Gene transcription regulation by ER at the single cell and allele level.

In this short review we discuss the current view of how the estrogen receptor (ER), a pivotal member of the nuclear receptor superfamily of transcription factors, regulates gene transcription at the single cell and allele level, focusing on in vitro cell line models. We discuss central topics and new trends in molecular biology including phenotypic heterogeneity, single cell sequencing, nuclear phase separated condensates, single cell imaging, and image analysis methods, with particular focus on the methodologies and results that have been reported in the last few years using microscopy-based techniques. These observations augment the results from biochemical assays that lead to a much more complex and dynamic view of how ER, and arguably most transcription factors, act to regulate gene transcription.

Analysis and Modeling of Early Estradiol-induced GREB1 Single Allele Gene Transcription at the Population Level.

Single molecule fluorescence in situ hybridization (smFISH) can be used to visualize transcriptional activation at the single allele level. We and others have applied this approach to better understand the mechanisms of activation by steroid nuclear receptors. However, there is limited understanding of the interconnection between the activation of target gene alleles inside the same nucleus and within large cell populations. Using the GREB1 gene as an early estrogen receptor (ER) response target, we applied smFISH to track E2-activated GREB1 allelic transcription over early time points to evaluate potential dependencies between alleles within the same nucleus. We compared two types of experiments where we altered the initial status of GREB1 basal transcription by treating cells with and without the elongation inhibitor flavopiridol (FV). E2 stimulation changed the frequencies of active GREB1 alleles in the cell population independently of FV pre-treatment. In FV treated cells, the response time to hormone was delayed, albeit still reaching at 90 minutes the same levels as in cells not treated by FV. We show that the joint frequencies of GREB1 activated alleles observed at the cell population level imply significant dependency between pairs of alleles within the same nucleus. We identify probabilistic models of joint alleles activations by applying a principle of maximum entropy. For pairs of alleles, we have then quantified statistical dependency by computing their mutual information. We have then introduced a stochastic model compatible with allelic statistical dependencies, and we have fitted this model to our data by intensive simulations. This provided estimates of the average lifetime for degradation of GREB1 introns and of the mean time between two successive transcription rounds. Our approach informs on how to extract information on single allele regulation by ER from within a large population of cells, and should be applicable to many other genes. AUTHOR SUMMARY: After application of a gene transcription stimulus, in this case the hormone 17 ß -estradiol, on large populations of cells over a short time period, we focused on quantifying and modeling the frequencies of GREB1 single allele activations. We have established an experimental and computational pipeline to analyze large numbers of high resolution smFISH images to detect and monitor active GREB1 alleles, that can be translatable to any target gene of interest. A key result is that, at the population level, activation of individual GREB1 alleles within the same nucleus do exhibit statistically significant dependencies which we quantify by the mutual information between activation states of pairs of alleles. After noticing that frequencies of joint alleles activations observed over our large cell populations evolve smoothly in time, we have defined a population level stochastic model which we fit to the observed time course of GREB1 activation frequencies. This provided coherent estimates of the mean time between rounds of GREB1 transcription and the mean lifetime of nascent mRNAs. Our algorithmic approach and experimental methods are applicable to many other genes.

Oral follicle-stimulating hormone receptor agonist affects granulosa cells differently than recombinant human FSH.

OBJECTIVE: To determine whether TOP5300, a novel oral follicle-stimulating hormone (FSH) receptor (FSHR) allosteric agonist, elicits a different cellular response than recombinant human FSH (rh-FSH) in human granulosa cells from patients undergoing in vitro fertilization. DESIGN: Basic science research with a preclinical allosteric FSHR agonist. SETTING: University hospital. PATIENT(S): Patients with infertility at a single academic fertility clinic were recruited under an Institutional Review Board-approved protocol. Primary granulosa cell cultures were established for 41 patients, of whom 8 had normal ovarian reserve (NOR), 17 were of advanced reproductive age (ARA), 12 had a diagnosis of polycystic ovary syndrome (PCOS), and 4 had a combination of diagnoses, such as ARA and PCOS. INTERVENTION(S): Primary granulosa-lutein (GL) cell cultures were treated with rh-FSH, TOP5300, or vehicle. MAIN OUTCOME MEASURE(S): Estradiol (E2) production using enzyme-linked immunosorbent assay, steroid pathway gene expression of StAR and aromatase using quantitative polymerase chain reaction, and FSHR membrane localization using immunofluorescence were measured in human GL cells. RESULT(S): TOP5300 consistently stimulated E2 production among patients with NOR, ARA, and PCOS. Recombinant FSH was the more potent ligand in GL cells from patients with NOR but was ineffective in cells from patients with ARA or PCOS. The lowest level of FSHR plasma membrane localization was seen in patients with ARA, although FSHR localization was more abundant in cells from patients with PCOS; the highest levels were present in cells from patients with NOR. The localization of FSHR was not affected by TOP5300 relative to rh-FSH in any patient group. TOP5300 stimulated greater expression of StAR and CYP19A1 across cells from all patients with NOR, ARA, and PCOS combined, although rh-FSH was unable to stimulate StAR and aromatase (CYP19A1) expression in cells from patients with PCOS. TOP5300-induced expression of StAR and CYP19A1 mRNA among patients with ARA and NOR was consistently lower than that observed in cells from patients with PCOS. CONCLUSION(S): TOP5300 appears to stimulate E2 production and steroidogenic gene expression from GL cells more than rh-FSH in PCOS, relative to patients with ARA and NOR. It does not appear that localization of FSHR at cell membranes is a limiting step for TOP5300 or rh-FSH stimulation of steroidogenic gene expression and E2 production.

Quality Control for Single Cell Imaging Analytics Using Endocrine Disruptor-Induced Changes in Estrogen Receptor Expression.

BACKGROUND: Diverse toxicants and mixtures that affect hormone responsive cells [endocrine disrupting chemicals (EDCs)] are highly pervasive in the environment and are directly linked to human disease. They often target the nuclear receptor family of transcription factors modulating their levels and activity. Many high-throughput assays have been developed to query such toxicants; however, single-cell analysis of EDC effects on endogenous receptors has been missing, in part due to the lack of quality control metrics to reproducibly measure cell-to-cell variability in responses. OBJECTIVE: We began by developing single-cell imaging and informatic workflows to query whether the single cell distribution of the estrogen receptor-α (ER), used as a model system, can be used to measure effects of EDCs in a sensitive and reproducible manner. METHODS: We used high-throughput microscopy, coupled with image analytics to measure changes in single cell ER nuclear levels on treatment with ∼100 toxicants, over a large number of biological and technical replicates. RESULTS: We developed a two-tiered quality control pipeline for single cell analysis and tested it against a large set of biological replicates, and toxicants from the EPA and Agency for Toxic Substances and Disease Registry lists. We also identified a subset of potentially novel EDCs that were active only on the endogenous ER level and activity as measured by single molecule RNA fluorescence in situ hybridization (RNA FISH). DISCUSSION: We demonstrated that the distribution of ER levels per cell, and the changes upon chemical challenges were remarkably stable features; and importantly, these features could be used for quality control and identification of endocrine disruptor toxicants with high sensitivity. When coupled with orthogonal assays, ER single cell distribution is a valuable resource for high-throughput screening of environmental toxicants. https://doi.org/10.1289/EHP9297.

The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics.

There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.

Morphological screening of mesenchymal mammary tumor organoids to identify drugs that reverse epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) has been implicated in conferring stem cell properties and therapeutic resistance to cancer cells. Therefore, identification of drugs that can reprogram EMT may provide new therapeutic strategies. Here, we report that cells derived from claudin-low mammary tumors, a mesenchymal subtype of triple-negative breast cancer, exhibit a distinctive organoid structure with extended "spikes" in 3D matrices. Upon a miR-200 induced mesenchymal-epithelial transition (MET), the organoids switch to a smoother round morphology. Based on these observations, we developed a morphological screening method with accompanying analytical pipelines that leverage deep neural networks and nearest neighborhood classification to screen for EMT-reversing drugs. Through screening of a targeted epigenetic drug library, we identified multiple class I HDAC inhibitors and Bromodomain inhibitors that reverse EMT. These data support the use of morphological screening of mesenchymal mammary tumor organoids as a platform to identify drugs that reverse EMT.

Oncogenic Mutations in PI3K/AKT/mTOR Pathway Effectors Associate with Worse Prognosis in BRAFV600E -Driven Papillary Thyroid Cancer Patients.

PURPOSE: The extent to which routine genomic sequencing can identify relevant secondary genomic alterations among BRAFV600E -mutant papillary thyroid carcinoma (PTC) is unknown. Such markers would prove highly valuable for prognostic purposes. EXPERIMENTAL DESIGN: We reviewed clinicopathologic data of 225 patients with BRAFV600E -mutant PTC and integrated them with genomic data derived from targeted next-generation sequencing (NGS) on tumor specimens. We defined patient subgroups based on bona fide secondary oncogenic events (separate from BRAFV600E ) and compared their clinical features and outcomes with those without additional oncogenic alterations. RESULTS: Additional oncogenic alterations were identified in 16% of tumors. Patients in the "BRAF+additional mutations" group were more likely to be at high American Thyroid Association (ATA) risk of recurrence (48.6% vs. 17.6%; P = 0.0009), had larger baseline tumor (2.7 vs. 1.9 cm; P = 0.0005) and more advanced stage at presentation (14.3% vs. 1.1% stage 4; P < 0.0001). Importantly, over a 65-month follow-up, disease-specific mortality (DSM) was increased when additional mutations were identified (13.8% vs. 1.4% in the BRAF-only group; P = 0.005). Separately, we identified a subcluster of patients harboring oncogenic mutations in key effectors of the PI3K/AKT/mTOR pathway, which were independently associated with DSM (OR = 47.9; 95% confidence interval, 3.5-1,246.5; P = 0.0043). CONCLUSIONS: Identification of additional PIK3/AKT/mTOR alterations in patients with BRAFV600E -mutant PTC provides important and actionable prognostic risk stratification. These data support genomic profiling of PTC tumors to inform prognosis and clinical strategy.

Targeted brachyury degradation disrupts a highly specific autoregulatory program controlling chordoma cell identity.

Chordomas are rare spinal tumors addicted to expression of the developmental transcription factor brachyury. In chordomas, brachyury is super-enhancer associated and preferentially downregulated by pharmacologic transcriptional CDK inhibition, leading to cell death. To understand the underlying basis of this sensitivity, we dissect the brachyury transcription regulatory network and compare the consequences of brachyury degradation with transcriptional CDK inhibition. Brachyury defines the chordoma super-enhancer landscape and autoregulates through binding its super-enhancer, and its locus forms a transcriptional condensate. Transcriptional CDK inhibition and brachyury degradation disrupt brachyury autoregulation, leading to loss of its transcriptional condensate and transcriptional program. Compared with transcriptional CDK inhibition, which globally downregulates transcription, leading to cell death, brachyury degradation is much more selective, inducing senescence and sensitizing cells to anti-apoptotic inhibition. These data suggest that brachyury downregulation is a core tenet of transcriptional CDK inhibition and motivates developing strategies to target brachyury and its autoregulatory feedback loop.

The Human Nose Organoid Respiratory Virus Model: an Ex Vivo Human Challenge Model To Study Respiratory Syncytial Virus (RSV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Pathogenesis and Evaluate Therapeutics.

There is an unmet need for preclinical models to understand the pathogenesis of human respiratory viruses and predict responsiveness to immunotherapies. Airway organoids can serve as an ex vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a noninvasive technique to generate human nose organoids (HNOs) as an alternative to biopsy-derived organoids. We made air-liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hypersecretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation), while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration-dependent manner. Thus, the HNO-ALI model can serve as an alternative to lung organoids to study respiratory viruses and test therapeutics. IMPORTANCE Preclinical models that recapitulate aspects of human airway disease are essential for the advancement of novel therapeutics and vaccines. Here, we report a versatile airway organoid model, the human nose organoid (HNO), that recapitulates the complex interactions between the host and virus. HNOs are obtained using noninvasive procedures and show divergent responses to SARS-CoV-2 and RSV infection. SARS-CoV-2 induces severe damage to cilia and the epithelium, no interferon-λ response, and minimal mucus secretion. In striking contrast, RSV induces hypersecretion of mucus and a profound interferon-λ response with ciliary damage. We also demonstrated the usefulness of our ex vivo HNO model of RSV infection to test the efficacy of palivizumab, an FDA-approved monoclonal antibody to prevent severe RSV disease in high-risk infants. Our study reports a breakthrough in both the development of a novel nose organoid model and in our understanding of the host cellular response to RSV and SARS-CoV-2 infection.