Estrogen Response Element Research Articles

The influence of estrogen response element ERα signaling in the control of feeding behaviors in male and female mice

Circulating 17β-estradiol (E2) controls energy homeostasis and feeding behaviors primarily by its nuclear receptor, estrogen receptor (ER) α. As such, it is important to understand the role of ERα signaling in the neuroendocrine control of feeding. Our previous data indicated that the loss of ERα signaling through estrogen response elements (ERE) alters food intake in a female mouse model. Hence, we hypothesize that ERE-dependent ERα is necessary for typical feeding behaviors in mice. To test this hypothesis, we examined feeding behaviors on low-fat diet (LFD) and high-fat diet (HFD) in three mouse strains: total ERα knockout (KO), ERα knockin/knockout (KIKO), which lack a functional DNA-binding domain, and their wild type (WT) C57 littermates comparing intact males and females and ovariectomized females with or without E2 replacement. All feeding behaviors were recorded using the Biological Data Acquisition monitoring system (Research Diets). In intact male mice, KO and KIKO consumed less than WT mice on LFD and HFD, while in intact female mice, KIKO consumed less than WT and KO. These differences were primarily driven by shorter meal duration in the KO and KIKO. In ovariectomized females, E2-treated WT and KIKO consumed more LFD than KO driven in part by an increase in meal frequency and a decrease in meal size. On HFD, WT consumed more than KO with E2, again due to effects on meal size and frequency. Collectively, these suggest that both ERE-dependent and -independent ERα signaling are involved in feeding behaviors in female mice depending on the diet consumed.

Genome-Wide Identification of the MADS-Box Gene Family during Male and Female Flower Development in Chayote (Sechium edule)

The MADS-box gene plays an important role in plant growth and development. As an important vegetable of Cucurbitaceae, chayote has great edible and medicinal value. So far, there is little molecular research on chayote, and there are no reports on the MADS-box transcription factor of chayote. In this study, the MADS-box gene family of chayote was analyzed for the first time, and a total of 70 MADS-box genes were identified, including 14 type I and 56 type II MICK MADS genes. They were randomly distributed on 13 chromosomes except for chromosome 11. The light response element, hormone response element and abiotic stress response element were found in the promoter region of 70 MADS genes, indicating that the MADS gene can regulate the growth and development of chayote, resist abiotic stress, and participate in hormone response; GO and KEGG enrichment analysis also found that SeMADS genes were mainly enriched in biological regulation and signal regulation, which further proved the important role of MADS-box gene in plant growth and development. The results of collinearity showed that segmental duplication was the main driving force of MADS gene expansion in chayote. RNA-seq showed that the expression levels of SeMADS06, SeMADS13, SeMADS26, SeMADS28, SeMADS36 and SeMADS37 gradually increased with the growth of chayote, indicating that these genes may be related to the development of root tubers of 'Tuershao'. The gene expression patterns showed that 12 SeMADS genes were specifically expressed in the male flower in 'Tuershao' and chayote. In addition, SeMADS03 and SeMADS52 may be involved in regulating the maturation of male flowers of 'Tuershao' and chayote. SeMADS21 may be the crucial gene in the development stage of the female flower of 'Tuershao'. This study laid a theoretical foundation for the further study of the function of the MADS gene in chayote in the future.

The androgen-thyroid hormone crosstalk in prostate cancer and the clinical implications.

There is increasing evidence that thyroid hormones (THs) work in an integrative fashion with androgen receptors (ARs) to regulate gonadal differentiation and reproductive function. Studies reveal that THs have interactions with the AR promoter region and increase AR expression. THs also have a role in the regulation of enzymes involved in the biosynthesis of androgens, such as 5α-reductase, which is essential in the conversion of testosterone into its active form, 5α-dihydrotestosterone. Additionally, the presence of androgen response elements in the promoter regions of TH-related genes, such as deiodinases and TH receptor isoforms, has been identified in some vertebrates, indicating a mutual interaction between THs and ARs. Since the androgen signaling pathway, mediated by ARs, plays a key role in the formation and progression of prostate cancer (PCa), the existence of crosstalk between THs and ARs supports the epidemiologic and experimental evidence indicating a relationship between the high incidence of PCa and hyperthyroidism. This article aims to review the role of androgen-TH crosstalk in PCa and its implication in clinical management. As life expectancy is growing these days, it can increase the number of patients with PCa and the critical relevance of the disease. In order to gain better knowledge about PCa and to improve clinical management, it is essential to get better insight into the key factors related to the formation and progression of this cancer.

Comprehensive Analysis of BR Receptor Expression under Hormone Treatment in the Rubber Tree (Hevea brasiliensis Muell. Arg.).

Brassinosteroids (BRs) are important for plant growth and development, with BRI1 and BAK1 kinases playing an important role in BR signal transduction. Latex from rubber trees is crucial for industry, medicine and defense use. Therefore, it is beneficial to characterize and analyze HbBRI1 and HbBAK1 genes to improve the quality of the resources obtained from Hevea brasiliensis (rubber tree). Based on bioinformatics predictions and rubber tree database, five HbBRI1s with four HbBAK1s were identified and named HbBRI1~HbBRL3 and HbBAK1a~HbBAK1d, respectively, which were clustered in two groups. HbBRI1 genes, except for HbBRL3, exclusively contain introns, which is convenient for responding to external factors, whereas HbBAK1b/c/d contain 10 introns and 11 exons, and HbBAK1a contains eight introns. Multiple sequence analysis showed that HbBRI1s include typical domains of the BRI1 kinase, indicating that HbBRI1s belong to BRI1. HbBAK1s that possess LRR and STK_BAK1_like domains illustrate that HbBAK1s belong to the BAK1 kinase. BRI1 and BAK1 play an important role in regulating plant hormone signal transduction. Analysis of the cis-element of all HbBRI1 and HbBAK1 genes identified hormone response, light regulation and abiotic stress elements in the promoters of HbBRI1s and HbBAK1s. The results of tissue expression patterns indicate that HbBRL1/2/3/4 and HbBAK1a/b/c are highly expressed in the flower, especially HbBRL2-1. The expression of HbBRL3 is extremely high in the stem, and the expression of HbBAK1d is extremely high in the root. Expression profiles with different hormones show that HbBRI1 and HbBAK1 genes are extremely induced by different hormone stimulates. These results provide theoretical foundations for further research on the functions of BR receptors, especially in response to hormone signals in the rubber tree.

Stabilization of estrogen receptor α by USP37 contributes to the progression of breast cancer.

Breast cancer is a major cause of cancer-related morbidity and mortality in women. Estrogen receptor-positive breast cancer accounts for roughly 70%-80% of breast tumors, and estrogen receptor alpha (ERα) has been considered as a key driver in promoting breast cancer progression. In the present study, we identified USP37 as a novel modulator in modulating ERα ubiquitination and stability. The expression of USP37 was upregulated in ERα-positive breast cancer and correlated with ERα protein level. High expression of USP37 was associated with unfavorable prognosis. USP37 depletion resulted in significantly decreased ERα protein level, ERα target genes expression as well as the estrogen response element activity in breast cancer cells. Further mechanistic study revealed the interaction between USP37 and ERα: USP37 regulated ERα signaling through modulating protein stability instead of gene expression, in which it stabilized ERα protein via inhibiting the K48-specific polyubiquitination process. Additionally, USP37 depletion led to growth inhibition and cell cycle arrest of ERα-positive breast cancer cells, which could be further rescued by ERα overexpression. Overall, our study proposed a novel post-translational mechanism of ERα in promoting breast cancer progression. Targeting USP37 may be proved to be a promising strategy for patients with ERα-positive breast cancer.

Genome-Wide Analysis, Characterization, Expression and Function of SERK Gene Family in Phyllostachys edulis

Somatic mmbryogenesis receptor-like kinase (SERK) is a kind of protein kinase widely distributed in plants. It plays a vital role in regulating plant immunity and responses to abiotic stress. The SERK gene family has not been systematically studied in moso bamboo (Phyllostachys edulis). In this study, we identified six PeSERK genes and classified them into four groups in moso bamboo. PeSERKs of each group shared a highly similar distribution of conserved domains. Cis-element analysis indicated that many stress and hormone response elements are distributed on the promoters of PeSERKs. Moreover, we analyzed the chromosomal locations and synteny of PeSERKs. A collinear gene pair, PeSERK1 and PeSERK3, shared a high similarity, 93%, and the expression analysis showed similar expression patterns. Compared to PeSERK3, PeSERK1 had a higher expression in all tissues examined and all stages of shoot development. PeSERK3 was expressed mainly in leaf sheaths but with a low expression in other tissues. The expressions of PeSERKs were analyzed in seedlings under abiotic and hormone treatments using qRT-PCR. Except for PeSERK1 and PeSERK3, the expressions of most genes were downregulated or had no big difference at 0 h of drought treatment. Under drought treatment, PeSERK1 and PeSERK3 had a similar expression trend of increasing first and then decreasing. However, the expression level of PeSERK3 was higher than PeSERK1 after 3 h of drought treatment. PeSERK3 might play a more vital role in the drought stress response than PeSERK1. This study provides a theoretical basis for the further study of the SERK response to stress conditions in moso bamboo.

Global analysis of basic leucine zipper transcription factors in trifoliate orange and the function identification of PtbZIP49 in salt tolerance

As one of the most widely distributed and highly conserved transcription factors in eukaryotes, basic leucine zipper proteins (bZIPs) are involved in a variety of biological processes in plants, but they are largely unknown in citrus. In this study, 56 bZIP family members were identified genome-wide from an important citrus rootstock, namely trifoliate orange (Poncirus trifoliata L. Raf.), and these putative bZIPs were named PtbZIP1–PtbZIP56. All PtbZIPs were classified into 13 subgroups by phylogenetic comparison with Arabidopsis thaliana bZIPs (AtbZIPs), and they were randomly distributed on nine known (50 genes) chromosomes and one unknown (6 genes) chromosome. Sequence analysis revealed the detailed characteristics of PtPZIPs, including their amino acid length, isoelectric point (pI), molecular weight (MW), predicted subcellular localization, gene structure, and conserved motifs. Prediction of promoter elements suggested the presence of drought, low-temperature, wound, and defense and stress responsive elements, as well as multiple hormone-responsive cis-acting elements. Spatiotemporal expression analysis showed the transcriptional patterns of PtbZIPs in different tissues and under dehydration, high salt, ABA, and IAA treatments. In addition, 21 PtbZIPs were predicted to have direct or indirect protein–protein interactions. Among these, PtbZIP49 was experimentally proven to interact with PtbZIP1 or PtbZIP11 by using a yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC). Subcellular localization analysis further revealed that PtbZIP1, PtbZIP11, and PtbZIP49 were localized in the nucleus. Moreover, PtbZIP49 was functionally identified as having an important role in salt stress via ectopic expression in A. thaliana and silenced in trifoliate orange using virus-induced gene silencing (VIGS). This study provided comprehensive information on PtbZIP transcription factors in citrus and highlights their potential functions in abiotic stress.

Cooperative action of separate interaction domains promotes high-affinity DNA binding of Arabidopsis thaliana ARF transcription factors

The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative binding to an inverted binding site. Most ARFs further contain a C-terminal PB1 domain that is capable of homotypic interactions and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how these domains contribute to DNA-binding specificity and affinity. So far, ARF-ARF and ARF-DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 auxin-responsive element (AuxRE). We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining binding affinity and kinetics across AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs toward composite DNA response elements is defined by dimerization equilibrium, identifying this as a key element in ARF-mediated transcriptional activity.

Genome-wide identification and expression analysis of the HVA22 gene family in cotton and functional analysis of GhHVA22E1D in drought and salt tolerance.

The HVA22 family of genes, induced by abscisic acid and stress, encodes a class of stress response proteins with a conserved TB2/DP1/HVA22 domain that are unique among eukaryotes. Previous studies have shown that HVA22s play an important role in plant responses to abiotic stresses. In the present study, 34, 32, 16, and 17 HVA22s were identified in G. barbadense, G. hirsutum, G. arboreum, and G. raimondii, respectively. These HVA22 genes were classified into nine subgroups, randomly distributed on the chromosomes. Synteny analysis showed that the amplification of the HVA22s were mainly due to segmental duplication or whole genome replication (WGD). Most HVA22s promoter sequences contain a large number of drought response elements (MYB), defense and stress response elements (TC-rich repeats), and hormone response elements (ABRE, ERE, SARE, etc.), suggesting that HVA22s may respond to adversity stresses. Expression profiling demonstrated that most GhHVA22s showed a constitutive expression pattern in G. hirsutum and could respond to abiotic stresses such as salt, drought, and low temperature. Overexpression of GhHVA22E1D (GH_D07G0564) in Arabidopsis thaliana enhances salt and drought tolerance in Arabidopsis. Virus-induced gene silencing of GhHVA22E1D reduced salt and drought tolerance in cotton. This indicates that GhHVA22E1D plays an active role in the plant response to salt stress and drought stress. GhHVA22E1D may act in plant response to adversity by altering the antioxidant capacity of plants. This study provides valuable information for the functional genomic study of the HVA22 gene family in cotton. It also provides a reference for further elucidation of the functional studies of HVA22 in plant resistance to abiotic stress response.

Identification of AREB/ABF Gene Family Involved in the Response of ABA under Salt and Drought Stresses in Jute (Corchorus olitorius L.).

The abscisic acid (ABA)-responsive element binding protein/ABRE-binding factor (AREB/ABF) subfamily members are essential to ABA signaling pathways and plant adaptation to various environmental stresses. Nevertheless, there are no reports on AREB/ABF in jute (Corchorus L.). Here, eight AREB/ABF genes were identified in the C. olitorius genome and classified into four groups (A-D) based on their phylogenetic relationships. A cis-elements analysis showed that CoABFs were widely involved in hormone response elements, followed by light and stress responses. Furthermore, the ABRE response element was involved in four CoABFs, playing an essential role in the ABA reaction. A genetic evolutionary analysis indicated that clear purification selection affects jute CoABFs and demonstrated that the divergence time was more ancient in cotton than in cacao. A quantitative real-time PCR revealed that the expression levels of CoABFs were upregulated and downregulated under ABA treatment, indicating that CoABF3 and CoABF7 are positively correlated with ABA concentration. Moreover, CoABF3 and CoABF7 were significantly upregulated in response to salt and drought stress, especially with the application of exogenous ABA, which showed higher intensities. These findings provide a complete analysis of the jute AREB/ABF gene family, which could be valuable for creating novel jute germplasms with a high resistance to abiotic stresses.

Abstract PD10-02: PD10-02 Novel ER fusion detection method to gain insight in fusion prevalence and endocrine resistant mechanisms

Abstract Breast cancer is a leading cause of female mortality and despite advancements in diagnostics and personalized therapeutics, metastatic disease largely remains incurable due to drug resistance. The druggable estrogen receptor (ER, ESR1), overexpressed in two-thirds of all breast cancer, evolves in 30% of tumors exposed to endocrine therapy consequently resulting in treatment resistance. A more recently discovered mechanism of ER mediated endocrine resistance is ER fusion proteins. ER fusions, found predominately in metastatic endocrine resistant disease, harbor ESR1 exons 1-6 fused to an in-frame gene partner due to an ESR1 intron 6 translocation break. Our lab has demonstrated that ER fusion proteins, which lack the C-terminal ligand-binding domain (LBD), recapitulate phenotypes of ER proteins harboring endocrine-resistant point mutations occurring in the LBD. Our research goals aim to 1) determine fusion prevalence and emergence, 2) understand fusion mechanisms of resistance and 3) explore alternative treatment options for our patients. The promiscuous nature of fusion partners hinders fusion detection by traditional sequencing and thus our research team has developed a novel ER fusion detection method, EnRich. The EnRich probe set (4,324 probes in total) is composed of two separate probe pools targeting at 2x tiling intronic and exonic ESR1, the upstream promoter region and select oncogenic genes. To optimize the EnRich pipeline, DNA was extracted from a frozen tumor sample and a PDX model harboring ESR1-DAB2 and ESR1-LPP fusions, respectively. ESR1-DAB2 and ESR1-LPP were accurately detected by quantifying discordant paired-end or split reads mapped to chromosome 6. In addition, liquid biopsies from 15 patients with ER positive advanced breast cancer were also assessed through EnRich to uncover unidentified ESR1 structural variants. Two patient samples were detected to harbor ESR1 fusions (ESR1-CCDC170, ESR1-AKAP12 and ESR1-YAP1) and were further validated in corresponding mRNA. These recurrent and novel fusions were supported with more than 10 reads each, indicating that the EnRich pipeline is an effective and accurate sequencing approach to understanding ER fusion prevalence. Our lab, furthermore, has studied ER fusion proteins mechanistically. We have stably overexpressed ER fusions in transgenic breast cancer cell lines engineered with shRNA targeting the endogenous wildtype ER (ESR1-WT). In this ESR1-WT depleted cellular context, we found that ER fusions (notably ESR1-SOX9 and ESR1-YAP1) demonstrate ER hyperactivation through an estrogen response element (ERE) assay compared to ESR1-WT and a truncated exon 1-6 ESR1 (ESR1∆CTD). Importantly, fusion ERE activity was robust in the absence of the ER ligand, estradiol, as well as in the presence of endocrine therapies, implying ER fusion proteins function in a ligand independent, endocrine resistant mechanism. ER fusion positive cell lines were also enriched in oncogenic phenotypes such as enhanced 3D growth, cell survival via colony formation, and migration in a wound scratch assay. These enhanced metastatic potentials of the ER fusions were observed in both invasive ductal and lobular carcinoma cell lines, albeit at varying magnitudes depending on the 3’ fusion partner and phenotype being assessed. Although the ER fusions harbored unique characteristics due to the C-terminal partner, transcriptomic profiling revealed that enhanced EMT and KRAS signaling signatures were shared among all fusions when compared to the ESR1-WT and ESR1∆CTD cell lines, which may serve as future exploitable drug targets. Comprehensive detection and functional evaluation of ER fusion proteins will provide clinicians and patients with better understanding of tumor endocrine-resistant prevalence and discovery of more effective treatment options. Citation Format: Megan E. Yates, Tiantong Liu, Jagmohan Hooda, Sichun Yang, Riyue Bao, Jennifer M. Atkinson, ADRIAN V. LEE, Steffi Oesterreich. PD10-02 Novel ER fusion detection method to gain insight in fusion prevalence and endocrine resistant mechanisms [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-02.

Genomic regulation of Krüppel-like-factor family members by corticosteroid receptors in the rat brain

Hippocampal mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) mediate glucocorticoid hormone (GC) action in the hippocampus. These receptors bind to glucocorticoid responsive elements (GREs) within target genes, eliciting transcriptional effects in response to stress and circadian variation. Until recently, little was known about the genome-wide targets of hippocampal MRs and GRs under physiological conditions. Following on from our genome-wide MR and GR ChIP-seq and Ribo-Zero RNA-seq studies on rat hippocampus, we investigated the Krüppel-like factors (KLFs) as targets of MRs and GRs throughout the brain under circadian variation and after acute stress. In particular, Klf2, Klf9 and Klf15 are known to be stress and/or GC responsive and play a role in neurobiological processes including synaptic plasticity and neuronal differentiation. We found increased binding of MR and GR to GREs within Klf2, Klf9 and Klf15 in the hippocampus, amygdala, prefrontal cortex, and neocortex after acute stress and resulting from circadian variation, which was accompanied by upregulation of corresponding hnRNA and mRNA levels. Adrenalectomy abolished transcriptional upregulation of specific Klf genes. These results show that MRs and GRs regulate Klf gene expression throughout the brain following exposure to acute stress or in response to circadian variation, likely alongside other transcription factors.

Extensive expertise in endocrinology: glucose-dependent insulinotropic peptide-dependent Cushing's syndrome.

Thirty years ago, we identified that cortisol secretion in some patients with unilateral adenoma or primary bilateral macronodular adrenal hyperplasia (PBMAH) was stimulated by food intake; this was secondary to the abnormal adrenocortical responsiveness to physiological post-prandial increase in glucose-dependent insulinotropic peptide (GIP). This resulted from the ectopic expression of non-mutated GIP receptor in the pathological adrenal tissues of those patients. Although ectopic GIP receptor (GIPR) was confirmed in a relatively limited number of cases to date, its elucidation leads to the identification of a wide diversity of aberrant G-protein-coupled receptors regulating steroidogenesis and cell proliferation in a high proportion of patients with PBMAH or cortisol-secreting adenomas. In addition, ectopic GIPR was identified in other endocrine tumors including somatotroph pituitary tumors with paradoxical growth hormone response to oral glucose, medullary thyroid carcinomas, and other neuroendocrine tumors. The first molecular pathogenic mechanism responsible for ectopic GIPR expression was elucidated in unilateral GIP-dependent adenomas in which somatic duplication and rearrangements in chromosome region 19q13.32 containing the GIPR locus lead to increased expression of GIPR which was enhanced by the activity of a glucocorticoid response element. Recently, germline lysine demythylase 1A (KDMIA) mutations combined with somatic chromosome 1p deletions were found to be specifically responsible for ectopic GIPR in sporadic or familial GIP-dependent PBMAH and can be associated with adrenal myelolipoma, monoclonal gammopathy of unknown significance (MGUS), or multiple myeloma. Screening for ectopic GIPR should be conducted in all patients with PBMAH; genetic studies to identify KDM1A mutations should be offered to such patients in order to detect affected members and provide early detection of PBMAH and other potential associated neoplasias. The elucidation of GIP-dependent Cushing's syndrome (CS) illustrates that careful bedside phenotyping of rare conditions can lead to identification of genetically determined diseases requiring personalized approaches to investigation and therapy.

Identification and in vitro enzymatic activity analysis of the AOP2 gene family associated with glucosinolate biosynthesis in Tumorous stem mustard (Brassica juncea var. tumida).

The major enzyme encoded by the glucosinolate biosynthetic gene AOP2 is involved in catalyzing the conversion of glucoiberin (GIB) into sinigrin (SIN) in Brassicaceae crops. The AOP2 proteins have previously been identified in several Brassicaceae species, but not in Tumorous stem mustard. As per this research, the five identified members of the AOP2 family from the whole genome of Brassica juncea named BjuAOP2.1-BjuAOP2.5 were found to be evenly distributed on five chromosomes. The subcellular localization results implied that BjuAOP2 proteins were mainly concentrated in the cytoplasm. Phylogenetic analysis of the AOP2 proteins from the sequenced Brassicaceae species in BRAD showed that BjuAOP2 genes were more closely linked to Brassica carinata and Brassica rapa than Arabidopsis. In comparison with other Brassicaceae plants, the BjuAOP2 members were conserved in terms of gene structures, protein sequences, and motifs. The light response and hormone response elements were included in the BjuAOP2 genes' cis-regulatory elements. The expression pattern of BjuAOP2 genes was influenced by the different stages of development and the type of tissue being examined. The BjuAOP2 proteins were used to perform the heterologous expression experiment. The results showed that all the five BjuAOP2 proteins can catalyze the conversion of GIB to SIN with different catalytic activity. These results provide the basis for further investigation of the functional study of BjuAOP2 in Tumorous stem mustard glucosinolate biosynthesis.

A possible role of the ecdysone receptor in modulating gonad-inhibiting hormone gene expression in the black tiger prawn, Penaeus monodon

Gonad-inhibiting hormone (GIH) is a neuronal eyestalk peptide that inhibits ovarian maturation in crustaceans. In this study, the aim was to investigate the transcriptional regulation of the GIH gene in the black tiger prawn (Penaeus monodon). An analysis of a 1158 bp DNA fragment 5′ upstream of the GIH gene revealed a putative half-site of the hormone response element (HRE) at −561 to −566 relative to the predicted transcription start site with an upstream imperfect palindromic sequence separated by one nucleotide. Deletion of the HRE from the GIH 5′ fragment increased the promoter activity in Sf9 cells, whereas the addition of 20-hydroxyecdysone (20E) reduced the promoter activity by approximately 60–70%. A candidate HRE-binding nuclear receptor, an ecdysone receptor (EcR) that serves to bind 20E in crustaceans, was therefore functionally characterized. EcR was expressed ubiquitously in female prawns, with the highest expression levels occurring in eyestalks, suggesting that EcR possibly stimulates vitellogenesis by binding to the HRE in the GIH 5′ regulatory sequence, thereby inhibiting GIH expression. This was verified by specific binding between a recombinant EcR-GST fusion protein and the HRE in the GIH 5′ DNA fragment. In addition, in vivo knockdown of EcR resulted in a 3-fold elevation of GIH expression levels in the eyestalks of adult female prawns. Taken together, the results obtained in this study indicate that EcR acts as an inhibitory factor in the regulation of the GIH gene.

Estrogen receptor beta in the central amygdala regulates the deleterious behavioral and neuronal consequences of repeated social stress in female rats

While over 95% of the population has reported experiencing extreme stress or trauma, females of reproductive age develop stress-induced neuropsychiatric disorders at twice the rate of males. This suggests that ovarian hormones may facilitate neural processes that increase stress susceptibility and underlie the heightened rates of these disorders, like depression and anxiety, that result from stress exposure in females. However, there is contradicting evidence in the literature regarding estrogen's role in stress-related behavioral outcomes. Estrogen signaling through estrogen receptor beta (ERβ) has been traditionally thought of as anxiolytic, but recent studies suggest estrogen exhibits distinct effects in the context of stress. Furthermore, ERβ is found abundantly in many stress-sensitive brain loci, including the central amygdala (CeA), in which transcription of the vital stress hormone, corticotropin releasing factor (CRF), can be regulated by an estrogen response element. Therefore, these experiments sought to identify the role of CeA ERβ activity during stress on behavioral outcomes in naturally cycling, adult, female Sprague-Dawley rats. Rats were exposed to an ethological model of vicarious social stress, witness stress (WS), in which they experienced the sensory and psychological aspects of an aggressive social defeat encounter between two males. Following WS, rats exhibited stress-induced anxiety-like behaviors in the marble burying taskand brain analysis revealed increased ERβ and CRF specifically within the CeA following exposure to stress cues. Subsequent experiments were designed to target this receptor in the CeA using microinjections of the ERβ antagonist, PHTPP, prior to each stress session. During WS, estrogen signaling through ERβ was responsible for the behavioral sensitization to repeated social stress. Sucrose preference, acoustic startle, and marble burying tasks determined that blocking ERβ in the CeA during WS prevented the development of depressive-, anxiety-like, and hypervigilant behaviors. Additionally, brain analysis revealed a long-term decrease of intra-CeA CRF expression in PHTPP-treated rats. These experiments indicate that ERβ signaling in the CeA, likely through its effects on CRF, contributes to the development of negative valence behaviors that result from exposure to repeated social stress in female rats.

GmERF54, an ERF Transcription Factor, Negatively Regulates the Resistance of Soybean to the Common Cutworm (Spodoptera litura Fabricius)

Soybean is attacked by various herbivorous insect pest species during the whole course of its life cycle in the field. It is important for soybean production to improve insect resistance by identifying and utilizing soybean endogenous insect-resistant genes. The ethylene-responsive transcription factor (ERF) plays a significant role in plant biotic and abiotic stresses; however, few studies focus on its role in insect resistance in soybean. Here, based on our previous common cutworm (CCW)-induced soybean transcriptome data, a soybean ERF gene GmERF54 was cloned, which responded to CCW feeding. Transcriptional analysis revealed that GmERF54 was ubiquitous in all soybean tissues and was expressed differently in insect-resistant and insect-susceptible soybean cultivars. RNA interference of GmERF54 increased the resistance to CCW, while the overexpression of GmERF54 decreased the resistance to CCW in transgenic soybean hairy roots compared with their controls. GmERF54 was localized to the nucleus, had transcriptional activation activity, and interacted with AP2/ERF GmPLT2. Several putative hormone response elements were predicted in the promoter sequence of GmERF54. Four putative elements were only found in the GmERF54 promoter sequence of insect-resistant cultivar Wanxianbaidongdou (WX), but not in the insect-susceptible cultivar Nannong 99-10 (99-10). GmERF54 promoter sequences of WX and 99-10 were cloned into the pCAMBIA1381z vector containing the β-glucuronidase (GUS) gene to generate GmERF54WX:GUS and GmERF5499-10:GUS recombinant vectors, respectively. GUS staining of soybean hairy roots containing GmERF54WX:GUS and GmERF5499-10:GUS showed that GmERF54 was induced by CCW attack and both MeJA (methyl jasmonate) and IAA (indole-3-acetic acid) treatments. Alleles in insect-resistant and insect-sensitive cultivars responded to these inductions differently. Overall, our results reveal that GmERF54 may be involved in the regulation of soybean resistance to CCW.

Genome-Wide Identification and Abiotic Stress Response Analysis of PP2C Gene Family in Woodland and Pineapple Strawberries

Protein phosphatase 2C (PP2C) is a negative regulator of serine/threonine residue protein phosphatase and plays an important role in abscisic acid (ABA) and abiotic-stress-mediated signaling pathways in plants. The genome complexity of woodland strawberry and pineapple strawberry is different due to the difference in chromosome ploidy. This study conducted a genome-wide investigation of the FvPP2C (Fragaria vesca) and FaPP2C (Fragaria ananassa) gene family. Fifty-six FvPP2C genes and 228 FaPP2C genes were identified from the woodland strawberry and pineapple strawberry genomes, respectively. FvPP2Cs were distributed on seven chromosomes, and FaPP2Cs were distributed on 28 chromosomes. The size of the FaPP2C gene family was significantly different from that of the FvPP2C gene family, but both FaPP2Cs and FvPP2Cs were localized in the nucleus, cytoplasm, and chloroplast. Phylogenetic analysis revealed that 56 FvPP2Cs and 228 FaPP2Cs could be divided into 11 subfamilies. Collinearity analysis showed that both FvPP2Cs and FaPP2Cs had fragment duplication, and the whole genome duplication was the main cause of PP2C gene abundance in pineapple strawberry. FvPP2Cs mainly underwent purification selection, and there were both purification selection and positive selection effects in the evolution of FaPP2Cs. Cis-acting element analysis found that the PP2C family genes of woodland and pineapple strawberries mainly contained light responsive elements, hormone responsive elements, defense and stress responsive elements, and growth and development-related elements. The results of quantitative real-time PCR (qRT-PCR) showed that the FvPP2C genes showed different expression patterns under ABA, salt, and drought treatment. The expression level of FvPP2C18 was upregulated after stress treatment, which may play a positive regulatory role in ABA signaling and abiotic stress response mechanisms. This study lays a foundation for further investigation on the function of the PP2C gene family.

Genome-Wide Identification, Classification, and Expression Analyses of the CsDGAT Gene Family in Cannabis sativa L. and Their Response to Cold Treatment.

Hempseed is a nutrient-rich natural resource, and high levels of hempseed oil accumulate within hemp seeds, consisting primarily of different triglycerides. Members of the diacylglycerol acyltransferase (DGAT) enzyme family play critical roles in catalyzing triacylglycerol biosynthesis in plants, often governing the rate-limiting step in this process. As such, this study was designed to characterize the Cannabis sativa DGAT (CsDGAT) gene family in detail. Genomic analyses of the C. sativa revealed 10 candidate DGAT genes that were classified into four families (DGAT1, DGAT2, DGAT3, WS/DGAT) based on the features of different isoforms. Members of the CsDGAT family were found to be associated with large numbers of cis-acting promoter elements, including plant response elements, plant hormone response elements, light response elements, and stress response elements, suggesting roles for these genes in key processes such as development, environmental adaptation, and abiotic stress responses. Profiling of these genes in various tissues and varieties revealed varying spatial patterns of CsDGAT expression dynamics and differences in expression among C. sativa varieties, suggesting that the members of this gene family likely play distinct functional regulatory functions CsDGAT genes were upregulated in response to cold stress, and significant differences in the mode of regulation were observed when comparing roots and leaves, indicating that CsDGAT genes may play positive roles as regulators of cold responses in hemp while also playing distinct roles in shaping the responses of different parts of hemp seedlings to cold exposure. These data provide a robust basis for further functional studies of this gene family, supporting future efforts to screen the significance of CsDGAT candidate genes to validate their functions to improve hempseed oil composition.

The SET oncoprotein promotes estrogen-induced transcription by facilitating establishment of active chromatin

SET is a multifunctional histone-binding oncoprotein that regulates transcription by an unclear mechanism. Here we show that SET enhances estrogen-dependent transcription. SET knockdown abrogates transcription of estrogen-responsive genes and their enhancer RNAs. In response to 17β-estradiol (E2), SET binds to the estrogen receptor α (ERα) and is recruited to ERα-bound enhancers and promoters at estrogen response elements (EREs). SET functions as a histone H2 chaperone that dynamically associates with H2A.Z via its acidic C-terminal domain and promotes H2A.Z incorporation, ERα, MLL1, and KDM3A loading and modulates histone methylation at EREs. SET depletion diminishes recruitment of condensin complexes to EREs and impairs E2-dependent enhancer-promoter looping. Thus, SET boosts E2-induced gene expression by establishing an active chromatin structure at ERα-bound enhancers and promoters, which is essential for transcriptional activation.