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.

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.

Proteasome Inhibitors Silence Oncogenes in Multiple Myeloma through Localized Histone Deacetylase 3 (HDAC3) Stabilization and Chromatin Condensation.

Proteasome inhibitors have become the standard of care for multiple myeloma (MM). Blocking protein degradation particularly perturbs the homeostasis of short-lived polypeptides such as transcription factors and epigenetic regulators. To determine how proteasome inhibitors directly impact gene regulation, we performed an integrative genomics study in MM cells. We discovered that proteasome inhibitors reduce the turnover of DNA-associated proteins and repress genes necessary for proliferation through epigenetic silencing. Specifically, proteasome inhibition results in the localized accumulation of histone deacetylase 3 (HDAC3) at defined genomic sites, which reduces H3K27 acetylation and increases chromatin condensation. The loss of active chromatin at super-enhancers critical for MM, including the super-enhancer controlling the proto-oncogene c-MYC, reduces metabolic activity and cancer cell growth. Epigenetic silencing is attenuated by HDAC3 depletion, suggesting a tumor-suppressive element of this deacetylase in the context of proteasome inhibition. In the absence of treatment, HDAC3 is continuously removed from DNA by the ubiquitin ligase SIAH2. Overexpression of SIAH2 increases H3K27 acetylation at c-MYC-controlled genes, increases metabolic output, and accelerates cancer cell proliferation. Our studies indicate a novel therapeutic function of proteasome inhibitors in MM by reshaping the epigenetic landscape in an HDAC3-dependent manner. As a result, blocking the proteasome effectively antagonizes c-MYC and the genes controlled by this proto-oncogene.

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.

Single-Cell Distribution Analysis of AR Levels by High-Throughput Microscopy in Cell Models: Application for Testing Endocrine-Disrupting Chemicals.

Cell-to-cell variation of protein expression in genetically homogeneous populations is a common biological trait often neglected during analysis of high-throughput (HT) screens and is rarely used as a metric to characterize chemicals. We have captured single-cell distributions of androgen receptor (AR) nuclear levels after perturbations as a means to evaluate assay reproducibility and characterize a small subset of chemicals. AR, a member of the nuclear receptor family of transcription factors, is the central regulator of male reproduction and is involved in many pathophysiological processes. AR protein levels and nuclear localization often increase following ligand binding, with dihydrotestosterone (DHT) being the natural agonist. HT AR immunofluorescence imaging was used in multiple cell lines to define single-cell nuclear values extracted from thousands of cells per condition treated with DHT or DMSO (control). Analysis of numerous biological replicates led to a quality control metric that takes into account the distribution of single-cell data, and how it changes upon treatments. Dose-response experiments across several cell lines showed a large range of sensitivity to DHT, prompting us to treat selected cell lines with 45 Environmental Protection Agency (EPA)-provided chemicals that include many endocrine-disrupting chemicals (EDCs); data from six of the compounds were then integrated with orthogonal assays. Our comprehensive results indicate that quantitative single-cell distribution analysis of AR protein levels is a valid method to detect the potential androgenic and antiandrogenic actions of environmentally relevant chemicals in a sensitive and reproducible manner.