Activation Function-2 Domain Research Articles

A Possible Novel Mechanism of Action of Genistein and Daidzein for Activating Thyroid Hormone Receptor-Mediated Transcription

Thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily that regulate their target genes for controlling organ development and functional maintenance. Soybean isoflavones, especially genistein and daidzein, modulate various hormone-mediated pathways. However, their effects on TRs have not yet been extensively studied. In this study, the effects of these isoflavones on TR action were evaluated using transient transfection-based reporter gene assays and molecular docking studies. Genistein and daidzein augmented T3-liganded TR-mediated transcription in a concentration-dependent manner. In the mammalian 2-hybrid study, these isoflavones augmented the recruitment of steroid receptor coactivator-1 and nuclear corepressor to liganded or unliganded TRs. Using a series of mutant TRs, we also showed that the activation function-2 domain of TRs was responsible for the augmentation by these isoflavones. CV-1 cells had expressed TRα, TRβ1, and ERα mRNAs. However, neither the overexpression nor the knocking down of ERα altered the augmentation of TR action by isoflavones, indicating that the effects of isoflavones are exerted through their direct action on TRs. In silico molecular docking studies showed that genistein and daidzein can directly bind to the TR-ligand-binding domain. These findings indicate that the augmentation of the TR-mediated transcription by genistein and daidzein is due to their direct binding to TR-ligand-binding domain to induce the recruitment of steroid receptor coactivator-1. Our study reports a novel mode of action of soybean isoflavones on TR function. The biological effects and the relevance of these isoflavones to human health may be partially attributable to the activation of thyroid hormone signaling.

Selective modulation of the androgen receptor AF2 domain rescues degeneration in spinal bulbar muscular atrophy.

Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by toxic gain-of-function of the androgen receptor (AR). In a prior study we showed that coregulator binding through the activation function-2 (AF2) domain of AR is essential to pathogenesis, suggesting that AF2 may be a druggable target for selective modulation of toxic AR activity. Here we screened previously identified AF2 modulators for their ability to rescue toxicity in a Drosophila model of SBMA. We identified two compounds, tolfenamic acid (TA) and 1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole (MEPB), as top candidates for rescuing lethality, locomotor function, and neuromuscular junction defects in SBMA flies. Pharmacokinetic analyses in mice showed a more favorable bioavailability and tissue retention of MEPB compared with TA in muscle, brain, and spinal cord. In a preclinical trial in a novel mouse model of SBMA, MEPB treatment yielded a dose-dependent rescue from loss of body weight, rotarod activity, and grip strength. In addition, MEPB ameliorated neuronal loss, neurogenic atrophy, and testicular atrophy, validating AF2 modulation as a potent androgen-sparing strategy for SBMA therapy.

Abstract P6-09-08: Identification of ESR1 mutation in breast cancers using targeted ultra-deep sequencing data analysis

Abstract Introduction: Estrogen Receptor 1 (ESR1) gene encodes an estrogen receptor, which regulates cell proliferation and promotes tumor progression in estrogen receptor(ER)-positive breast cancer (BC). Therefore, endocrine therapy that inhibiting ER downstream signal, is the most effective treatment strategy in ER-positive BC. However, about 25% of patients with primary disease and almost all patients with metastases will present with or eventually develop endocrine resistance. And genetic alteration of ESR1 is now identified as the endocrine resistance mechanism. However, a few data from clinical trials or public data base exists and could not reflect real world clinic. Therefore, we aimed to identify the frequency and type of ESR1 genetic alterations in BCs through this large scaled study. Methods: We performed targeted ultra-deep sequencing (CancerSCAN™) using BC tissue specimens. This sequencing was covered entire coding area of ESR1 gene and also detected copy number alteration and translocation of ESR1. Results: Targeted ultra-deep sequencing of ESR1 was performed using 618 BC tissues. Of 618 tissue samples, 253(40.9%) were MBCs, 362(58.6%) were early BCs (EBCs) and 3 were not identified. In terms of subtypes, 220 ER-positive BCs, 122 ER-positive and HER2-positive BCs, 119 HER2-positive and 153 triple-negative BCs (TNBCs) were included. BCs from patients under 40 year-old were 277(44.8%)(Median: 43.0, range: 23.5 -75.6). ESR1 genetic alterations were identified in 21 BCs (5 EBCs and 16 MBCs). In EBCs, 3 cases were observed in TNBCs and 2 cases were in ER-positive BCs (2.6% and 1.2%, respectively). All five EBC were treatment naïve status. Of 16 cases of ESR1 alterations in MBCs, 10 cases of ESR1 alterations were detected in ER-positive BCs (17.6%), 5cases in ER and HER2-positive BCs(6.7%) and 1 in HER2-positive BCs (1.2%). All ER-positive MBCs were treated with more than one line of endocrine therapy. Most commonly detected genetic alteration was single nucleotide variant (SNV) (15 of 21, 71.4%). Thirteen were in ligand binding domain and two cases occurred in activation function-1 (AF-1) domain (P79A and G145S). D538G and V392I were most frequently mutated loci followed by Y537N (3, 3 and 2 cases, respectively) and only metastatic ER-positive BCs harbored ESR1 activating mutation. Four copy number (CN) amplification in 2 ER-positive and 2 ER and HER2-positive BCs, one CN deletion in TNBC and one ESR1 fusion in ER and HER2-positive BC were also detected (19.0%, 4.8% and 4.8%, respectively). In frame ESR1 fusion was occurred between ESR1 and NPHS1 genes. Conclusion: In this experimental study, ESR1 genetic alterations were frequently detected in ER-positive MBC but ER-negative or EBC also harbored. The type of genetic alterations varied including SNVs, CN alterations and translocation and ESR1-NPHS1 fusion is the novel genetic alteration that has not been reported. To identify the role of ESR1 genetic alteration in ER-negative BCs and novel translocation, further functional validation would be warranted (Clinical trials.gov Number :NCT02591966). Citation Format: Kim J-Y, Park K, Park W-Y, Nam SJ, Kim SW, Lee JE, Lee SK, Jung HH, Yu JH, Ahn JS, Im Y-H, Park YH. Identification of ESR1 mutation in breast cancers using targeted ultra-deep sequencing data analysis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-09-08.

Development of Bag-1L as a therapeutic target in androgen receptor-dependent prostate cancer.

Targeting the activation function-1 (AF-1) domain located in the N-terminus of the androgen receptor (AR) is an attractive therapeutic alternative to the current approaches to inhibit AR action in prostate cancer (PCa). Here we show that the AR AF-1 is bound by the cochaperone Bag-1L. Mutations in the AR interaction domain or loss of Bag-1L abrogate AR signaling and reduce PCa growth. Clinically, Bag-1L protein levels increase with progression to castration-resistant PCa (CRPC) and high levels of Bag-1L in primary PCa associate with a reduced clinical benefit from abiraterone when these tumors progress. Intriguingly, residues in Bag-1L important for its interaction with the AR AF-1 are within a potentially druggable pocket, implicating Bag-1L as a potential therapeutic target in PCa.

A novel activation domain is essential for CIA5-mediated gene regulation in response to CO2 changes in Chlamydomonas reinhardtii

The inducible CO2 concentrating mechanism (CCM) of microalgae is essential for their acclimation to highly variable aquatic inorganic carbon levels. The key transcriptional regulator, CIA5, affects expression of thousands of genes in Chlamydomonas reinhardtii, but the molecular characteristics of this important protein are poorly understood. This study identifies a functional activation domain in CIA5 and demonstrates the functionality of a mini-CIA5 including only the zinc-binding domain and the identified activation domain. A highly conserved 130aa region from CIA5 exhibits auto-activation in yeast and appears responsible for the markedly slow migration of CIA5 when analyzed by SDS-PAGE. This 130aa region or either half of this region also effectively replaced the activation domain of a modified designer Transcription Activator-like Element (dTALE) in targeted activation of an endogenous Chlamydomonas gene. Additionally, a mini-CIA5 combining the conserved zinc-binding domain with the 130aa putative activation domain complemented the growth phenotype of the cia5 mutant and triggered CO2-regulated gene expression patterns similar to wild-type cells or cia5 complemented with the full-length CIA5. Although the mini-CIA5 complementation did not fully restore wild-type growth rates or full gene induction/repression amplitudes, especially in very low CO2, this newly identified activation domain combined with the previously described zinc-binding domain are demonstrated to be the key essential components of CIA5 that permit rapid CIA5-mediated responses to changes in CO2 concentrations.

Nucleosome distortion as a possible mechanism of transcription activation domain function.

After more than three decades since the discovery of transcription activation domains (ADs) in gene-specific activators, the mechanism of their function remains enigmatic. The widely accepted model of direct recruitment by ADs of co-activators and basal transcriptional machinery components, however, is not always compatible with the short size yet very high degree of sequence randomness and intrinsic structural disorder of natural and synthetic ADs. In this review, we formulate the basis for an alternative and complementary model, whereby sequence randomness and intrinsic structural disorder of ADs are necessary for transient distorting interactions with promoter nucleosomes, triggering promoter nucleosome translocation and subsequently gene activation.

F-box only protein 9 is an E3 ubiquitin ligase of PPARγ.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical regulator of carbohydrate and lipid metabolism, adipocyte differentiation and inflammatory response. Post-translational modification of PPARγ and its degradation involve several pathways, including the ubiquitin–proteasome system. Here, we identified F-box only protein 9 (FBXO9) as an E3 ubiquitin ligase of PPARγ. We screened interacting partners of PPARγ using immunoprecipitation and mass spectrometric analysis and identified FBXO9 as an E3 ubiquitin ligase of PPARγ. FBXO9 directly interacted with PPARγ through the activation function-1 domain and ligand-binding domain. FBXO9 decreased the protein stability of PPARγ through induction of ubiquitination. We found that the F-box motif of FBXO9 was required for its ubiquitination function. The activity of PPARγ was significantly decreased by FBXO9 overexpression. Furthermore, FBXO9 overexpression in 3T3-L1 adipocytes resulted in decreased levels of endogenous PPARγ and suppression of adipogenesis. These results suggest that FBXO9 is an important enzyme that regulates the stability and activity of PPARγ through ubiquitination.

LB-S&T-08 EFFICACY AND SAFETY OF A NEW HERBAL FORMULA (KBMSI-2) IN THE TREATMENT OF ERECTILE DYSFUNCTION: A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED CLINICAL TRIAL

castration-resistant prostate cancer (CRPC) emphasizes the continued importance of the AR signaling axis in the disease. Despite the use of this new generation of therapies, some men with CRPC do not respond and resistance to these therapies typically develops for those that do. Mechanisms attributed to the emergence of this non-responsive CRPC include the expression of ligand binding domain (LBD)-null constitutively active splice variants of the AR (AR-SV), hyperactive AR, and others. These AR-SV expressing CRPCs require alternate approaches to inhibit both the full length and AR-SVs. The objective of this work is to develop selective androgen receptor degraders (SARDs) that degrade all forms of the AR and provide advanced treatment options to men with CRPC. METHODS: AR ligand binding, transactivation, fluorescence polarization, and Western blot assays were performed to screen novel SARDs. Prostate cancer cell line gene expression, proliferation, cell line and patient-derived xenografts (PDX) were performed to evaluate the efficacy of SARDs. Molecular mechanistic studies were performed to understand the mechanism of action. RESULTS: A novel series of highly potent SARDs with unique pharmacology have been discovered. These compounds selectively bind to the LBD, inhibit, and degrade the AR at nanomolar concentrations. The SARDs degrade AR-V7 and other variants that lack the LBD and inhibit the proliferation of ARand AR-SVdependent PCa cells with potencies better than that of comparators. SARDs robustly inhibit the growth of the LNCaP androgen-dependent prostate cancer (PCa) xenograft, the 22RV1 CRPC xenograft, and ARand AR-SVpositive CRPC patient-derived xenografts (PDX). 22RV1 and PDX Pr-3001 are dependent on AR-SV expression and the observed inhibition of growth is indicative of the SARDs0 ability to inhibit the AR-SV activity in vivo. Quenching of the steady-state fluorescence spectrum supports an interaction between the SARDs and the AR activation function domain (AF-1), making these molecules first-in-class dual-interacting AR antagonists and degraders. Ongoing studies are revealing the mechanism of action for these SARDs. CONCLUSIONS: Novel highly potent SARDs that interact with both the AF-1 and LBD of the AR were discovered and characterized as a potential next-generation treatment option for CRPC.

Allosteric Pathways in the Multi-Domain Thyroid Hormone Receptor

Allostery is an important regulatory mechanism in biology. Such mechanisms fine-tune the response of the nuclear hormone receptors (NRs) to extracellular stimuli. The structural topology of NRs consists of a mostly unstructured N-terminal (NTD)/activation function-1 domain (AF-1), a DNA binding domain (DBD) that recognizes and binds specific DNA sequences and a C-terminal ligand binding domain (LBD). Besides DNA, these domains also interact with specific factors that are subunits of the basal transcriptional machinery. Through multiple sources of data, we have observed that NRs are not simply docking sites for cellular molecules. Instead, we have characterized multiple allosteric pathways that link ligand, DNA and coactivator-binding sites. Here, we will present data from biophysical and cell-based studies on the thyroid-hormone receptor (TR). TR is a type II NR and functions as a heterodimer with the retinoid X receptor (RXR). Thyroid hormone (T3) is essential for development, differentiation, and metabolic balance in higher organisms and humans. Improper functioning of TR has been associated with pathophysiological conditions such as prostate cancer, colon cancer and hypothyroidism. Yet, the mechanism of transcriptional regulation by TR is not completely understood. Here, we present data that describes specific allosteric communication between the multiple TR domains. Specifically, we show how the AF1 influences DNA binding by the DBD and that the DNA-DBD interactions influence AF1 structural conformations. Furthermore, we show that there are direct interactions between the DBD and LBD and these are dependent on the DNA-bound state of the DBD. Our study on TR allostery also links the DNA and coactivator. Finally, propose a novel ‘frustrated-fit’ model to explain the negative cooperativity between TR and RXR ligands in the TR:RXR heterodimeric complex. For our studies, we utilize a combination of crystallography, experimental and theoretical biophysics and cell-based reporter gene assays.

Functional characterization of the hGRαT556I causing Chrousos syndrome.

Chrousos syndrome is a rare pathologic condition characterized by generalized, partial resistance of target tissues to glucocorticoids and caused by inactivating mutations of the human glucocorticoid receptor (hGR) gene. A novel case of Chrousos syndrome has been reported in a patient with adrenal incidentaloma, who harboured a heterozygous point mutation in the hGR gene, which resulted in threonine (T) to isoleucine (I) substitution at amino acid position 556 in the ligand-binding domain of the receptor. To delineate the molecular mechanisms through which the mutant receptor hGRαT556I causes Chrousos syndrome. Compared with the wild-type receptor, the mutant receptor hGRαT556I demonstrated 50% reduction in its ability to transactivate glucocorticoid-responsive genes and in the affinity for the ligand, 30% increase in the ability to transrepress the nuclear factor-κB-target genes and a 3,4-fold delay in the cytoplasmic-to-nuclear translocation. The mutant receptor hGRαT556I did not exert a dominant negative effect upon the hGRα-mediated transcriptional activity; it preserved its ability to bind to DNA and interacted with the glucocorticoid receptor-interacting protein 1 coactivator mostly through its activation function-1 domain. Structural biology studies revealed that the T556I mutation caused disruption of the hydrogen bond formed by the T556 with the =O group of P637 backbone, which resulted in a significant relocation of the P637-bearing loop. This conformational alteration affected the local 3D arrangement of the receptor and hence the electrostatic surface of the region. The hGRαT556I causes Chrousos syndrome by impairing multiple steps of the glucocorticoid signal transduction pathway.

Automatic generation of LUTs for Hardware Neural Networks

One very important component of Hardware implementations of Artificial Neural Networks (HNN) is the activation function. A common solution for activation function design in HNN is the Look-Up Table (LUT) representation that has acceptable area constraints and usually achieves acceptable execution time. Tools and techniques for automatic generation of LUTs have been developed but have some problems as high complexity, high dependency of external tools or languages and the optimization techniques that are not efficient enough for critical applications. In order to solve some of the presented problems this paper describes a method for automatic LUT generation that overcomes some deficiencies of existing methods using a simple approach. This method was evaluated initially considering the xor function and the sine function for evaluation purposes. After these initial tests, two other ANN based robotic systems were tested. These robotic systems will be implemented in hardware in the near future and they demonstrate a different behavior using different domains of the same activation function. Proposed method achieved good results that are directly related to how neural networks define the activation function domain.

Methyltransferase-like 17 physically and functionally interacts with estrogen receptors.

Estrogen exerts its physiological and pathological functions through two estrogen receptors (ERs), ERα and ERβ, which act as transcription factors. Coregulators, including coactivators and corepressors, have been shown to be crucial for regulation of ER transcriptional activity. Although many coregulators have been identified to regulate activities of ERs, novel coregulators are still needed to be investigated. Here, we show that human methyltransferase-like 17 (METTL17), whose function is unknown, physically interacts with ERα and ERβ, and functionally acts as a coactivator for ERs. METTL17 interacts with ER in vitro and in yeast and mammalian cells. Activation function-1 (AF1) and AF2 domains of ERs are responsible for the interaction between METTL17 and ERs. Knockdown of METTL17 reduces transcriptional activities of ERα and ERβ in breast cancer cells, whereas METTL17 overexpression increases ERα and ERβ transcriptional activities. Inhibition of METTL17 expression decreases mRNA and protein levels of ER target genes, including PR, cathepsin D, and pS2. Moreover, METTL17 knockdown reduces breast cancer cell growth. These results indicate that METTL17 is a novel coactivator of ERs and may play a role in breast tumorigenesis.

Liver X Receptor (LXR) in yellow catfish Pelteobagrus fulvidraco: Molecular characterization, mRNA tissue expression and transcriptional regulation by insulin in vivo and in vitro.

Liver X Receptor (LXR) plays a pivotal role in metabolic regulation in mammals, but little is known about its function in fish. In this study, two lxra isoforms, namely lxra1 and lxra2, were isolated. Their molecular characterization, tissues distribution and transcriptional regulation by insulin in vivo and in vitro were determined. lxrα1 and lxrα2 cDNA covered 2775bp and 3093bp, encoding 446 and 515 amino acid residues, respectively. The protein sequence of yellow catfish lxra included characteristic feature of mammalian lxrs, including the DNA binding (DBD) (containing P-box), ligand binding (LBD) and activation function-2 (AF-2) domains, D-box, and D (hinge) region. Phylogenetic analysis revealed that yellow catfish lxra grouped with lxra of zebrafish but was distant from those of medaka and stickleback. lxrβ clades was absent in teleosts in phylogenetic tree, proving gene loss of lxrβ in teleosts during evolution. The two lxra isoforms (lxra1 and lxra2) mRNAs were ubiquitously expressed in 11 tested tissues. Compared to lxra2, lxra1 mRNA expression was predominant in all tested tissues. The expression of lxrα1 was the highest in testis, then in liver, and the lowest in other tissues. lxrα2 expression was the highest in liver, then in testis, and the lowest in ovary. Insulin significantly stimulated the mRNA expression of lxra1 in vitro and in vivo, while the expression of lxra2 remained unchanged after insulin treatment. The present study serves to increase our understanding into the function of lxra in fish.

Peptidylprolyl Isomerase Pin1 Directly Enhances the DNA Binding Functions of Estrogen Receptor α

The transcriptional activity of estrogen receptor α (ERα), the key driver of breast cancer proliferation, is enhanced by multiple cellular interactions, including phosphorylation-dependent interaction with Pin1, a proline isomerase, which mediates cis-trans isomerization of the N-terminal Ser(P)(118)-Pro(119) in the intrinsically disordered AF1 (activation function 1) domain of ERα. Because both ERα and Pin1 have multiple cellular partners, it is unclear how Pin1 assists in the regulation of ERα transactivation mechanisms and whether the functional effects of Pin1 on ERα signaling are direct or indirect. Here, we tested the specific action of Pin1 on an essential step in ERα transactivation, binding to specific DNA sites. DNA binding analysis demonstrates that stable overexpression of Pin1 increases endogenous ERα DNA binding activity when activated by estrogen but not by tamoxifen or EGF. Increased DNA binding affinity is a direct effect of Pin1 on ERα because it is observed in solution-based assays with purified components. Further, our data indicate that isomerization is required for Pin1-modulation of ERα-DNA interactions. In an unbiased in vitro DNA binding microarray with hundreds of thousands of permutations of ERα-binding elements, Pin1 selectively enhances the binding affinity of ERα to consensus DNA elements. These studies reveal that Pin1 isomerization of phosphorylated ERα can directly regulate the function of the adjacent DNA binding domain, and this interaction is further modulated by ligand binding in the ligand-binding domain, providing evidence for Pin1-dependent allosteric regulation of ERα function.

Abstract PD6-4: ESR1 gene fusions implicated in endocrine therapy resistance of ER+ breast cancer

Abstract Background. We recently identified an in-frame ESR1 translocation in a tumor and PDX pair from a patient with endocrine therapy resistant advanced disease. The fusion gene preserved the N-terminal 365aa of ESR1 containing the intact activation function 1 (AF1), DNA-binding domain (DBD) and hinge, followed by the C-terminal transactivation domain of YAP1. The ESR1-365>YAP1 fusion drove estrogen-independent and anti-estrogen-resistant tumor cell growth (Cell Reports 4:1116, 2013). We therefore sought to further investigate the role of ESR1 fusion genes in breast cancer. Methods. 711 cases from TCGA, 82 ER+ cases from neoadjuvant endocrine therapy trials and 25 ER+ systemic relapse samples were screened for fusion genes using RNAseq. Fusion genes involving ESR1 were subsequently assayed for estrogen response element (ERE)-mediated transcriptional activity and ability to drive estradiol independent breast cancer cell growth. Results. Multiple ESR1 fusions were identified in mostly luminal B cancers. ESR1 fusions resulted most frequently from rearrangements involving another gene on Chromosome (Chr) 6 but also fusions with genes on other chromosomes. The fusions involving partners on Chr6 that retained an intact N terminal AF1 and DNA binding domain (DBD) of ESR1 (≥1-253aa) included an Out-of-Frame (OF) fusion event with CCDC170 (a gene immediately centromeric to ESR1), POLH (in-frame or IF), AKAP12 (IF), PCMT1 (OF), SYNE1 (OF) and GPR126 (OF). Also identified were inter-chromosomal translocations involving Chr12p-NOP2 (IF) and ChrX-PCDH11X (IF) and Chr7q-AKR1D1 (OF). The ESR1 fusions with transcriptional potential (because of a retained DNA binding domain) included variable 5’ ESR1 exons that preserved 253aa, 365aa, 412aa or 458aa of ESR1 sequence: in all cases disrupting ligand binding though loss of C terminal sequence. The effect of the IF and OF 3’ sequences on the transcriptional activity of the relevant ESR1 fragment was highly variable. The most transcriptionally active fusion (more active than the relevant ESR1 fragment alone) was the ESR1-365>YAP1 as well as ESR1-365>PCDH11X and ESR1-253>CCDC170. This is remarkable since PCDH11X is not considered a transcription factor and the CCDC170 sequence was OF – in both cases suggesting the activity of the fusion was due to "neomorphic" properties. Two fusions, NOP2 and POLH were inactive in the ERE reporter assay, yet both stimulated estradiol independent growth. In these cases we suggest that the ESR1 locus can function as a "promoter trap" which allows the identification of genes with previously unknown functions in endocrine therapy resistance. Conclusions: In luminal-type breast cancer the ESR1 gene was fused to multiple 3’ partners with remarkably heterogeneous functions. The two most transcriptionally active, ESR1-365>YAP1 and ESR1-365>PCDH11X, were both identified in endocrine therapy refractory advanced disease – suggesting a role in fatal disease progression. Citation Format: Jieya Shao, Jin Zhang, Robert J Crowder, Rodrigo Goncalves, Chanpheng Phommaly, Breast AWG and Network, Charles M Perou, Christopher A Maher, E Aubrey Thompson, Matthew J Ellis. ESR1 gene fusions implicated in endocrine therapy resistance of ER+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-4.

Cloning of multiple ERα mRNA variants in killifish (Fundulus heteroclitus), and differential expression by tissue type, stage of reproduction, and estrogen exposure in fish from polluted and unpolluted environments

To test the hypothesis that alternative splicing could be an adaptive mechanism for populations subject to multi-generational estrogenic exposures, we compared estrogen receptor alpha (ERα) splicing variants in two populations of killifish (Fundulus heteroclitus): one resident in an estrogenic polluted environment (New Bedford Harbor, NBH, MA, USA) and one from a relatively uncontaminated reference site (Scorton Creek, SC, MA, USA). In total we identified 19 ERα variants, each with deletions of one or more coding exons. Four of the variants with potential functional relevance were analyzed by qPCR to test for population differences in expression by tissue type, site, sex, seasonal reproductive status and estrogen treatment. Significantly, a 5′-truncated short form variant (ERαS) was highly expressed in liver and ovary, and was associated with seasonal reproductive activity in SC but not NBH fish. Both ERαS and the full-length long variant (ERαL) were estrogen-inducible (ERαS>ERαL) but the induction response was lower in NBH than in SC fish. In contrast, NBH killifish were hyper-responsive to estrogen as measured by expression of two other estrogen responsive genes: vitellogenin (Vtg) and aromatase B (AroB). Most strikingly, two ERα deletion variants (Δ6 and Δ6–8), lacking ligand binding and activation function domains, were identified in a subset of NBH fish, where they were associated with reduced responsiveness to estrogen treatment. Together, these results support the hypothesis that alternative splicing of the esr1 gene of killifish could be an autoregulatory mechanism by which estrogen modulates the differential expression of ERα, and suggests a novel and adaptive mechanistic response to xenoestrogenic exposure.

A TFG–TEC nuclear localization mutant forms complexes with the wild-type TFG–TEC oncoprotein and suppresses its activity

Human EMCs (extraskeletal myxoid chondrosarcomas) are soft tissue tumours characterized by specific chromosomal abnormalities. Recently, a proportion of EMCs were found to harbour a characteristic translocation, t(3;9)(q11-12;q22), involving the TFG (TRK-fused gene) at 3q11-12 and the TEC (translocated in extraskeletal chondrosarcoma) gene at 9q22. The present study used both in vitro and in vivo systems to show that the TFG-TEC protein self-associates, and that this is dependent upon the CC (coiled-coil) domain (amino acids 97-124), the AF1 (activation function 1) domain (amino acids 275-562) and the DBD (DNA-binding domain) (amino acids 563-655). The TFG-TEC protein also associated with a mutant NLS-TFG-TEC (AAAA) protein, which harbours mutations in the NLS (nuclear localization signal). Subcellular localization assays showed that the NLS mutant TFG-TEC (AAAA) protein interfered with the nuclear localization of wild-type TFG-TEC. Most importantly, the mutant protein inhibited TFG-TEC-mediated transcriptional activation in vivo. Thus mutations in the TFG-TEC NLS yield a dominant-negative protein. These results show that the biological functions of the TFG-TEC oncogene can be modulated by a dominant-negative mutant.

Ligand Binding Reduces SUMOylation of the Peroxisome Proliferator-activated Receptor γ (PPARγ) Activation Function 1 (AF1) Domain

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear receptor regulating adipogenesis, glucose homeostasis and inflammatory responses. The activity of PPARγ is controlled by post-translational modifications including SUMOylation and phosphorylation that affects its biological and molecular functions. Several important aspects of PPARγ SUMOylation including SUMO isoform-specificity and the impact of ligand binding on SUMOylation remain unresolved or contradictory. Here, we present a comprehensive study of PPARγ1 SUMOylation. We show that PPARγ1 can be modified by SUMO1 and SUMO2. Mutational analyses revealed that SUMOylation occurs exclusively within the N-terminal activation function 1 (AF1) domain predominantly at lysines 33 and 77. Ligand binding to the C-terminal ligand-binding domain (LBD) of PPARγ1 reduces SUMOylation of lysine 33 but not of lysine 77. SUMOylation of lysine 33 and lysine 77 represses basal and ligand-induced activation by PPARγ1. We further show that lysine 365 within the LBD is not a target for SUMOylation as suggested in a previous report, but it is essential for full LBD activity. Our results suggest that PPARγ ligands negatively affect SUMOylation by interdomain communication between the C-terminal LBD and the N-terminal AF1 domain. The ability of the LBD to regulate the AF1 domain may have important implications for the evaluation and mechanism of action of therapeutic ligands that bind PPARγ.

Transactivating function (AF) 2–mediated AF-1 activity of estrogen receptor α is crucial to maintain male reproductive tract function

Estrogen receptor alpha (ERα) is a ligand-dependent transcription factor containing two transcriptional activation function (AF) domains. AF-1 is in the N terminus of the receptor protein, and AF-2 activity is dependent on helix 12 of the C-terminal ligand-binding domain. We recently showed that two point mutations converting leucines 543 and 544 to alanines in helix 12 (AF2ER) minimized estrogen-dependent AF-2 transcriptional activation. A characteristic feature of AF2ER is that the estrogen antagonists ICI182780 and tamoxifen (TAM) act as agonists through intact AF-1, but not through mutated AF-2. Here we report the reproductive phenotype of male AF2ER knock-in (AF2ERKI) mice and demonstrate the involvement of ERα in male fertility. The AF2ERKI male homozygotes are infertile because of seminiferous tubular dysmorphogenesis in the testis, similar to ERα KO males. Sperm counts and motility did not differ at age 6 wk in AF2ERKI and WT mice, but a significant testis defect was observed in adult AF2ERKI male mice. The expression of efferent ductal genes involved in fluid reabsorption was significantly lower in AF2ERKI males. TAM treatment for 3 wk beginning at age 21 d activated AF-2-mutated ERα (AF2ER) and restored expression of efferent ductule genes. At the same time, the TAM treatment reversed AF2ERKI male infertility compared with the vehicle-treated group. These results indicate that the ERα AF-2 mutation results in male infertility, suggesting that the AF-1 is regulated in an AF-2-dependent manner in the male reproductive tract. Activation of ERα AF-1 is capable of rescuing AF2ERKI male infertility.

Binding of the N-terminal Region of Coactivator TIF2 to the Intrinsically Disordered AF1 Domain of the Glucocorticoid Receptor Is Accompanied by Conformational Reorganizations

Control of gene transcription by glucocorticoid receptors (GRs) is important for many physiological processes. Like other steroid hormone receptors, the regulation of target genes by GR is mediated by two transactivation domains: activation function 1 (AF1) in the N-terminal domain and AF2 in the C-terminal ligand-binding domain (LBD). Full receptor activity requires both AF1 and -2 plus assorted coregulatory proteins. Crystal structures of the ligand-bound LBD have provided insight regarding how AF2 interacts with specific coactivators. However, despite its being the major activation domain of GRs, knowledge of AF1 structure/function has languished. This is mainly because of the highly disorganized structure of the GR N-terminal domain. This lack of AF1 structure is shared by all members of the steroid/nuclear receptor superfamily for which it has been examined and AF1 is thought to allow productive interactions with assorted cofactors via protein-induced changes in secondary/tertiary structures. To date, there are no reports of a classical coactivator altering the secondary/tertiary structure of the GR AF1 domain. Earlier, we reported an N-terminal fragment of the p160 coactivator TIF2, called TIF2.0, that binds the GR N-terminal domain and alters GR transcriptional activity. We therefore proposed that TIF2.0 binding to AF1 changes both its conformation and transcriptional activity. We now report that TIF2.0 interacts with the GR AF1 domain to increase the amount of α-helical structure in the complex. Furthermore, TIF2 coactivator activity is observed in the absence of the GR LBD in a manner that requires the AF1 domain. This contrasts with previous models where TIF2 receptor interaction domains binding to GR LBD somehow alter AF1 conformation. Our results establish for the first time that coactivators can modify the structure of the AF1 domain directly via the binding of a second region of the coactivator and suggest a molecular explanation for how coactivators increase the transcriptional activity of GR-agonist complexes.