RNA-Mediated Feedback Control of Transcriptional Condensates.

Regulation of biological processes typically incorporates mechanisms that initiate and terminate the process and, where understood, these mechanisms often involve feedback control. Regulation of transcription is a fundamental cellular process where the mechanisms involved in initiation have been studied extensively, but those involved in arresting the process are poorly understood. Modeling of the potential roles of RNA in transcriptional control suggested a non-equilibrium feedback control mechanism where low levels of RNA promote condensates formed by electrostatic interactions whereas relatively high levels promote dissolution of these condensates. Evidence from in vitro and in vivo experiments support a model where RNAs produced during early steps in transcription initiation stimulate condensate formation, whereas the burst of RNAs produced during elongation stimulate condensate dissolution. We propose that transcriptional regulation incorporates a feedback mechanism whereby transcribed RNAs initially stimulate but then ultimately arrest the process.

Hotspot mutations in the structured ENL YEATS domain link aberrant transcriptional condensates and cancer.

Growing evidence suggests prevalence of transcriptional condensates on chromatin, yet their mechanisms of formation and functional significance remain largely unclear. In human cancer, a series of mutations in the histone acetylation reader ENL create gain-of-function mutants with increased transcriptional activation ability. Here, we show that these mutations, clustered in ENL's structured acetyl-reading YEATS domain, trigger aberrant condensates at native genomic targets through multivalent homotypic and heterotypic interactions. Mechanistically, mutation-induced structural changes in the YEATS domain, ENL's two disordered regions of opposing charges, and the incorporation of extrinsic elongation factors are all required for ENL condensate formation. Extensive mutagenesis establishes condensate formation as a driver of oncogenic gene activation. Furthermore, expression of ENL mutants beyond the endogenous level leads to non-functional condensates. Our findings provide new mechanistic and functional insights into cancer-associated condensates and support condensate dysregulation as an oncogenic mechanism.

Stress-induced nuclear condensation of NELF drives transcriptional downregulation.

In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.

Mediator Condensates Localize Signaling Factors to Key Cell Identity Genes.

The gene expression programs that define the identity of each cell are controlled by master transcription factors (TFs) that bind cell-type-specific enhancers, as well as signaling factors, which bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here, we present evidence that signaling factors for the WNT, TGF-ß, and JAK/STAT pathways use their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator ß-catenin interacts both with components of condensates and DNA-binding factors to selectively occupy super-enhancer-associated genes. We propose that the cell-type specificity of the response to signaling is mediated in part by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity.

Nonspecific Interactions in Transcription Regulation and Organization of Transcriptional Condensates.

Eukaryotic cells are characterized by a high degree of compartmentalization of their internal contents, which ensures precise and controlled regulation of intracellular processes. During many processes, including different stages of transcription, dynamic membraneless compartments termed biomolecular condensates are formed. Transcription condensates contain various transcription factors and RNA polymerase and are formed by high- and low-specificity interactions between the proteins, DNA, and nearby RNA. This review discusses recent data demonstrating important role of nonspecific multivalent protein-protein and RNA-protein interactions in organization and regulation of transcription.

Phase-separation antagonists potently inhibit transcription and broadly increase nucleosome density.

Biomolecular condensates are self-organized membraneless bodies involved in many critical cellular activities, including ribosome biogenesis, protein synthesis, and gene transcription. Aliphatic alcohols are commonly used to study biomolecular condensates, but their effects on transcription are unclear. Here, we explore the impact of the aliphatic dialcohol, 1,6-hexanediol (1,6-HD), on Pol II transcription and nucleosome occupancy in budding yeast. As expected, 1,6-HD, a reagent effective in disrupting biomolecular condensates, strongly suppressed the thermal stress-induced transcription of Heat Shock Factor 1-regulated genes that have previously been shown to physically interact and coalesce into intranuclear condensates. Surprisingly, the isomeric dialcohol, 2,5-HD, typically used as a negative control, abrogated Heat Shock Factor 1-target gene transcription under the same conditions. Each reagent also abolished the transcription of genes that do not detectably coalesce, including Msn2/Msn4-regulated heat-inducible genes and constitutively expressed housekeeping genes. Thus, at elevated temperature (39 °C), HDs potently inhibit the transcription of disparate genes and as demonstrated by chromatin immunoprecipitation do so by abolishing occupancy of RNA polymerase in chromatin. Concurrently, histone H3 density increased at least twofold within all gene coding and regulatory regions examined, including quiescent euchromatic loci, silent heterochromatic loci, and Pol III-transcribed loci. Our results offer a caveat for the use of HDs in studying the role of condensates in transcriptional control and provide evidence that exposure to these reagents elicits a widespread increase in nucleosome density and a concomitant loss of both Pol II and Pol III transcription.

RNA polymerase II clusters form in line with surface condensation on regulatory chromatin.

It is essential for cells to control which genes are transcribed into RNA. In eukaryotes, two major control points are recruitment of RNA polymerase II (Pol II) into a paused state, and subsequent pause release toward transcription. Pol II recruitment and pause release occur in association with macromolecular clusters, which were proposed to be formed by a liquid-liquid phase separation mechanism. How such a phase separation mechanism relates to the interaction of Pol II with DNA during recruitment and transcription, however, remains poorly understood. Here, we use live and super-resolution microscopy in zebrafish embryos to reveal Pol II clusters with a large variety of shapes, which can be explained by a theoretical model in which regulatory chromatin regions provide surfaces for liquid-phase condensation at concentrations that are too low for canonical liquid-liquid phase separation. Model simulations and chemical perturbation experiments indicate that recruited Pol II contributes to the formation of these surface-associated condensates, whereas elongating Pol II is excluded from these condensates and thereby drives their unfolding.

Pluripotency transcription factors at the focus: the phase separation paradigm in stem cells.

The transcription factors (TFs) OCT4, SOX2 and NANOG are key players of the gene regulatory network of pluripotent stem cells. Evidence accumulated in recent years shows that even small imbalances in the expression levels or relative concentrations of these TFs affect both, the maintenance of pluripotency and cell fate decisions. In addition, many components of the transcriptional machinery including RNA polymerases, cofactors and TFs such as those required for pluripotency, do not distribute homogeneously in the nucleus but concentrate in multiple foci influencing the delivery of these molecules to their DNA-targets. How cells control strict levels of available pluripotency TFs in this heterogeneous space and the biological role of these foci remain elusive. In recent years, a wealth of evidence led to propose that many of the nuclear compartments are formed through a liquid-liquid phase separation process. This new paradigm early penetrated the stem cells field since many key players of the pluripotency circuitry seem to phase-separate. Overall, the formation of liquid compartments may modulate the kinetics of biochemical reactions and consequently regulate many nuclear processes. Here, we review the state-of-the-art knowledge of compartmentalization in the cell nucleus and the relevance of this process for transcriptional regulation, particularly in pluripotent stem cells. We also highlight the recent advances and new ideas in the field showing how compartmentalization may affect pluripotency preservation and cell fate decisions.

ENL mutation and AML: a new model that reveals oncogenic condensate's function in leukemogenesis.

Precise regulation of gene expression is essential for proper development and the maintenance of homeostasis in organisms. Studies have shown that some transcriptional regulatory proteins influence gene expression through the formation of dynamic, locally concentrated assemblies known as condensates, while dysregulation of transcriptional condensates was associated with several cancers, such as Ewing sarcoma and AML [Wang Y et al. (2023) Nat Chem Biol 19, 1223-1234; Chandra B et al. (2022) Cancer Discov 12, 1152-1169]. Mutations in the histone acetylation "reader" eleven-nineteen-leukemia (ENL) have been shown to form discrete condensates at endogenous genomic targets, but it remains unclear how ENL mutations drive tumorigenesis and whether it is correlated with their condensate formation property. Liu et al. now show, using a conditional knock-in mouse model, that ENL YEATS domain mutation is a bona fide oncogenic driver for AML. This mutant ENL forms condensates in hematopoietic stem/progenitor cells at the genomic loci of key leukemogenic genes, including Meis1 and Hoxa cluster genes, and disrupting condensate formation via mutagenesis impairs its chromatin and oncogenic function. Furthermore, they show that small-molecule inhibition of the acetyl-binding activity displaces ENL mutant condensates from oncogenic target loci, and this inhibitor significantly impairs the onset and progression of AML driven by mutant ENL in vivo.

Heat Shock Factor 1 forms nuclear condensates and restructures the yeast genome before activating target genes.

In insects and mammals, 3D genome topology has been linked to transcriptional states yet whether this link holds for other eukaryotes is unclear. Using both ligation proximity and fluorescence microscopy assays, we show that in Saccharomyces cerevisiae, Heat Shock Response (HSR) genes dispersed across multiple chromosomes and under the control of Heat Shock Factor (Hsf1) rapidly reposition in cells exposed to acute ethanol stress and engage in concerted, Hsf1-dependent intergenic interactions. Accompanying 3D genome reconfiguration is equally rapid formation of Hsf1-containing condensates. However, in contrast to the transience of Hsf1-driven intergenic interactions that peak within 10-20 min and dissipate within 1 hr in the presence of 8.5% (v/v) ethanol, transcriptional condensates are stably maintained for hours. Moreover, under the same conditions, Pol II occupancy of HSR genes, chromatin remodeling, and RNA expression are detectable only later in the response and peak much later (>1 hr). This contrasts with the coordinate response of HSR genes to thermal stress (39°C) where Pol II occupancy, transcription, histone eviction, intergenic interactions, and formation of Hsf1 condensates are all rapid yet transient (peak within 2.5-10 min and dissipate within 1 hr). Therefore, Hsf1 forms condensates, restructures the genome and transcriptionally activates HSR genes in response to both forms of proteotoxic stress but does so with strikingly different kinetics. In cells subjected to ethanol stress, Hsf1 forms condensates and repositions target genes before transcriptionally activating them.

Extra- and intranuclear heat perception and triggering mechanisms in plants.

The escalating impact of global warming on crop yield and quality poses a significant threat to future food supplies. Breeding heat-resistant crop varieties holds promise, but necessitates a deeper understanding of the molecular mechanisms underlying plant heat tolerance. Recent studies have shed light on the initial events of heat perception in plants. In this review, we provide a comprehensive summary of the recent progress made in unraveling the mechanisms of heat perception and response in plants. Calcium ion (Ca2+), hydrogen peroxide (H2O2), and nitric oxide (NO) have emerged as key participants in heat perception. Furthermore, we discuss the potential roles of the NAC transcription factor NTL3, thermo-tolerance 3.1 (TT3.1), and Target of temperature 3 (TOT3) as thermosensors associated with the plasma membrane. Additionally, we explore the involvement of cytoplasmic HISTONE DEACETYLASE 9 (HDA9), mRNA encoding the phytochrome-interacting factor 7 (PIF7), and chloroplasts in mediating heat perception. This review also highlights the role of intranuclear transcriptional condensates formed by phytochrome B (phyB), EARLY FLOWERING 3 (ELF3), and guanylate-binding protein (GBP)-like GTPase 3 (GBPL3) in heat perception. Finally, we raise the unresolved questions in the field of heat perception that require further investigation in the future.

Ethanol stress induces transient restructuring of the yeast genome yet stable formation of Hsf1 transcriptional condensates.

In mammals, 3D genome topology has been linked to transcriptional states yet whether this link holds for other eukaryotes is unclear. Here we show that in budding yeast, Heat Shock Response (HSR) genes under the control of Heat Shock Factor (Hsf1) rapidly reposition in cells exposed to acute ethanol stress and engage in concerted, Hsf1-dependent intergenic interactions. Accompanying 3D genome reconfiguration is equally rapid formation of Hsf1-containing condensates. However, in contrast to the transience of Hsf1-driven intergenic interactions that peak within 10 min and dissipate within 1 h, Hsf1 condensates are stably maintained for hours. Moreover, under the same conditions, Pol II occupancy of HSR genes and RNA expression are detectable only later in the response and peak much later (>1 h). This contrasts with the coordinate response of HSR genes to thermal stress where Pol II occupancy, transcription, intergenic interactions, and formation of Hsf1 condensates are all rapid yet transient (peak within 2.5-10 min and dissipate within 1 h). Collectively, our data suggest that different stimuli drive distinct transcription, topologic, and phase-separation phenomena dependent on the same transcription factor and that transcription factor-containing condensates represent only part of the ensemble required for gene activation.

Heterotypic transcriptional condensates formed by prion-like paralogous proteins canalize flowering transition in tomato.

BACKGROUND: Paralogs that arise from gene duplications during genome evolution enable genetic redundancy and phenotypic robustness. Variation in the coding or regulatory sequence of paralogous transcriptional regulators diversifies their functions and relationships, which provides developmental robustness against genetic or environmental perturbation. The fate transition of plant shoot stem cells for flowering and reproductive success requires a robust transcriptional control. However, how paralogs function and interact to achieve such robustness is unknown. RESULTS: Here, we explore the genetic relationship and protein behavior of ALOG family transcriptional factors with diverse transcriptional abundance in shoot meristems. A mutant spectrum covers single and higher-order mutant combinations of five ALOG paralogs and creates a continuum of flowering transition defects, showing gradually enhanced precocious flowering, along with inflorescence simplification from wild-type-like to progressively fewer flowers until solitary flower with sterile floral organs. Therefore, these paralogs play unequal roles and act together to achieve a robust genetic canalization. All five proteins contain prion-like intrinsically disordered regions (IDRs) and undergo phase separation. Accumulated mutations following gene duplications lead to IDR variations among ALOG paralogs, resulting in divergent phase separation and transcriptional regulation capabilities. Remarkably, they retain the ancestral abilities to assemble into a heterotypic condensate that prevents precocious activation of the floral identity gene ANANTHA. CONCLUSIONS: Our study reveals a novel genetic canalization mechanism enabled by heterotypic transcriptional condensates formed by paralogous protein interactions and phase separation, uncovering the molecular link between gene duplication caused IDR variation and robust transcriptional control of stem cell fate transition.

Metabolic modulation of transcription: The role of one-carbon metabolism.

While it is well known that expression levels of metabolic enzymes regulate the metabolic state of the cell, there is mounting evidence that the converse is also true, that metabolite levels themselves can modulate gene expression via epigenetic modifications and transcriptional regulation. Here we focus on the one-carbon metabolic pathway, which provides the essential building blocks of many classes of biomolecules, including purine nucleotides, thymidylate, serine, and methionine. We review the epigenetic roles of one-carbon metabolic enzymes and their associated metabolites and introduce an interactive computational resource that places enzyme essentiality in the context of metabolic pathway topology. Therefore, we briefly discuss examples of metabolic condensates and higher-order complexes of metabolic enzymes downstream of one-carbon metabolism. We speculate that they may be required to the formation of transcriptional condensates and gene expression control. Finally, we discuss new ways to exploit metabolic pathway compartmentalization to selectively target these enzymes in cancer.

FUS oncofusion protein condensates recruit mSWI/SNF chromatin remodeler via heterotypic interactions between prion-like domains.

Fusion transcription factors generated by genomic translocations are common drivers of several types of cancers including sarcomas and leukemias. Oncofusions of the FET (FUS, EWSR1, and TAF15) family proteins result from the fusion of the prion-like domain (PLD) of FET proteins to the DNA-binding domain (DBD) of certain transcription regulators and are implicated in aberrant transcriptional programs through interactions with chromatin remodelers. Here, we show that FUS-DDIT3, a FET oncofusion protein, undergoes PLD-mediated phase separation into liquid-like condensates. Nuclear FUS-DDIT3 condensates can recruit essential components of the global transcriptional machinery such as the chromatin remodeler SWI/SNF. The recruitment of mammalian SWI/SNF (mSWI/SNF) is driven by heterotypic PLD-PLD interactions between FUS-DDIT3 and core subunits of SWI/SNF, such as the catalytic component BRG1. Further experiments with single-molecule correlative force-fluorescence microscopy support a model wherein the fusion protein forms condensates on DNA surface and enrich BRG1 to activate transcription by ectopic chromatin remodeling. Similar PLD-driven co-condensation of mSWI/SNF with transcription factors can be employed by other oncogenic fusion proteins with a generic PLD-DBD domain architecture for global transcriptional reprogramming.

Primordial super-enhancers: heat shock-induced chromatin organization in yeast.

Specialized mechanisms ensure proper expression of critically important genes such as those specifying cell identity or conferring protection from environmental stress. Investigations of the heat shock response have been critical in elucidating basic concepts of transcriptional control. Recent studies demonstrate that in response to thermal stress, heat shock-responsive genes associate with high levels of transcriptional activators and coactivators and those in yeast intensely interact across and between chromosomes, coalescing into condensates. In mammalian cells, cell identity genes that are regulated by super-enhancers (SEs) are also densely occupied by transcriptional machinery that form phase-separated condensates. We suggest that the stress-remodeled yeast nucleome bears functional and structural resemblance to mammalian SEs, and will reveal fundamental mechanisms of gene control by transcriptional condensates.

Evidence for and against Liquid-Liquid Phase Separation in the Nucleus.

Enclosed by two membranes, the nucleus itself is comprised of various membraneless compartments, including nuclear bodies and chromatin domains. These compartments play an important though still poorly understood role in gene regulation. Significant progress has been made in characterizing the dynamic behavior of nuclear compartments and liquid-liquid phase separation (LLPS) has emerged as a prominent mechanism governing their assembly. However, recent work reveals that certain nuclear structures violate key predictions of LLPS, suggesting that alternative mechanisms likely contribute to nuclear organization. Here, we review the evidence for and against LLPS for several nuclear compartments and discuss experimental strategies to identify the mechanism(s) underlying their assembly. We propose that LLPS, together with multiple modes of protein-nucleic acid binding, drive spatiotemporal organization of the nucleus and facilitate functional diversity among nuclear compartments.