with perineural invasion ( P = 0.030), menstrual status ( P = 0.016) and histological grade ( P = 0.001, P = 0.003, P = 0.025; respectively) of BC patients. There was no significant association between the genotypes of rs4784227 and disease-free survival (DFS) or overall survival (OS) of breast cancer patients ( P > 0.05).
Conclusions
The rs4784227 polymorphism of CASC16 may affect susceptibility to breast cancer and is associated with perineural invasion, menstrual status and histological grade in BC patients. Additionally, our results could not confirm that this polymorphism was related to breast cancer prognosis.
Acute liver failure (ALF) is a medical emergency due to massive hepatocyte loss. In such a harsh condition, maintaining transcriptional regulation in the remaining hepatocytes while activating similar transcription factor networks in liver progenitor cells (LPC) to ensure essential liver functions are two critical processes to rescue patients from liver failure and death. In this review, we discuss the formation and functions of transcription networks in ALF and liver development. We focus on a hierarchical network of transcription factors that responds to different pathophysiological circumstances: (1) Under normal circumstances, pioneer factor forkhead box (FOX) A2 coordinates the constitutive hepatic transcription factors, such as hepatic nuclear factor (HNF) 4α and CCAAT-enhancer binding protein (C/EBP) α, which ensure normal liver function; (2) When the expression of both HNF4α and C/EBPα in hepatocytes are disrupted by severe inflammation, retinoic acid receptor (RAR) is the alternative transcription factor that compensates for their absence; (3) In the condition of massive hepatic necrosis, a similar transcription network including FOXA2 and HNF4α, is activated as a "rescue network" in LPC to maintain vital liver functions when hepatocytes fail, and thus ensures survival. Expression of these master transcription factors in hepatocytes and LPC is tightly regulated by hormone signals and inflammation. The performance of this hierarchical transcription network, in particularly the "rescue network" described above, significantly affects the clinical outcome of ALF.
Transcription of the proto-oncogene c-fos is stimulated by 17β-estradiol in estrogen responsive human and rat cells. To understand the molecular mechanisms of estrogen
regulation of c-fos gene transcription, the human c-fos gene promoter, with 2.25 Kb of 5′-flanklng DNA, was cloned upstream of the bacterial CAT gene and tested for estrogen regulation
by transient transfection in HeLa cells. When an expression vector coding for the human estrogen receptor was cotransfected
with the fos -CAT reporter, the promoter was found to respond to 17β-estradiol. An element responsible for estrogen induction was mapped
in a 240 bp region localized 1060 to 1300 bases upstream of the startsite of transcription of the gene. Sequence analysis
revealed, clustered in a 19 bp sub-region, a sequence corresponding to an imperfectly palindromic ERE: CGGCAGCGTGACC and two sequences: CTGAG and GTGAC, homologous to the core sequence of AP-1 transcription factor binding sites. A synthetic
oligonucleotide reproducing this sub-region binds ‘in vitro’ both the estrogen receptor and AP-1 factor(s) and confers estrogen-responsivity
to the HSV-tk gene promoter. Transcriptional activation by the estrogen receptor is prevented by mutations in the fos ERE that hamper binding of the receptor in vitro. Activation of the c-fos gene promoter in HeLa cells requires the DNA binding domain of the estrogen receptor, and can be achieved independently by
the TAF-1 and the TAF-2 transcriptional activation functions of this molecule. A receptor mutant lacking the hormone binding
domain can activate the c-fos gene promoter in the absence of estrogen.
Using chimeric recombinants transfected into HeLa cells and a transient expression assay, we demonstrate that the 5'-flanking region of the pS2 gene from position -428 to position -324 exhibits both constitutive and estrogen-inducible enhancer activity. The estrogen-inducible activity, but not the constitutive activity, was inhibited by antiestrogens. ICI 164,384 behaved as a pure antagonist, whereas hydroxytamoxifen was a partial agonist-antagonist. The estrogen-responsive element of the pS2 gene has been narrowed down by site-directed deletion mutagenesis to a 13-base-pair (position -405 to position -393) imperfectly palindromic sequence, which in isolation can confer estrogen inducibility to the heterologous rabbit beta-globin gene promoter. On the other hand, the sequences responsible for the constitutive enhancer activity are spread over the entire region.
As a step toward elucidating the physiological role of insulin-like growth factor-I (IGF-I) in mediating estrogen action, we sought to determine the molecular basis of the phenomenon. In HepG2 cells expressing exogenous estrogen receptors (ER), a reporter gene plasmid containing 600 base pairs of the chicken IGF-I promoter enhanced expression of luciferase 8.6-fold in response to 10(-6) M 17 beta-estradiol, indicating that the IGF-I promoter is a target of estrogen regulation. Although no conventional estrogen-responsive element was identified within the promoter fragment, the AP-1 motif located therein was shown to be essential; the estrogen-responsive enhancement of the Fos-Jun binding to the AP-1 motif, which takes place by means of post-translational modification, mediates the estrogen action. A direct or indirect interaction between the estrogen-ER complex and the Fos-Jun complex seems to facilitate the Fos-Jun binding to the target DNA. Although ER binding to the target DNA was not considered to be involved in the signaling pathway, the DNA binding domain-deficient ER did not mediate the phenomenon, providing support for the existence of a unique function of the DNA binding domain of ER in facilitating some protein-protein interaction. In conclusion, our present observations demonstrate that the chicken IGF-I gene promoter is controlled by estrogen through a unique pathway involving Fos, Jun, and the DNA binding domain of ER.
Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5, 5'-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H2O2 to reinvestigate the mechanism of DMPO/.OH formation from the SOD and SOD mutants. The relative ratios of DMPO/.17OH and DMPO/.16OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H217O2 to H216O2. The reaction of the WT SOD with H217O2 in bicarbonate/CO2 buffer yielded 63% DMPO/.17OH and 37% DMPO/.16OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H217O2: DMPO/.17OH (64%); DMPO/.16OH (36%) from A4V and DMPO/.17OH (62%); and DMPO/.16OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/.OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/.OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/.OH or DEPMPO/.OH formed nor in the relative incorporation of oxygen from H2O2 or water.
We have compared the DNase I hypersensitivity of the regulatory region of two estrogen-regulated genes, pS2 and cathepsin D in hormone-dependent and -independent breast carcinoma cell lines. This strategy allowed the identification of two important control regions, one in pS2 and the other in cathepsin D genes. In the hormone-dependent MCF7 cell line, within the pS2 gene 5'-flanking region, we detected two major DNase I hypersensitive sites, induced by estrogens and/or IGFI: pS2-HS1, located in the proximal promoter and pS2-HS4, located -10.5 Kb from the CAP site, within a region that has not been cloned. The presence of these two DNase I hypersensitive sites correlates with pS2 expression. Interestingly in MCF7 cells, estrogens and IGFI induced indistinguishable chromatin structural changes over the pS2 regulatory region, suggesting that the two transduction-pathways converge to a unique chromatin target. In two cell lines that do not express pS2, MDA MB 231, a hormone-independent cell line that lacks the estrogen receptor alpha, and HE5, a cell line derived from MDA MB 231 by transfection that expresses estrogen receptor alpha, there was only one hormone-independent DNase I hypersensitive site. This site, pS2-HS2, was located immediately upstream of pS2-HS1. In MCF7 cells, two major DNase I hypersensitive sites were present in the 5'-flanking sequences of the cathepsin D gene, which is regulated by estrogens in these cells. These sites, catD-HS2 and catD-HS3, located at positions -2.3 Kb and -3.45 Kb, respectively, were both hormone-independent. A much weaker site, catD-HS1, covered the proximal promoter. In MDA MB 231 cells, that express cathepsin D constitutively, we detected an additional strong hormone-independent DNase I hypersensitive site, catD-HS4, located at position -4.3 Kb. This region might control the constitutive over-expression of cathepsin D in hormone-independent breast cancer cells. All together, these data demonstrate that a local reorganization of the chromatin structure over pS2 and cathepsin D promoters accompanies the establishment of the hormone-independent phenotype of the cells.
Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/ threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. In this study, we demonstrate that Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene.
The phosphatidylinositol-3-OH-kinase (PI(3)K) effector protein kinase B regulates certain insulin-responsive genes, but the transcription factors regulated by protein kinase B have yet to be identified. Genetic analysis in Caenorhabditis elegans has shown that the Forkhead transcription factor daf-16 is regulated by a pathway consisting of insulin-receptor-like daf-2 and PI(3)K-like age-1. Here we show that protein kinase B phosphorylates AFX, a human orthologue of daf-16, both in vitro and in vivo. Inhibition of endogenous PI(3)K and protein kinase B activity prevents protein kinase B-dependent phosphorylation of AFX and reveals residual protein kinase B-independent phosphorylation that requires Ras signalling towards the Ral GTPase. In addition, phosphorylation of AFX by protein kinase B inhibits its transcriptional activity. Together, these results delineate a pathway for PI(3)K-dependent signalling to the nucleus.
The liver-specific vitellogenin B1 promoter is efficiently activated by estrogen within a nucleosomal environment after microinjection into Xenopus laevis oocytes, consistent with the hypothesis that significant nucleosome remodeling over this promoter is not a prerequisite for the activation by the estrogen receptor (ERalpha). This observation lead us to investigate determinants other than ERalpha of chromatin structure and transcriptional activation of the vitellogenin B1 promoter in this system and in vitro. We find that the liver-enriched transcription factor HNF3 has an important organizational role for chromatin structure as demonstrated by DNase I-hypersensitive site mapping. Both HNF3 and the estrogen receptor activate transcription synergistically and are able to interact with chromatin reconstituted in vitro with three positioned nucleosomes. We propose that HNF3 is the cellular determinant which establishes a promoter environment favorable to a rapid transcriptional activation by the estrogen receptor.
Estrogen antagonists inhibit cell cycle progression in estrogen-responsive cells, but the molecular mechanisms are not fully defined. Antiestrogen-mediated G(0)/G(1) arrest is associated with decreased cyclin D1 gene expression, inactivation of cyclin D1-cyclin dependent kinase (Cdk) 4 complexes, and decreased phosphorylation of the retinoblastoma protein (pRb). We now show that treatment of MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 results in inhibition of cyclin E-Cdk2 activity prior to a decrease in the G(1) to S phase transition. This decrease was dependent on p21(WAF1/Cip1) since treatment with antisense oligonucleotides to p21 attenuated the effect. Recruitment of p21 to cyclin E-Cdk2 complexes was in turn dependent on decreased cyclin D1 expression since it was apparent following treatment with antisense cyclin D1 oligonucleotides. To define where within the G(0) to S phase continuum antiestrogen-treated cells arrested, we assessed the relative abundance and phosphorylation state of pocket protein-E2F complexes. While both pRb and p107 levels were significantly decreased, p130 was increased 4-fold and was accompanied by the formation of p130.E2F4 complexes and the accumulation of hyperphophorylated E2F4, putative markers of cellular quiescence. Thus, ICI 182780 inhibits both cyclin D1-Cdk4 and cyclin E-Cdk2 activity, resulting in the arrest of MCF-7 cells in a state with characteristics of quiescence (G(0)), as opposed to G(1) arrest.
Most breast cancers, even those that are initially responsive to tamoxifen, ultimately become resistant. The molecular basis for this resistance, which in some patients is thought to involve stimulation of tumor growth by tamoxifen, is unclear. Tamoxifen induces cellular oxidative stress, and because changes in cell redox state can activate signaling pathways leading to the activation of activating protein-1 (AP-1), we investigated whether tamoxifen-resistant growth in vivo is associated with oxidative stress and/or activation of AP-1 in a xenograft model system where resistance is caused by tamoxifen-stimulated growth.
Control estrogen-treated, tamoxifen-sensitive, and tamoxifen-resistant MCF-7 xenograft tumors were assessed for oxidative stress by measuring levels of antioxidant enzyme (e.g., superoxide dismutase [SOD], glutathione S-transferase [GST], and hexose monophosphate shunt [HMS]) activity, glutathione, and lipid peroxidation. AP-1 protein levels, phosphorylated c-jun levels, and phosphorylated Jun NH(2)-terminal kinase (JNK) levels were examined by western blot analyses, and AP-1 DNA-binding and transcriptional activities were assessed by electrophoretic mobility shift assays and a reporter gene system. All statistical tests are two-sided.
Compared with control estrogen-treated tumors, tamoxifen resistant tumors had statistically significantly increased SOD (more than threefold; P=.004) and GST (twofold; P=.004) activity and statistically significantly reduced glutathione levels (greater than twofold; P<.001) and HMS activity (10-fold; P<.001). Lipid peroxides were not significantly different between control and tamoxifen-resistant tumors. We observed no differences in AP-1 protein components or DNA-binding activity. However, AP-1-dependent transcription (P=.04) and phosphorylated c-Jun and JNK levels (P<.001) were statistically significantly increased in the tamoxifen-resistant tumors.
Our results suggest that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with oxidative stress and the subsequent antioxidant response and with increased phosphorylated JNK and c-Jun levels and AP-1 activity, which together could contribute to tumor growth.
Estrogen acting through the estrogen receptor (ER) is able to regulate cell growth and differentiation of a variety of normal tissues and hormone-responsive tumors. Ligand-activated ER binds DNA and transactivates the promoters of estrogen target genes. In addition, ligand-activated ER can interact with other factors to alter the physiology and growth of cells. Using a yeast two-hybrid screen, we have identified an interaction between ER alpha and the proapoptotic forkhead transcription factor FKHR. The ER alpha-FKHR interaction depends on beta-estradiol and is reduced significantly in the absence of hormone or the presence of Tamoxifen. A glutathione S-transferase pull-down assay was used to confirm the interaction and localized two interaction sites, one in the forkhead domain and a second in the carboxyl terminus. The FKHR interaction was specific to ER alpha and was not detected with other ligand-activated steroid receptors. The related family members, FKHRL1 and AFX, also bound to ER alpha in the presence of beta-estradiol. FKHR augmented ER alpha transactivation through an estrogen response element. Conversely, ER alpha repressed FKHR-mediated transactivation through an insulin response sequence, and cell cycle arrest induced by FKHRL1 in MCF7 cells was abrogated by estradiol. These results suggest a novel mechanism of estrogen action that involves regulation of the proapoptotic forkhead transcription factors.
The estrogen receptor (ER) is a ligand-activated enhancer protein that is a member of the steroid/nuclear receptor superfamily.
Two genes encode mammalian ER: ERα and ERβ. ER binds to specific DNA sequences called estrogen response elements (EREs) with
high affinity and transactivates gene expression in response to estradiol (E2). The purpose of this review is to summarize how natural and synthetic variations in the ERE sequence impact the affinity
of ER–ERE binding and E2-induced transcriptional activity. Surprisingly, although the consensus ERE sequence was delineated in 1989, there are only
seven natural EREs for which both ERα binding affinity and transcriptional activation have been examined. Even less information
is available regarding how variations in ERE sequence impact ERβ binding and transcriptional activity. Review of data from
our own laboratory and those in the literature indicate that ERα binding affinity does not relate linearly with E2-induced transcriptional activation. We suggest that the reasons for this discord include cellular amounts of coactivators
and adaptor proteins that play roles both in ER binding and transcriptional activation; phosphorylation of ER and other proteins
involved in transcriptional activation; and sequence-specific and protein-induced alterations in chromatin architecture.
The E2F transcription factor family is known to play a key role in the timely expression of genes required for cell cycle progression and proliferation, but only a few E2F target genes have been identified. We explored the possibility that E2F regulators play a broader role by identifying additional genes bound by E2F in living human cells. A protocol was developed to identify genomic binding sites for DNA-binding factors in mammalian cells that combines immunoprecipitation of cross-linked protein-DNA complexes with DNA microarray analysis. Among approximately 1200 genes expressed during cell cycle entry, we found that the promoters of 127 were bound by the E2F4 transcription factor in primary fibroblasts. A subset of these targets was also bound by E2F1. Most previously identified target genes known to have roles in DNA replication and cell cycle control and represented on the microarray were confirmed by this analysis. We also identified a remarkable cadre of genes with no previous connection to E2F regulation, including genes that encode components of the DNA damage checkpoint and repair pathways, as well as factors involved in chromatin assembly/condensation, chromosome segregation, and the mitotic spindle checkpoint. Our data indicate that E2F directly links cell cycle progression with the coordinate regulation of genes essential for both the synthesis of DNA as well as its surveillance.
Previously, identification of promoters regulated by mammalian transcription factors has relied upon overexpression studies. Here we present the identification of a large set of promoters that are bound by E2F in physiological conditions. Probing a human CpG microarray with chromatin immunoprecipitated using an antibody to E2F4, we have identified 68 unique target loci; 15% are bidirectional promoters and 25% recruit E2F via a mechanism distinct from the defined consensus site. Interestingly, although E2F has been shown previously to regulate genes involved in cell cycle progression, many of the new E2F target genes encode proteins involved in DNA repair or recombination. We suggest that human CpG microarrays, in combination with chromatin immunoprecipitation, will allow rapid identification of target promoters for many mammalian transcription factors.
Chromatin immunoprecipitation followed by cDNA microarray hybridization (ChIP-array) has become a popular procedure for studying genome-wide protein-DNA interactions and transcription regulation. However, it can only map the probable protein-DNA interaction loci within 1-2 kilobases resolution. To pinpoint interaction sites down to the base-pair level, we introduce a computational method, Motif Discovery scan (MDscan), that examines the ChIP-array-selected sequences and searches for DNA sequence motifs representing the protein-DNA interaction sites. MDscan combines the advantages of two widely adopted motif search strategies, word enumeration and position-specific weight matrix updating, and incorporates the ChIP-array ranking information to accelerate searches and enhance their success rates. MDscan correctly identified all the experimentally verified motifs from published ChIP-array experiments in yeast (STE12, GAL4, RAP1, SCB, MCB, MCM1, SFF, and SWI5), and predicted two motif patterns for the differential binding of Rap1 protein in telomere regions. In our studies, the method was faster and more accurate than several established motif-finding algorithms. MDscan can be used to find DNA motifs not only in ChIP-array experiments but also in other experiments in which a subgroup of the sequences can be inferred to contain relatively abundant motif sites. The MDscan web server can be accessed at http://BioProspector.stanford.edu/MDscan/.
Estrogen binds to receptors that translocate to the plasma membrane and to the nucleus. The rapid, non-genomic actions of this sex steroid are attributed to membrane action, while gene transcription occurs through nuclear receptor function. However, gene transcription can also result from estrogen signaling initiated at the membrane, but the relative importance of this mechanism is not known. In vascular endothelial cells (EC), estradiol (E(2)) activates several kinase cascades, including phosphatidylinositol 3-phosphate (PI3K)/Akt, a signaling pathway that impacts EC biology. We determined here by DNA microarray that 40-min exposure to E(2) significantly increased 250 genes in EC, up-regulation that was substantially prevented by the PI3K inhibitor, LY294002. This coincided with maximum E(2)-induced PI3K activity at 15-30 min. An important vascular gene strongly up-regulated by E(2) in our array produces cyclooxygenase-2 (Cox-2). In cultured EC, E(2) induced both Cox-2 gene expression and new Cox-2 protein synthesis by 40 and 60 min, respectively, and rapidly stimulated the secretion of prostaglandins PGI(2) and PGE(2). The up-regulation of gene expression reflected transcriptional transactivation, shown using Cox-2 promoter/luciferase reporters in the EC. Soluble inhibitors or dominant negative constructs for PI3K and Akt prevented all these actions of E(2). Functionally, EC migration was induced by the sex steroid, and this was significantly reversed by NS-398, a Cox-2 inhibitor. Gene transcription and cell biological effects of estrogen emanate from rapid and specific signaling, integrating cell surface and nuclear actions of this steroid.
The Mouse Genome Analysis Consortium aligned the human and mouse genome sequences for a variety of purposes, using alignment programs that suited the various needs. For investigating issues regarding genome evolution, a particularly sensitive method was needed to permit alignment of a large proportion of the neutrally evolving regions. We selected a program called BLASTZ, an independent implementation of the Gapped BLAST algorithm specifically designed for aligning two long genomic sequences. BLASTZ was subsequently modified, both to attain efficiency adequate for aligning entire mammalian genomes and to increase its sensitivity. This work describes BLASTZ, its modifications, the hardware environment on which we run it, and several empirical studies to validate its results.
Recognition of gene starts is a difficult and yet unsolved problem. We present a program, Dragon Gene Start Finder (DGSF),
which assesses the gene start in mammalian genomes and predicts a region which should overlap with the first exon of the gene
or be in its proximity. The program has been rigorously tested on human chromosomes 4, 21 and 22, and in a strand specific
search achieves an overall sensitivity of ∼65% and a positive predictive value of ∼78%. The sensitivity for the CpG-island
related promoters is >88%. DGSF is free for academic and non-profit users at http://sdmc.lit.org.sg/promoter/dragonGSF1_0/genestart.htm; the download version of the program integrated within the TRANSPLORERTM package can be obtained from Biobase GmbH, at http://www.biobase.de/.
Estrogen receptors (ERs) are nuclear transcription factors that regulate gene expression in response to estrogen and estrogen-like compounds. Identification of estrogen-regulated genes in target cells is an essential step toward understanding the molecular mechanisms of estrogen action. Using cDNA microarray examinations, 19 genes were identified as induced by 17 beta-estradiol in MCF-7 cells, 10 of which have been reported previously to be estrogen responsive or to be linked with ER status. Five known estrogen-regulated genes, E2IG4, IGFBP4, SLC2A1, XBP1 and B4GALT1, and AFG3L1, responded quickly to estrogen treatment. A novel estrogen-responsive gene was identified and named EEIG1for early estrogen-induced gene 1. EEIG1 was clearly induced by 17 beta-estradiol within 2 h of treatment, and was widely responsive to a group of estrogenic compounds including natural and synthetic estrogens and estrogenic environmental compounds. EEIG1 was expressed in ER-positive but not in ER-negative breast cancer cell lines. EEIG1 expression was repressed by antiestrogens 4-OH-tamoxifen and ICI 182,780 but not by protein synthesis inhibitors cycloheximide and puromycin. These results provide evidence that some estrogenic compounds differentially enhance the transcription of estrogen-regulated genes and suggest a role for EEIG1 in estrogen action.
In breast tumours and breast cancer cell (BCC) lines, microarray analyses have revealed that a series of genes are expressed in close association with the oestrogen receptor-alpha (ER-alpha) gene, ESR1. Three of them, GATA3, HNF3A (also known as FOXA1), and XBP1 encode transcription factors. Here, we present these factors and we discuss their potential involvement in the ER-alpha-mediated actions in BCC. We notably show the relations that exist, or that might exist, between these factors and the oestrogen-inducible trefoil factor TFF1.
Although estrogen receptors (ERs) recognize 15-bp palindromic estrogen response elements (EREs) with maximal affinity in vitro, few near-consensus sequences have been characterized in estrogen target genes. Here we report the design of a genome-wide screen for high-affinity EREs and the identification of approximately 70000 motifs in the human and mouse genomes. EREs are enriched in regions proximal to the transcriptional start sites, and approximately 1% of elements appear conserved in the flanking regions (-10 kb to +5 kb) of orthologous human and mouse genes. Conserved and nonconserved elements were also found, often in multiple occurrences, in more than 230 estrogen-stimulated human genes previously identified from expression studies. In genes containing known EREs, we also identified additional distal elements, sometimes with higher in vitro binding affinity and/or better conservation between the species considered. Chromatin immunoprecipitation experiments in breast cancer cell lines indicate that most novel elements present in responsive genes bind ERalpha in vivo, including some EREs located up to approximately 10 kb from transcriptional start sites. Our results demonstrate that near-consensus EREs occur frequently in both genomes and that whereas chromatin structure likely modulates access to binding sites, far upstream elements can be evolutionarily conserved and bind ERs in vivo.
Mutations in MECP2 are associated with Rett syndrome, an X-linked neurodevelopmental disorder. To identify genes targeted by Mecp2, we sequenced 100 in vivo Mecp2-binding sites in mouse brain. Several sequences mapped to an imprinted gene cluster on chromosome 6, including Dlx5 and Dlx6, whose transcription was roughly two times greater in brains of Mecp2-null mice compared with those of wild-type mice. The maternally expressed gene DLX5 showed a loss of imprinting in lymphoblastoid cells from individuals with Rett syndrome. Because Dlx5 regulates production of enzymes that synthesize gamma-aminobutyric acid (GABA), loss of imprinting of Dlx5 may alter GABAergic neuron activity in individuals with Rett syndrome. In mouse brain, Dlx5 imprinting was relaxed, yet Mecp2-mediated silent-chromatin structure existed at the Dlx5-Dlx6 locus in brains of wild-type, but not Mecp2-null, mice. Mecp2 targeted histone deacetylase 1 to a sharply defined, approximately 1-kb region at the Dlx5-Dlx6 locus and promoted repressive histone methylation at Lys9 at this site. Chromatin immunoprecipitation-combined loop assays showed that Mecp2 mediated the silent chromatin-derived 11-kb chromatin loop at the Dlx5-Dlx6 locus. This loop was absent in chromatin of brains of Mecp2-null mice, and Dlx5-Dlx6 interacted with far distant sequences, forming distinct active chromatin-associated loops. These results show that formation of a silent-chromatin loop is a new mechanism underlying gene regulation by Mecp2.
Estradiol (E2) induces transforming growth factor (TGF) gene expression in MCF-7 cells and previous studies have identified a 53 bp (252 to 200) sequence containing two imperfect estrogen responsive elements (EREs) that contrib- ute to E2 responsiveness. Deletion analysis of the TGF gene promoter in this study identified a second upstream region of the promoter (623 to 549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans- acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor (ER)/Sp1 complex interacting with an Sp1(N)30 ERE half-site (½) motif in which both ER and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ER or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2- responsive Sp1(N) x ERE½ motif identified in the TGF gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.
Motivation:
When running experiments that involve multiple high density oligonucleotide arrays, it is important to remove sources of variation between arrays of non-biological origin. Normalization is a process for reducing this variation. It is common to see non-linear relations between arrays and the standard normalization provided by Affymetrix does not perform well in these situations.
Results:
We present three methods of performing normalization at the probe intensity level. These methods are called complete data methods because they make use of data from all arrays in an experiment to form the normalizing relation. These algorithms are compared to two methods that make use of a baseline array: a one number scaling based algorithm and a method that uses a non-linear normalizing relation by comparing the variability and bias of an expression measure. Two publicly available datasets are used to carry out the comparisons. The simplest and quickest complete data method is found to perform favorably.
Availability:
Software implementing all three of the complete data normalization methods is available as part of the R package Affy, which is a part of the Bioconductor project http://www.bioconductor.org.
Supplementary information:
Additional figures may be found at http://www.stat.berkeley.edu/~bolstad/normalize/index.html
Human chromosome 21 is the smallest and one of the most intensively studied autosomes. The generation of high quality genetic and physical maps for the long arm has enabled the research community to accelerate gene discovery and the identification of disease loci on the chromosome. However, the emerging pattern from large-scale transcriptional mapping from many groups suggests that the majority of the 600-1000 genes predicted to reside on the chromosome are clustered in two distinct regions of the long arm, on distal 21q22.1 and 21q22.3. Here, we report the mapping of the gene for receptor interacting protein 140 (RIP140) on 21q11 by means of YACs, PACs and hybrid cell lines. We have placed RIP140 within 100 kb of D21S13, in a region of the chromosome where only one other gene has been described to date. The association of the RIP140 protein with the superfamily of nuclear receptors may be of significance in studies of trisomy 21 (Down syndrome) and Alzheimers disease, since a modifier locus has been speculated to reside on 21q11.
The 5'-flanking region of the human lactoferrin gene was isolated from a human placental genomic library. This genomic clone contains a 16-kilobase pair (kbp) insert and produces seven fragments when digested with the SacI restriction enzyme. We sequenced one of the fragments that comprises 1294 bp of the 5'-flanking sequence, 79 bp of the first exon, and 690 bp of the first intron. A major transcription start site was mapped by primer extension. The region immediately upstream from the transcription initiation site following the first exon is abundant in G and C nucleotides. In the promoter and 5'-flanking region within a 300-bp stretch (-465 to -165) of the DNA, we found a noncanonical TATA box (ATAAA), CAAT-like sequence (CAAC) and sequences homologous to the consensus SP1 binding site, Pu.1/Sp.1 binding element (PU box), two half-palindromic estrogen response elements (EREs; GGTCA), an imperfect ERE (GGTCAAGGCGATC), and a sequence resembling the chicken ovalbumin upstream promoter transcription factor (COUP-TF) binding site (GTCTCACAGGTCA). The COUP-TF binding site and the imperfect ERE shared five nucleotides (GGTCA). With the exception of the two half-palindromic EREs, the elements with very well matched sequences were also found in the corresponding positions in the mouse lactoferrin gene. The synthetic oligonucleotide, including the 26 bp of COUP/ERE sequence, was cloned before the SV40 promoter in a chloramphenicol acetyltransferase reporter construct. These chimeric plasmids were transiently transfected into human endometrium carcinoma RL95-2 cells to assess hormone responsiveness. We found that the COUP/ERE element acted as an enhancer in response to estrogen stimulation. In vitro DNase I footprinting analysis showed binding of the estrogen receptor on the imperfect ERE. In contrast to the mouse lactoferrin COUP/ERE element, COUP-TF does not interact with this element, as demonstrated by band shift assay and site-directed mutagenesis. Therefore, the molecular mechanisms of the estrogen action that govern the lactoferrin gene expression differ between mouse and human.
The estrogen receptor complex is a known trans-acting factor that regulates transcription of specific genes through an interaction with a specific estrogen-responsive cis-acting element (ERE). In previous studies we have shown that in estrogen-responsive human breast cancer cells estrogen rapidly activates c-myc expression. This activated expression occurs through enhanced transcription and does not require the synthesis of new protein intermediates; therefore, an ERE is present in the human c-myc gene regulatory region. To localize the ERE, constructs containing varying lengths of the c-myc 5'-flanking region ranging from -2327 to +25 (relative to the P1 promoter) placed adjacent to the chloramphenicol acetyl transferase reporter gene (CAT) were prepared. They were used in transient transfection studies in MCF-7 and HeLa cells co-transfected with an estrogen receptor expression vector. These studies reveal that all constructs containing the P2 promoter region exhibited estrogen-regulated CAT expression and that a 116-bp region upstream and encompassing the P2 TATA box is necessary for this activity. Analysis of this 116-bp region failed to identify a cis-acting element with sequences resembling the consensus ERE; however, co-transfection studies with mutant estrogen receptor expression vectors showed that the DNA-binding domain of the receptor is essential for estrogen-regulated CAT gene expression. We have also observed that anti-estrogen receptor complexes can weakly trans-activate from this 116-bp region but fail to do so from the ERE-containing ApoVLDLII-CAT construct. To explain these results we propose a new mechanism of estrogen trans-activation in the c-myc gene promoter.
A conserved region in the hormone-dependent activation domain AF2 of nuclear receptors plays an important role in transcriptional activation. We have characterized a novel nuclear protein, RIP140, that specifically interacts in vitro with this domain of the estrogen receptor. This interaction was increased by estrogen, but not by anti-estrogens and the in vitro binding capacity of mutant receptors correlates with their ability to stimulate transcription. RIP140 also interacts with estrogen receptor in intact cells and modulates its transcriptional activity in the presence of estrogen, but not the anti-estrogen 4-hydroxytamoxifen. In view of its widespread expression in mammalian cells, RIP140 may interact with other members of the superfamily of nuclear receptors and thereby act as a potential co-activator of hormone-regulated gene transcription.
Cathepsin D, a lysosomal proteinase, is induced by estrogens in mammary cancer cells where its concentration is correlated with a higher risk of metastasis. Its gene expression is stimulated by estrogens in MCF7 cells, and we have shown that a short proximal promoter fragment from -365 to -122 is required for this induction. We now characterize, at -261, a nonconsensus estrogen-responsive element (ERE) (E2) with two differences in the distal half of its palindrome, which confers estradiol responsiveness to the heterologous Herpes simplex virus thymidine kinase promoter in transient transfection experiments. This ERE is located in a 21-base pair sequence: 5'GGGCCGGGCTGACCCCGC GGG3', containing a GC-rich region in its 3'-part, which is almost perfectly repeated at -362 (the E1 site). The E2 site was necessary but not sufficient to mediate an estrogen response and required cooperation with the homologous E1 element and/or with general transcription sites located downstream. In vitro, the E2 site but not the E1 site was protected by estrogen receptor (ER) against DNAse I digestion, and gel shift experiments suggested an interaction with the ER as a dimer. Moreover, we showed in vivo that ER DNA binding domain was required to mediate estrogen induction from the cathepsin D ERE. We conclude that estradiol induction of cathepsin D is mediated by interaction of the ER with a nonconsensus ERE that requires synergy with other elements located upstream and/or downstream of this central ERE.
The estrogen receptor is a transcription factor which, when bound to estradiol, binds DNA and regulates expression of estrogen-responsive
genes. A 160-kilodalton estrogen receptor-associated protein, ERAP160, was identified that exhibits estradiol-dependent binding
to the receptor. Mutational analysis of the receptor shows that its ability to activate transcription parallels its ability
to bind ERAP160. Antiestrogens are unable to promote ERAP160 binding and can block the estrogen-dependent interaction of the
receptor and ERAP160 in a dose-dependent manner. This evidence suggests that ERAP160 may mediate estradiol-dependent transcriptional
activation by the estrogen receptor. Furthermore, the ability of antiestrogens to block estrogen receptor-ERAP160 complex
formation could account for their therapeutic effects in breast cancer.
Treatment of MCF-7 human breast cancer cells with 10 nM 17 beta-estradiol (E2) resulted in a 2-fold induction of heat shock protein (Hsp) 27 mRNA levels, and this response persisted for up to 24 h. The 5'-promoter region of the gene was further investigated to identify genomic sequences associated with E2 responsiveness. An Sp1 and half-palindromic estrogen response element (ERE) separated by 10 nucleotides, GGGCGGG(N)10GGTCA, were identified at -105 to -84, and formation of the Sp1/estrogen receptor (ER) complex was investigated by in vitro assays using synthetic Hsp 27-[32P]Sp1/ERE oligonucleotides in a gel mobility shift assay and transient transfection studies using short (-108/-84) and long (-108/+23) 5'-promoter sequences linked to a thymidine kinase promoter and the bacterial chloramphenicol acetyl transferase (CAT) reporter gene (Hsp-CATs and Hsp-CATl, respectively). Incubation of nuclear extracts from MCF-7 cells with an Hsp 27-[32P]Sp1/ERE oligonucleotide results in formation of an Sp1/ER complex. The formation of this complex was inhibited by coincubation with unlabeled Sp1/ERE, ERE, and Sp1 oligonucleotides and by preincubation with ER or Sp1 antibodies (immunodepletion). In addition, the complex was supershifted by coincubation with ER antibodies. Mutation of either Sp1 or ERE sites also decreases formation of the retarded band. E2 induced CAT activity in MCF-7 cells transiently transfected with either Hsp-CATs or Hsp-CATl plasmids. It was also demonstrated that E2 did not significantly induce CAT activity in MCF-7 cells transiently transfected with Hsp-CATl-containing mutations in both the Sp1 and ERE sites. The results of this study demonstrate that an Sp1/ER complex is involved in E2-induced Hsp 27 gene expression.
The transcription factor HNF3 and linker histones H1 and H5 possess winged-helix DNA-binding domains, yet HNF3 and other fork head-related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild-type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site-directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome-binding activities that are not predicted from studies of factor interactions with free DNA.
The winged helix transcription factors HNF-3/FKH (forkhead homologs) activate endodermal-derived and acute-phase gene expression and control gut development in Drosophila. Trefoil factor family (TFFs) peptides are vertebrate products secreted by mucin-producing epithelial cells of the gastrointestinal tract involved in restitution and repair of the mucosa. They are positively regulated in ulcerative and neoplastic conditions. We describe a consensus sequence in human and rodent TFF promoters close to the TATAA box showing striking similarity to the binding site of the HNF-3/FKH family. In gel retardation assays, HNF-3 alpha and beta bound predominantly to the site in TFF1 (formerly pS2) and, to a lesser extent, to the sites in TFF2 or TFF3. Mutations generated in this motif severely impaired transcription of TFF1 reporter genes. Cotransfection with expression vectors of HNF-3alpha and beta, but not the related HFH 11A and B, specifically activated the wild-type TFF1 reporter genes. Activation of endogenous expression of TFF1 by HNF-3 alpha and beta gene products was more than 1000 fold in the pancreatic cell line Capan-2 and fivefold in the gastric cell line MKN-45, whereas the intestinal cell lines HUTU 80 and HT-29 displayed no effect. Thus, HNF-3/FKH factors contribute causally to cell-specific regulation of TFF genes and may explain the acute-phase response of TFF peptides.
17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (-252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGFalpha gene promoter in this study identified a second upstream region of the promoter (-623 to -549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans-acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE(1/2) motif identified in the TGFalpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.
The human EBAG9 was previously identified as an estrogen responsive gene using CpG-genomic binding site cloning (Watanate et al., (1998) Mol. Cell. Biol. 18: 442-449). Recently it was revealed that the EBAG9 is identical with RCAS1 which is a cancer cell surface antigen implicated in immune escape. Here, we isolated and analyzed the 5'-flanking region of human EBAG9 gene. We determined transcription initiation site, which has a homology with an initiator element YYCAYYYY, and found that TATA motif was absent. Deletion analysis of the 5'-flanking region using MCF-7 breast cancer cells indicated that the sequences -86 to -36 containing the ERE had the basal level of promoter activity and the upstream GC-rich region positively regulated the activity. EBAG9 promoter luciferase reporters containing the ERE could respond to estrogen, and electrophoretic mobility shift assay showed that ERalpha bound to the ERE. Moreover, fluorescent in situ hybridization analysis has shown that the human EBAG9 gene is located at chromosome 8q23 which is frequently amplified in tumors. These findings suggest that the human EBAG9 might be involved in carcinogenesis as an estrogen responsive gene.
Many cofactors bind the hormone-activated estrogen receptor (ER), yet the specific regulators of endogenous ER-mediated gene transcription are unknown. Using chromatin immunoprecipitation (ChIP), we find that ER and a number of coactivators rapidly associate with estrogen responsive promoters following estrogen treatment in a cyclic fashion that is not predicted by current models of hormone activation. Cycles of ER complex assembly are followed by transcription. In contrast, the anti-estrogen tamoxifen (TAM) recruits corepressors but not coactivators. Using a genetic approach, we show that recruitment of the p160 class of coactivators is sufficient for gene activation and for the growth stimulatory actions of estrogen in breast cancer supporting a model in which ER cofactors play unique roles in estrogen signaling.
We describe an approach to detect the frequency of interaction between any two genomic loci. Generation of a matrix of interaction
frequencies between sites on the same or different chromosomes reveals their relative spatial disposition and provides information
about the physical properties of the chromatin fiber. This methodology can be applied to the spatial organization of entire
genomes in organisms from bacteria to human. Using the yeast Saccharomyces cerevisiae, we could confirm known qualitative features of chromosome organization within the nucleus and dynamic changes in that organization
during meiosis. We also analyzed yeast chromosome III at the G1stage of the cell cycle. We found that chromatin is highly flexible throughout. Furthermore, functionally distinct AT- and
GC-rich domains were found to exhibit different conformations, and a population-average 3D model of chromosome III could be
determined. Chromosome III emerges as a contorted ring.
The transcription factors HNF3 (FoxA) and GATA-4 are the earliest known to bind the albumin gene enhancer in liver precursor cells in embryos. To understand how they access sites in silent chromatin, we assembled nucleosome arrays containing albumin enhancer sequences and compacted them with linker histone. HNF3 and GATA-4, but not NF-1, C/EBP, and GAL4-AH, bound their sites in compacted chromatin and opened the local nucleosomal domain in the absence of ATP-dependent enzymes. The ability of HNF3 to open chromatin is mediated by a high affinity DNA binding site and by the C-terminal domain of the protein, which binds histones H3 and H4. Thus, factors that potentiate transcription in development are inherently capable of initiating chromatin opening events.
Recent studies of beta-globin gene expression have concentrated on the analysis of factor binding and chromatin structure within the endogenous locus. These studies have more precisely defined the extent and nature of the active chromosomal domain and the elements that organize it. Surprisingly, the beta-globin locus control region (LCR), although critical for high-level gene expression, plays little role in the overall architecture of the active locus. Analysis of the effects of targeted deletion of the beta-globin LCR, along with emerging knowledge of the behavior of the erythroid transcription factor NF-E2, leads to a new perspective on factor binding and LCR function.
Selective estrogen receptor modulators (SERMs) mimic estrogen action in certain tissues while opposing it in others. The therapeutic
effectiveness of SERMs such as tamoxifen and raloxifene in breast cancer depends on their antiestrogenic activity. In the
uterus, however, tamoxifen is estrogenic. Here, we show that both tamoxifen and raloxifene induce the recruitment of corepressors
to target gene promoters in mammary cells. In endometrial cells, tamoxifen, but not raloxifene, acts like estrogen by stimulating
the recruitment of coactivators to a subset of genes. The estrogen-like activity of tamoxifen in the uterus requires a high
level of steroid receptor coactivator 1 (SRC-1) expression. Thus cell type– and promoter-specific differences in coregulator
recruitment determine the cellular response to SERMs.
The sequences of the human chromosomes 21 and 22 indicate that there are approximately 770 well-characterized and predicted
genes. In this study, empirically derived maps identifying active areas of RNA transcription on these chromosomes have been
constructed with the use of cytosolic polyadenylated RNA obtained from 11 human cell lines. Oligonucleotide arrays containing
probes spaced on average every 35 base pairs along these chromosomes were used. When compared with the sequence annotations
available for these chromosomes, it is noted that as much as an order of magnitude more of the genomic sequence is transcribed
than accounted for by the predicted and characterized exons.
The progesterone receptor (PR) gene is activated by estrogen in MCF-7 human breast cancer cells. Although the human PR gene does not contain an estrogen response element (ERE), we have identified a putative activating protein-1 (AP-1) site at +90 in the PR gene that was hypersensitive to deoxyribonuclease I cleavage in genomic Southern analysis, bound purified Fos and Jun, formed a complex with Fos/Jun heterodimers present in MCF-7 nuclear extracts in gel mobility shift assays, and functioned as an estrogen-responsive enhancer in transient cotransfection assays. When the +90 AP-1 site was mutated in the context of the PR gene, estrogen responsiveness was significantly decreased. Purified estrogen receptor (ER) enhanced binding of Fos and Jun to the +90 AP-1 site and bound to an adjacent imperfect ERE half-site. Mutating this ERE half-site diminished the binding of ER, Fos, and Jun and decreased transcription. Chromatin immunoprecipitation assays demonstrated that the ER, Fos, and Jun were present at the +90 AP-1 site in the endogenous PR gene only after treatment of MCF-7 cells with estrogen. These studies suggest that the cooperative interaction of the ER with Fos and Jun proteins helps confer estrogen responsiveness to the endogenous PR gene.
When running experiments that involve multiple high density oligonucleotide arrays, it is important to remove sources of variation between arrays of non-biological origin. Normalization is a process for reducing this variation. It is common to see non-linear relations between arrays and the standard normalization provided by Affymetrix does not perform well in these situations.
We present three methods of performing normalization at the probe intensity level. These methods are called complete data methods because they make use of data from all arrays in an experiment to form the normalizing relation. These algorithms are compared to two methods that make use of a baseline array: a one number scaling based algorithm and a method that uses a non-linear normalizing relation by comparing the variability and bias of an expression measure. Two publicly available datasets are used to carry out the comparisons. The simplest and quickest complete data method is found to perform favorably.
Software implementing all three of the complete data normalization methods is available as part of the R package Affy, which is a part of the Bioconductor project http://www.bioconductor.org.
Additional figures may be found at http://www.stat.berkeley.edu/~bolstad/normalize/index.html
To investigate the molecular basis of beta-globin gene activation, we analyzed factor recruitment and histone modification at the adult beta-globin gene in wild-type (WT)/locus control region knockout (DeltaLCR) heterozygous mice and in murine erythroleukemia (MEL) cells. Although histone acetylation and methylation (Lys 4) are high before and after MEL differentiation, recruitment of the erythroid-specific activator NF-E2 to the promoter and preinitiation complex (PIC) assembly occur only after differentiation. We reported previously that targeted deletion of the LCR reduces beta-globin gene expression to 1%-4% of WT without affecting promoter histone acetylation. Here, we report that NF-E2 is recruited equally efficiently to the adult beta-globin promoters of the DeltaLCR and WT alleles. Moreover, the LCR deletion reduces PIC assembly only twofold, but has a dramatic effect on Ser 5 phosphorylation of RNA polymerase II and transcriptional elongation. Our results suggest at least three distinct stages in beta-globin gene activation: (1) an LCR-independent chromatin opening stage prior to NF-E2 recruitment to the promoter and PIC assembly; (2) an intermediate stage in which NF-E2 binding (LCR-independent) and PIC assembly (partially LCR-dependent) occur; and (3) an LCR-dependent fully active stage characterized by efficient pol II elongation. Thus, in its native location the LCR functions primarily downstream of activator recruitment and PIC assembly.
Transcriptional activation of a gene involves an orchestrated recruitment of components of the basal transcription machinery and intermediate factors, concomitant with an alteration in local chromatin structure generated by posttranslational modifications of histone tails and nucleosome remodeling. We provide here a comprehensive picture of events resulting in transcriptional activation of a gene, through evaluating the estrogen receptor-alpha (NR3A1) target pS2 gene promoter in MCF-7 cells. This description integrates chromatin remodeling with a kinetic evaluation of cyclical networks of association of 46 transcription factors with the promoter, as determined by chromatin immunoprecipitation assays. We define the concept of a "transcriptional clock" that directs and achieves the sequential and combinatorial assembly of a transcriptionally productive complex on a promoter. Furthermore, the unanticipated findings of key roles for histone deacetylases and nucleosome-remodeling complexes in limiting transcription implies that transcriptional activation is a cyclical process that requires both activating and repressive epigenetic processes.
Using high-density oligonucleotide arrays representing essentially all nonrepetitive sequences on human chromosomes 21 and 22, we map the binding sites in vivo for three DNA binding transcription factors, Sp1, cMyc, and p53, in an unbiased manner. This mapping reveals an unexpectedly large number of transcription factor binding site (TFBS) regions, with a minimal estimate of 12,000 for Sp1, 25,000 for cMyc, and 1600 for p53 when extrapolated to the full genome. Only 22% of these TFBS regions are located at the 5' termini of protein-coding genes while 36% lie within or immediately 3' to well-characterized genes and are significantly correlated with noncoding RNAs. A significant number of these noncoding RNAs are regulated in response to retinoic acid, and overlapping pairs of protein-coding and noncoding RNAs are often coregulated. Thus, the human genome contains roughly comparable numbers of protein-coding and noncoding genes that are bound by common transcription factors and regulated by common environmental signals.
The transcriptional regulatory networks that specify and maintain human tissue diversity are largely uncharted. To gain insight into this circuitry, we used chromatin immunoprecipitation combined with promoter microarrays to identify systematically the genes occupied by the transcriptional regulators HNF1alpha, HNF4alpha, and HNF6, together with RNA polymerase II, in human liver and pancreatic islets. We identified tissue-specific regulatory circuits formed by HNF1alpha, HNF4alpha, and HNF6 with other transcription factors, revealing how these factors function as master regulators of hepatocyte and islet transcription. Our results suggest how misregulation of HNF4alpha can contribute to type 2 diabetes.
The gene encoding AIB1, an estrogen receptor coactivator, is amplified in a subset of human breast cancers. Here we show that overexpression of AIB1 in transgenic mice (AIB1-tg) leads to mammary hypertrophy, hyperplasia, abnormal postweaning involution, and the development of malignant mammary tumors. Tumors are also increased in other organs, including the pituitary and uterus. AIB1 overexpression increases mammary IGF-I mRNA and serum IGF-I protein levels. In addition, IGF-I receptor and downstream signaling molecules are activated in primary mammary epithelial cells and mammary tumor cells derived from AIB1-tg mice. Knockdown of AIB1 expression in cultured AIB1-tg mammary tumor cells leads to reduced IGF-I mRNA levels and increased apoptosis, suggesting that an autocrine IGF-I loop underlies the mechanism of AIB1-induced oncogenesis.