Estrogen receptor gets a grip on RNA.

The nuclear hormone receptor estrogen receptor alpha (ERα) is a well-known transcription factor present in many breast cancers, where it promotes cancer progression. In this issue of Cell, Xu et al. report that ERα is also an RNA-binding protein and that its post-transcriptional activity enables cancer cell fitness and survival.

Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains.

Gene expression is controlled by transcription factors (TFs) that consist of DNA-binding domains (DBDs) and activation domains (ADs). The DBDs have been well characterized, but little is known about the mechanisms by which ADs effect gene activation. Here, we report that diverse ADs form phase-separated condensates with the Mediator coactivator. For the OCT4 and GCN4 TFs, we show that the ability to form phase-separated droplets with Mediator in vitro and the ability to activate genes in vivo are dependent on the same amino acid residues. For the estrogen receptor (ER), a ligand-dependent activator, we show that estrogen enhances phase separation with Mediator, again linking phase separation with gene activation. These results suggest that diverse TFs can interact with Mediator through the phase-separating capacity of their ADs and that formation of condensates with Mediator is involved in gene activation.

Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions.

The estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER-binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Moreover, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1.

The Nuclear Matrix Protein SAFB Cooperates with Major Satellite RNAs to Stabilize Heterochromatin Architecture Partially through Phase Separation.

Interphase chromatin is hierarchically organized into higher-order architectures that are essential for gene functions, yet the biomolecules that regulate these 3D architectures remain poorly understood. Here, we show that scaffold attachment factor B (SAFB), a nuclear matrix (NM)-associated protein with RNA-binding functions, modulates chromatin condensation and stabilizes heterochromatin foci in mouse cells. SAFB interacts via its R/G-rich region with heterochromatin-associated repeat transcripts such as major satellite RNAs, which promote the phase separation driven by SAFB. Depletion of SAFB leads to changes in 3D genome organization, including an increase in interchromosomal interactions adjacent to pericentromeric heterochromatin and a decrease in genomic compartmentalization, which could result from the decondensation of pericentromeric heterochromatin. Collectively, we reveal the integrated roles of NM-associated proteins and repeat RNAs in the 3D organization of heterochromatin, which may shed light on the molecular mechanisms of nuclear architecture organization.

SAFB2 Enables the Processing of Suboptimal Stem-Loop Structures in Clustered Primary miRNA Transcripts.

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.

Drosophila estrogen-related receptor directs a transcriptional switch that supports adult glycolysis and lipogenesis.

Metabolism and development must be closely coupled to meet the changing physiological needs of each stage in the life cycle. The molecular mechanisms that link these pathways, however, remain poorly understood. Here we show that the Drosophila estrogen-related receptor (dERR) directs a transcriptional switch in mid-pupae that promotes glucose oxidation and lipogenesis in young adults. dERR mutant adults are viable but display reduced locomotor activity, susceptibility to starvation, elevated glucose, and an almost complete lack of stored triglycerides. Molecular profiling by RNA-seq, ChIP-seq, and metabolomics revealed that glycolytic and pentose phosphate pathway genes are induced by dERR, and their reduced expression in mutants is accompanied by elevated glycolytic intermediates, reduced TCA cycle intermediates, and reduced levels of long chain fatty acids. Unexpectedly, we found that the central pathways of energy metabolism, including glycolysis, the tricarboxylic acid cycle, and electron transport chain, are coordinately induced at the transcriptional level in mid-pupae and maintained into adulthood, and this response is partially dependent on dERR, leading to the metabolic defects observed in mutants. Our data support the model that dERR contributes to a transcriptional switch during pupal development that establishes the metabolic state of the adult fly.

Estrogen-related receptors are targetable ROS sensors.

Excessive reactive oxygen species (ROS) can cause oxidative stress and consequently cell injury contributing to a wide range of diseases. Addressing the critical gaps in our understanding of the adaptive molecular events downstream ROS provocation holds promise for the identification of druggable metabolic vulnerabilities. Here, we unveil a direct molecular link between the activity of two estrogen-related receptor (ERR) isoforms and the control of glutamine utilization and glutathione antioxidant production. ERRα down-regulation restricts glutamine entry into the TCA cycle, while ERRγ up-regulation promotes glutamine-driven glutathione production. Notably, we identify increased ERRγ expression/activation as a hallmark of oxidative stress triggered by mitochondrial disruption or chemotherapy. Enhanced tumor antioxidant capacity is an underlying feature of human breast cancer (BCa) patients that respond poorly to treatment. We demonstrate that pharmacological inhibition of ERRγ with the selective inverse agonist GSK5182 increases antitumor efficacy of the chemotherapeutic paclitaxel on poor outcome BCa tumor organoids. Our findings thus underscore the ERRs as novel redox sensors and effectors of a ROS defense program and highlight the potential therapeutic advantage of exploiting ERRγ inhibitors for the treatment of BCa and other diseases where oxidative stress plays a central role.

Promoter-Enhancer Communication Occurs Primarily within Insulated Neighborhoods.

Metazoan chromosomes are sequentially partitioned into topologically associating domains (TADs) and then into smaller sub-domains. One class of sub-domains, insulated neighborhoods, are proposed to spatially sequester and insulate the enclosed genes through self-association and chromatin looping. However, it has not been determined functionally whether promoter-enhancer interactions and gene regulation are broadly restricted to within these loops. Here, we employed published datasets from murine embryonic stem cells (mESCs) to identify insulated neighborhoods that confine promoter-enhancer interactions and demarcate gene regulatory regions. To directly address the functionality of these regions, we depleted estrogen-related receptor ß (Esrrb), which binds the Mediator co-activator complex, to impair enhancers of genes within 222 insulated neighborhoods without causing mESC differentiation. Esrrb depletion reduces Mediator binding, promoter-enhancer looping, and expression of both nascent RNA and mRNA within the insulated neighborhoods without significantly affecting the flanking genes. Our data indicate that insulated neighborhoods represent functional regulons in mammalian genomes.

Transcriptional Burst Initiation and Polymerase Pause Release Are Key Control Points of Transcriptional Regulation.

Transcriptional regulation occurs via changes to rates of different biochemical steps of transcription, but it remains unclear which rates are subject to change upon biological perturbation. Biochemical studies have suggested that stimuli predominantly affect the rates of RNA polymerase II (Pol II) recruitment and polymerase release from promoter-proximal pausing. Single-cell studies revealed that transcription occurs in discontinuous bursts, suggesting that features of such bursts like frequency and intensity could also be regulated. We combined Pol II chromatin immunoprecipitation sequencing (ChIP-seq) and single-cell transcriptional measurements to show that an independently regulated burst initiation step is required before polymerase recruitment can occur. Using a number of global and targeted transcriptional regulatory perturbations, we showed that biological perturbations regulated both burst initiation and polymerase pause release rates but seemed not to regulate polymerase recruitment rate. Our results suggest that transcriptional regulation primarily acts by changing the rates of burst initiation and polymerase pause release.

Transcription decouples estrogen-dependent changes in enhancer-promoter contact frequencies and spatial proximity.

How enhancers regulate their target genes in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we use the activation of estrogen receptor-dependent enhancers in a breast cancer cell line to study enhancer-promoter communication at two loci. This allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine how both enhancer-promoter spatial proximity assayed by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation, change dynamically during enhancer-driven gene activation. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while spatial proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate that enhancer-promoter contact frequencies are transcription independent whereas altered enhancer-promoter proximity depends on transcription. Our results emphasize that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one.

Gasdermin E suppresses tumour growth by activating anti-tumour immunity.

Cleavage of the gasdermin proteins to produce pore-forming amino-terminal fragments causes inflammatory cell death (pyroptosis)1. Gasdermin E (GSDME, also known as DFNA5)-mutated in familial ageing-related hearing loss2-can be cleaved by caspase 3, thereby converting noninflammatory apoptosis to pyroptosis in GSDME-expressing cells3-5. GSDME expression is suppressed in many cancers, and reduced GSDME levels are associated with decreased survival as a result of breast cancer2,6, suggesting that GSDME might be a tumour suppressor. Here we show that 20 of 22 tested cancer-associated GSDME mutations reduce GSDME function. In mice, knocking out Gsdme in GSDME-expressing tumours enhances, whereas ectopic expression in Gsdme-repressed tumours inhibits, tumour growth. This tumour suppression is mediated by killer cytotoxic lymphocytes: it is abrogated in perforin-deficient mice or mice depleted of killer lymphocytes. GSDME expression enhances the phagocytosis of tumour cells by tumour-associated macrophages, as well as the number and functions of tumour-infiltrating natural-killer and CD8+ T lymphocytes. Killer-cell granzyme B also activates caspase-independent pyroptosis in target cells by directly cleaving GSDME at the same site as caspase 3. Uncleavable or pore-defective GSDME proteins are not tumour suppressive. Thus, tumour GSDME acts as a tumour suppressor by activating pyroptosis, enhancing anti-tumour immunity.

Exploring the Spatial Landscape of the Estrogen Receptor Proximal Proteome With Antibody-Based Proximity Labeling.

Estrogen receptor α (ERα) drives the transcription of genes involved in breast cancer (BC) progression, relying on coregulatory protein recruitment for its transcriptional and biological activities. Mutation of ERα as well as aberrant recruitment of its regulatory proteins contribute to tumor adaptation and drug resistance. Therefore, understanding the dynamic changes in ERα protein interaction networks is crucial for elucidating drug resistance mechanisms in BC. Despite progress in studying ERα-associated proteins, capturing subcellular transient interactions remains challenging and, as a result, significant number of important interactions remain undiscovered. In this study, we employed biotinylation by antibody recognition (BAR), an innovative antibody-based proximity labeling (PL) approach, coupled with mass spectrometry to investigate the ERα proximal proteome and its changes associated with resistance to aromatase inhibition, a key therapy used in the treatment of ERα-positive BC. We show that BAR successfully detected most of the known ERα interactors and mainly identified nuclear proteins, using either an epitope tag or endogenous antibody to target ERα. We further describe the ERα proximal proteome rewiring associated with resistance applying BAR to a panel of isogenic cell lines modeling tumor adaptation in the clinic. Interestingly, we find that ERα associates with some of the canonical cofactors in resistant cells and several proximal proteome changes are due to increased expression of ERα. Resistant models also show decreased levels of estrogen-regulated genes. Sensitive and resistant cells harboring a mutation in the ERα (Y537C) revealed a similar proximal proteome. We provide an ERα proximal protein network covering several novel ERα-proximal partners. These include proteins involved in highly dynamic processes such as sumoylation and ubiquitination difficult to detect with traditional protein interaction approaches. Overall, we present BAR as an effective approach to investigate the ERα proximal proteome in a spatial context and demonstrate its application in different experimental conditions.

The marionette mechanism of domain-domain communication in the antagonist, agonist, and coactivator responses of the estrogen receptor.

The human estrogen receptor α (hERα) is involved in the regulation of growth, development, and tissue homeostasis. Agonists that bind to the receptor's ligand-binding domain (LBD) lead to recruitment of coactivators and the enhancement of gene expression. In contrast, antagonists bind to the LBD and block the binding of coactivators thus decreasing gene expressions. In this work, we carry out simulations using the AWSEM (Associative memory, Water mediated, Structure and Energy Model)-Suite force field along with the 3SPN.2C force field for DNA to predict the structure of hERα and study its dynamics when binding to DNA and coactivators. Using simulations of antagonist-bound hERα and agonist-bound hERα by themselves and also along with bound DNA and coactivators, principal component analyses and free energy landscape analyses capture the pathway of domain-domain communication for agonist-bound hERα. This communication is mediated through the hinge domains that are ordinarily intrinsically disordered. These disordered segments manipulate the hinge domains much like the strings of a marionette as they twist in different ways when antagonists or agonists are bound to the ligand-binding domain.

Novel Pan-ERR Agonists Ameliorate Heart Failure Through Enhancing Cardiac Fatty Acid Metabolism and Mitochondrial Function.

BACKGROUND: Cardiac metabolic dysfunction is a hallmark of heart failure (HF). Estrogen-related receptors ERRα and ERRγ are essential regulators of cardiac metabolism. Therefore, activation of ERR could be a potential therapeutic intervention for HF. However, in vivo studies demonstrating the potential usefulness of ERR agonist for HF treatment are lacking, because compounds with pharmacokinetics appropriate for in vivo use have not been available. METHODS: Using a structure-based design approach, we designed and synthesized 2 structurally distinct pan-ERR agonists, SLU-PP-332 and SLU-PP-915. We investigated the effect of ERR agonist on cardiac function in a pressure overload-induced HF model in vivo. We conducted comprehensive functional, multi-omics (RNA sequencing and metabolomics studies), and genetic dependency studies both in vivo and in vitro to dissect the molecular mechanism, ERR isoform dependency, and target specificity. RESULTS: Both SLU-PP-332 and SLU-PP-915 significantly improved ejection fraction, ameliorated fibrosis, and increased survival associated with pressure overload-induced HF without affecting cardiac hypertrophy. A broad spectrum of metabolic genes was transcriptionally activated by ERR agonists, particularly genes involved in fatty acid metabolism and mitochondrial function. Metabolomics analysis showed substantial normalization of metabolic profiles in fatty acid/lipid and tricarboxylic acid/oxidative phosphorylation metabolites in the mouse heart with 6-week pressure overload. ERR agonists increase mitochondria oxidative capacity and fatty acid use in vitro and in vivo. Using both in vitro and in vivo genetic dependency experiments, we show that ERRγ is the main mediator of ERR agonism-induced transcriptional regulation and cardioprotection and definitively demonstrated target specificity. ERR agonism also led to downregulation of cell cycle and development pathways, which was partially mediated by E2F1 in cardiomyocytes. CONCLUSIONS: ERR agonists maintain oxidative metabolism, which confers cardiac protection against pressure overload-induced HF in vivo. Our results provide direct pharmacologic evidence supporting the further development of ERR agonists as novel HF therapeutics.

Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells.

Mouse embryonic stem cells (mESCs) are biased toward producing embryonic rather than extraembryonic endoderm fates. Here, we identify the mechanism of this barrier and report that the histone deacetylase Hdac3 and the transcriptional corepressor Dax1 cooperatively limit the lineage repertoire of mESCs by silencing an enhancer of the extraembryonic endoderm-specifying transcription factor Gata6. This restriction is opposed by the pluripotency transcription factors Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC transcriptional network.

Investigating the shared genetic architecture of uterine leiomyoma and breast cancer: A genome-wide cross-trait analysis.

Little is known regarding the shared genetic architecture or causality underlying the phenotypic association observed for uterine leiomyoma (UL) and breast cancer (BC). Leveraging summary statistics from the hitherto largest genome-wide association study (GWAS) conducted in each trait, we investigated the genetic overlap and causal associations of UL with BC overall, as well as with its subtypes defined by the status of estrogen receptor (ER). We observed a positive genetic correlation between UL and BC overall (rg = 0.09, p = 6.00 × 10-3), which was consistent in ER+ subtype (rg = 0.06, p = 0.01) but not in ER- subtype (rg = 0.06, p = 0.08). Partitioning the whole genome into 1,703 independent regions, local genetic correlation was identified at 22q13.1 for UL with BC overall and with ER+ subtype. Significant genetic correlation was further discovered in 9 out of 14 functional categories, with the highest estimates observed in coding, H3K9ac, and repressed regions. Cross-trait meta-analysis identified 9 novel loci shared between UL and BC. Mendelian randomization demonstrated a significantly increased risk of BC overall (OR = 1.09, 95% CI = 1.01-1.18) and ER+ subtype (OR = 1.09, 95% CI = 1.01-1.17) for genetic liability to UL. No reverse causality was found. Our comprehensive genome-wide cross-trait analysis demonstrates a shared genetic basis, pleiotropic loci, as well as a putative causal relationship between UL and BC, highlighting an intrinsic link underlying these two complex female diseases.

Estrogen receptor activation remodels TEAD1 gene expression to alleviate hepatic steatosis.

Sex-based differences in obesity-related hepatic malignancies suggest the protective roles of estrogen. Using a preclinical model, we dissected estrogen receptor (ER) isoform-driven molecular responses in high-fat diet (HFD)-induced liver diseases of male and female mice treated with or without an estrogen agonist by integrating liver multi-omics data. We found that selective ER activation recovers HFD-induced molecular and physiological liver phenotypes. HFD and systemic ER activation altered core liver pathways, beyond lipid metabolism, that are consistent between mice and primates. By including patient cohort data, we uncovered that ER-regulated enhancers govern central regulatory and metabolic genes with clinical significance in metabolic dysfunction-associated steatotic liver disease (MASLD) patients, including the transcription factor TEAD1. TEAD1 expression increased in MASLD patients, and its downregulation by short interfering RNA reduced intracellular lipid content. Subsequent TEAD small molecule inhibition improved steatosis in primary human hepatocyte spheroids by suppressing lipogenic pathways. Thus, TEAD1 emerged as a new therapeutic candidate whose inhibition ameliorates hepatic steatosis.

HOXA9 forms a repressive complex with nuclear matrix-associated protein SAFB to maintain acute myeloid leukemia.

HOXA9 is commonly upregulated in acute myeloid leukemia (AML), in which it confers a poor prognosis. Characterizing the protein interactome of endogenous HOXA9 in human AML, we identified a chromatin complex of HOXA9 with the nuclear matrix attachment protein SAFB. SAFB perturbation phenocopied HOXA9 knockout to decrease AML proliferation, increase differentiation and apoptosis in vitro, and prolong survival in vivo. Integrated genomic, transcriptomic, and proteomic analyses further demonstrated that the HOXA9-SAFB (H9SB)-chromatin complex associates with nucleosome remodeling and histone deacetylase (NuRD) and HP1γ to repress the expression of factors associated with differentiation and apoptosis, including NOTCH1, CEBPδ, S100A8, and CDKN1A. Chemical or genetic perturbation of NuRD and HP1γ-associated catalytic activity also triggered differentiation, apoptosis, and the induction of these tumor-suppressive genes. Importantly, this mechanism is operative in other HOXA9-dependent AML genotypes. This mechanistic insight demonstrates the active HOXA9-dependent differentiation block as a potent mechanism of disease maintenance in AML that may be amenable to therapeutic intervention by targeting the H9SB interface and/or NuRD and HP1γ activity.

Growth characteristics of HCT116 xenografts lacking asparagine synthetase vary according to sex.

BACKGROUND: Sex-related differences in colorectal (CRC) incidence and mortality are well-documented. However, the impact of sex on metabolic pathways that drive cancer growth is not well understood. High expression of asparagine synthetase (ASNS) is associated with inferior survival for female CRC patients only. Here, we used a CRISPR/Cas9 technology to generate HCT116 ASNS-/- and HCT 116 ASNS+/+ cancer cell lines. We examine the effects of ASNS deletion on tumor growth and the subsequent rewiring of metabolic pathways in male and female Rag2/IL2RG mice. RESULTS: ASNS loss reduces cancer burden in male and female tumor-bearing mice (40% reduction, q < 0.05), triggers metabolic reprogramming including gluconeogenesis, but confers a survival improvement (30 days median survival, q < 0.05) in female tumor-bearing mice alone. Transcriptomic analyses revealed upregulation of G-protein coupled estrogen receptor (GPER1) in tumors from male and female mice with HCT116 ASNS-/- xenograft. Estradiol activates GPER1 in vitro in the presence of ASNS and suppresses tumor growth. CONCLUSIONS: Our study indicates that inferior survival for female CRC patients with high ASNS may be due to metabolic reprogramming that sustains tumor growth. These findings have translational relevance as ASNS/GPER1 signaling could be a future therapeutic target to improve the survival of female CRC patients.

G-protein-coupled estrogen receptor 1 promotes peritoneal metastasis of gastric cancer through nicotinamide adenine dinucleotide kinase 1-mediated redox modulation.

Adipose tissue is the second most important site of estrogen production, where androgens are converted into estrogen by aromatase. While gastric cancer patients often develop adipocyte-rich peritoneal metastasis, the underlying mechanism remains unclear. In this study, we identified the G-protein-coupled estrogen receptor (GPER1) as a promoter of gastric cancer peritoneal metastasis. Functional in vitro studies revealed that ß-Estradiol (E2) or the GPER1 agonist G1 inhibited anoikis in gastric cancer cells. Additionally, genetic overexpression or knockout of GPER1 significantly inhibited or enhanced gastric cancer cell anoikis in vitro and peritoneal metastasis in vivo, respectively. Mechanically, GPER1 knockout disrupted the NADPH pool and increased reactive oxygen species (ROS) generation. Conversely, overexpression of GPER1 had the opposite effects. GPER1 suppressed nicotinamide adenine dinucleotide kinase 1(NADK1) ubiquitination and promoted its phosphorylation, which were responsible for the elevated expression of NADK1 at protein levels and activity, respectively. Moreover, genetic inhibition of NADK1 disrupted NADPH and redox homeostasis, leading to high levels of ROS and significant anoikis, which inhibited lung and peritoneal metastasis in cell-based xenograft models. In summary, our study suggests that inhibiting GPER1-mediated NADK1 activity and its ubiquitination may be a promising therapeutic strategy for peritoneal metastasis of gastric cancer.