How transcription factor binding controls transcriptional bursting dynamics: A single-molecule view.

In this issue, Pomp et al.1 simultaneously tracked transcriptional bursts of yeast gene GAL10 and transient binding of transcription factor Gal4 at the gene using novel methods. Dynamic exchange and infrequent long binding of Gal4 together enable prolonged transcriptional bursts of GAL10.

Single-Molecule Measurement of Protein Interaction Dynamics within Biomolecular Condensates.

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

Meet the authors: Lizhen Chen, Shasha Chong, and Zhijie "Jason" Liu.

Here, Molecular Cell talks to first and co-corresponding author Lizhen Chen and co-corresponding authors Shasha Chong and Zhijie "Jason" Liu about their paper, ''Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions'' (in this issue of Molecular Cell) and their scientific journeys until now.

Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions.

Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.

Visualizing Protein Localizations in Fixed Cells: Caveats and the Underlying Mechanisms.

Fluorescence microscopy techniques have been widely adopted in biology for their ability to visualize the structure and dynamics of a wide range of cellular and subcellular processes. The specificity and sensitivity that these techniques afford have made them primary tools in the characterization of protein localizations within cells. Many of the fluorescence microscopy techniques require cells to be fixed via chemical or alternative methods before being imaged. However, some fixation methods have been found to induce the redistribution of particular proteins in the cell, resulting in artifacts in the characterization of protein localizations and functions under physiological conditions. Here, we review the ability of commonly used cell fixation methods to faithfully preserve the localizations of proteins that bind to chromatin, undergo liquid-liquid phase separation (LLPS), and are involved in the formation of various membrane-bound organelles. We also review the mechanisms underlying various fixation artifacts and discuss potential alternative fixation methods to minimize the artifacts while investigating different proteins and cellular structures. Overall, fixed-cell fluorescence microscopy is a very powerful tool in biomedical research; however, each experiment demands the careful selection of an appropriate fixation method to avoid potential artifacts and may benefit from live-cell imaging validation.

Phase Separation in Biology and Disease; Current Perspectives and Open Questions.

In the past almost 15 years, we witnessed the birth of a new scientific field focused on the existence, formation, biological functions, and disease associations of membraneless bodies in cells, now referred to as biomolecular condensates. Pioneering studies from several laboratories [reviewed in1-3] supported a model wherein biomolecular condensates associated with diverse biological processes form through the process of phase separation. These and other findings that followed have revolutionized our understanding of how biomolecules are organized in space and time within cells to perform myriad biological functions, including cell fate determination, signal transduction, endocytosis, regulation of gene expression and protein translation, and regulation of RNA metabolism. Further, condensates formed through aberrant phase transitions have been associated with numerous human diseases, prominently including neurodegeneration and cancer. While in some cases, rigorous evidence supports links between formation of biomolecular condensates through phase separation and biological functions, in many others such links are less robustly supported, which has led to rightful scrutiny of the generality of the roles of phase separation in biology and disease.4-7 During a week-long workshop in March 2022 at the Telluride Science Research Center (TSRC) in Telluride, Colorado, ∼25 scientists addressed key questions surrounding the biomolecular condensates field. Herein, we present insights gained through these discussions, addressing topics including, roles of condensates in diverse biological processes and systems, and normal and disease cell states, their applications to synthetic biology, and the potential for therapeutically targeting biomolecular condensates.

Mutant NPM1 Hijacks Transcriptional Hubs to Maintain Pathogenic Gene Programs in Acute Myeloid Leukemia.

Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of biological functions. In 30% of acute myeloid leukemia (AML), the terminal exon of NPM1 is often found mutated, resulting in the addition of a nuclear export signal and a shift of the protein to the cytoplasm (NPM1c). AMLs carrying this mutation have aberrant expression of the HOXA/B genes, whose overexpression leads to leukemogenic transformation. Here, for the first time, we comprehensively prove that NPM1c binds to a subset of active gene promoters in NPM1c AMLs, including well-known leukemia-driving genes-HOXA/B cluster genes and MEIS1. NPM1c sustains the active transcription of key target genes by orchestrating a transcription hub and maintains the active chromatin landscape by inhibiting the activity of histone deacetylases. Together, these findings reveal the neomorphic function of NPM1c as a transcriptional amplifier for leukemic gene expression and open up new paradigms for therapeutic intervention. SIGNIFICANCE: NPM1 mutation is the most common mutation in AML, yet the mechanism of how the mutant protein results in AML remains unclear. Here, for the first time, we prove mutant NPM1 directly binds to active chromatin regions and hijacks the transcription of AML-driving genes. See related article by Uckelmann et al., p. 746. This article is highlighted in the In This Issue feature, p. 517.

Fixation can change the appearance of phase separation in living cells.

Fixing cells with paraformaldehyde (PFA) is an essential step in numerous biological techniques as it is thought to preserve a snapshot of biomolecular transactions in living cells. Fixed-cell imaging techniques such as immunofluorescence have been widely used to detect liquid-liquid phase separation (LLPS) in vivo. Here, we compared images, before and after fixation, of cells expressing intrinsically disordered proteins that are able to undergo LLPS. Surprisingly, we found that PFA fixation can both enhance and diminish putative LLPS behaviors. For specific proteins, fixation can even cause their droplet-like puncta to artificially appear in cells that do not have any detectable puncta in the live condition. Fixing cells in the presence of glycine, a molecule that modulates fixation rates, can reverse the fixation effect from enhancing to diminishing LLPS appearance. We further established a kinetic model of fixation in the context of dynamic protein-protein interactions. Simulations based on the model suggest that protein localization in fixed cells depends on an intricate balance of protein-protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. Consistent with simulations, live-cell single-molecule imaging experiments showed that a fast overall rate of fixation relative to protein-protein interaction dynamics can minimize fixation artifacts. Our work reveals that PFA fixation changes the appearance of LLPS from living cells, presents a caveat in studying LLPS using fixation-based methods, and suggests a mechanism underlying the fixation artifact.

A typical human cell is a crowded soup of thousands of different proteins. One way that the cell organizes this complex mix of contents is by creating separate droplets within the cell, like oil in water. These droplets can form through a process known as liquid-liquid phase separation, or LLPS, where specific proteins gather in high concentrations to carry out their cellular roles. The critical role of LLPS in cellular organization means that it is widely studied by biologists. To detect LLPS, researchers often subject the cells to treatments designed to hold all the proteins in place, creating a snapshot of their natural state. This process, known as fixing, allows scientists to easily label a protein with a fluorescent tag, take pictures of the cells, and look at whether the protein forms droplets in its natural state. This is often easier to do than imaging cells live, but it relies on LLPS being well-preserved upon fixation. To test if this is true, Irgen-Gioro, Yoshida et al. looked at protein droplets in live cells, and then fixed the cells to check whether the appearance of the droplets had changed. The images taken showed that fixation could alter the size and number of droplets depending on the protein being studied. To explain why the effects of fixing change depending on the protein, Irgen-Gioro, Yoshida et al. hypothesized that a faster fixation ­ relative to how quickly proteins can bind and unbind to their droplets ­ can better preserve the LLPS droplets. They verified their idea using a microscopy technique in which they imaged single molecules, allowing them to see how different fixation speeds relative to protein binding affected the droplets. The work of Irgen-Gioro, Yoshida et al. identifies an important caveat to using fixation for the study of LLPS in cells. Their findings suggest that researchers should be cautious when interpreting the results of such studies. Given that LLPS in cells is an area of research with a lot of interest, these results could benefit a broad range of biological and medical fields. In the future, Irgen-Gioro, Yoshida et al.'s findings could prompt scientists to develop new fixing methods that better preserve LLPS in cells.

Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription.

Gene activation by mammalian transcription factors (TFs) requires multivalent interactions of their low-complexity domains (LCDs), but how such interactions regulate transcription remains unclear. It has been proposed that extensive LCD-LCD interactions culminating in liquid-liquid phase separation (LLPS) of TFs is the dominant mechanism underlying transactivation. Here, we investigated how tuning the amount and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. Quantitative single-cell and single-molecule imaging reveals that the oncogenic TF EWS::FLI1 requires a narrow optimum of LCD-LCD interactions to activate its target genes associated with GGAA microsatellites. Increasing LCD-LCD interactions toward putative LLPS represses transcription of these genes in patient-derived cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS::FLI1 into a well-documented LLPS compartment, the nucleolus, inhibits EWS::FLI1-driven transcription and oncogenic transformation. Our findings show how altering the balance of LCD-LCD interactions can influence transcriptional regulation and suggest a potential therapeutic strategy for targeting disease-causing TFs.

TRIM8 modulates the EWS/FLI oncoprotein to promote survival in Ewing sarcoma.

Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif-containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared with >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion oncoprotein-specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF-driven cancers.

Towards Decoding the Sequence-Based Grammar Governing the Functions of Intrinsically Disordered Protein Regions.

A substantial portion of the proteome consists of intrinsically disordered regions (IDRs) that do not fold into well-defined 3D structures yet perform numerous biological functions and are associated with a broad range of diseases. It has been a long-standing enigma how different IDRs successfully execute their specific functions. Further putting a spotlight on IDRs are recent discoveries of functionally relevant biomolecular assemblies, which in some cases form through liquid-liquid phase separation. At the molecular level, the formation of biomolecular assemblies is largely driven by weak, multivalent, but selective IDR-IDR interactions. Emerging experimental and computational studies suggest that the primary amino acid sequences of IDRs encode a variety of their interaction behaviors. In this review, we focus on findings and insights that connect sequence-derived features of IDRs to their conformations, propensities to form biomolecular assemblies, selectivity of interaction partners, functions in the context of physiology and disease, and regulation of function. We also discuss directions of future research to facilitate establishing a comprehensive sequence-function paradigm that will eventually allow prediction of selective interactions and specificity of function mediated by IDRs.

Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection.

The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid-based tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the U.S. Centers for Disease Control and Prevention takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply, and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the "gold-standard" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own laboratories. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.

Impaired cell fate through gain-of-function mutations in a chromatin reader.

Modifications of histone proteins have essential roles in normal development and human disease. Recognition of modified histones by 'reader' proteins is a key mechanism that mediates the function of histone modifications, but how the dysregulation of these readers might contribute to disease remains poorly understood. We previously identified the ENL protein as a reader of histone acetylation via its YEATS domain, linking it to the expression of cancer-driving genes in acute leukaemia1. Recurrent hotspot mutations have been found in the ENL YEATS domain in Wilms tumour2,3, the most common type of paediatric kidney cancer. Here we show, using human and mouse cells, that these mutations impair cell-fate regulation by conferring gain-of-function in chromatin recruitment and transcriptional control. ENL mutants induce gene-expression changes that favour a premalignant cell fate, and, in an assay for nephrogenesis using murine cells, result in undifferentiated structures resembling those observed in human Wilms tumour. Mechanistically, although bound to largely similar genomic loci as the wild-type protein, ENL mutants exhibit increased occupancy at a subset of targets, leading to a marked increase in the recruitment and activity of transcription elongation machinery that enforces active transcription from target loci. Furthermore, ectopically expressed ENL mutants exhibit greater self-association and form discrete and dynamic nuclear puncta that are characteristic of biomolecular hubs consisting of local high concentrations of regulatory factors. Such mutation-driven ENL self-association is functionally linked to enhanced chromatin occupancy and gene activation. Collectively, our findings show that hotspot mutations in a chromatin-reader domain drive self-reinforced recruitment, derailing normal cell-fate control during development and leading to an oncogenic outcome.

Imaging dynamic and selective low-complexity domain interactions that control gene transcription.

Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity sequence domains (LCDs), but how these LCDs drive transactivation remains unclear. We used live-cell single-molecule imaging to reveal that TF LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the RNA Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for drugs targeting gene regulatory interactions implicated in disease.

Mechanism of transcriptional bursting in bacteria.

Transcription of highly expressed genes has been shown to occur in stochastic bursts. But the origin of such ubiquitous phenomenon has not been understood. Here, we present the mechanism in bacteria. We developed a high-throughput, in vitro, single-molecule assay to follow transcription on individual DNA templates in real time. We showed that positive supercoiling buildup on a DNA segment by transcription slows down transcription elongation and eventually stops transcription initiation. Transcription can be resumed upon gyrase binding to the DNA segment. Furthermore, using single-cell mRNA counting fluorescence in situ hybridization (FISH), we found that duty cycles of transcriptional bursting depend on the intracellular gyrase concentration. Together, these findings prove that transcriptional bursting of highly expressed genes in bacteria is primarily caused by reversible gyrase dissociation from and rebinding to a DNA segment, changing the supercoiling level of the segment.

Probing allostery through DNA.

Allostery is well documented for proteins but less recognized for DNA-protein interactions. Here, we report that specific binding of a protein on DNA is substantially stabilized or destabilized by another protein bound nearby. The ternary complex's free energy oscillates as a function of the separation between the two proteins with a periodicity of ~10 base pairs, the helical pitch of B-form DNA, and a decay length of ~15 base pairs. The binding affinity of a protein near a DNA hairpin is similarly dependent on their separation, which-together with molecular dynamics simulations-suggests that deformation of the double-helical structure is the origin of DNA allostery. The physiological relevance of this phenomenon is illustrated by its effect on gene expression in live bacteria and on a transcription factor's affinity near nucleosomes.

Imaging chromophores with undetectable fluorescence by stimulated emission microscopy.

Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging. However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. Here we use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, and report a new contrast mechanism for optical microscopy. In a pump-probe experiment, on photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, the intensity of which is concurrently increased. We extract the miniscule intensity increase with shot-noise-limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz frequency. The signal is generated only at the laser foci owing to the nonlinear dependence on the input intensities, providing intrinsic three-dimensional optical sectioning capability. In contrast, conventional one-beam absorption measurement exhibits low sensitivity, lack of three-dimensional sectioning capability, and complication by linear scattering of heterogeneous samples. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging.

An extensive ylide thermodynamic stability scale predicted by first-principle calculations.

Thermodynamic stabilities of ylides are measured by the ease of the carbanion formation through the removal of a proton from their precursors. A full-spectrum scale of ylide thermodynamic stability is important to understand the reactivities and selectivities in ylide chemistry. In the present study is reported the first theoretical protocol for predicting the acidities of structurally unrelated ylide precursors in DMSO whose reliability has been tested against almost all the available experimental data. The ONIOM/G3B3//HF//CPCM/Bondi method is found to be the optimal protocol to handle the N-, P-, and S-containing ylides, whereas the B3LYP/6-311+G(3df,2p)//B3LYP/6-31G(d)//HF//CPCM/Bondi method can be used to deal with those systems for which the ONIOM/G3B3 method is not feasible. Extensive calculations on about 80 experimentally characterized ylide precursors show that this theoretical protocol can reliably predict the pK(a) values of diverse structurally unrelated ylide precursors in DMSO with an error bar of ca. 1.6-1.9 pK(a) units. With the authorized theoretical protocol in hand, we have developed an extensive scale of ylide thermodynamic stability that may find applications in synthetic organic chemistry.

Ring opening versus ring expansion in rearrangement of bicyclic cyclobutylcarbinyl radicals.

Ring opening and expansion of multicyclic cyclobutylcarbinyl radicals provides an appealing method for the construction of heavily substituted ring systems in a stereocontrollable fashion. Here we conducted the first, systematic study on the regioselectivity in the rearrangement of various synthetically relevant cyclobutylcarbinyl radicals. It was found that a two-layer ONIOM method, namely ONIOM(QCISD(T)/6-311+G(2d,2p):B3LYP/6-311+G(2df,2p)), could accurately predict the free energy barriers of the ring openings of cyclobutylcarbinyl radicals with a precision of 0.3 kcal/mol. By using this powerful tool we found that the regiochemistry for the ring opening of monocyclic cyclobutylcarbinyl radicals could be easily predicted by the relative stability of the two possible carbon radical products. A linear correlation was found between the activation and reaction free energies. This observation indicated that the ring opening of cyclobutylcarbinyl radicals was strongly affected by the thermodynamic factors. On the basis of the above results we extended our study to the rearrangement of bicyclic cyclobutylcarbinyl radicals that could undergo both ring opening and expansion. It was found that for bicyclic cyclobutylcarbinyl radicals whose radical center was located at the bridge methyl group, ring expansion was the favored rearrangement pathway unless a strongly radical-stabilizing substituent was placed in the cyclobutyl ring adjacent to the bridge methyl group. On the other hand, for bicyclic cyclobutylcarbinyl radicals whose radical center was located at the 2-position, ring opening was the favored rearrangement pathway unless a strongly radical-stabilizing substituent was placed in the cyclobutyl ring at the bridge position.

O-H bond dissociation enthalpies of oximes: a theoretical assessment and experimental implications.

By using a multilayer composite ab initio method ONION-G3B3, we calculated O-H bond dissociation enthalpies (BDEs) of 58 oximes that were measured experimentally. Experimental BDEs derived from thermal decomposition kinetics and calorimetric measurements were found to be consistent with the theory. However, the electrochemical method was found to give questionably high BDEs possibly due to errors in the measurement of pKa's or redox potentials. Subsequently, the performances of a variety of DFT functionals including B3LYP, B3P86, B3PW91, BHandH, BHandHLYP, BMK, PBE1PBE, MPW1KCIS, mPWPW91, MPW1B95, and MPW1K were tested to calculate oxime O-H BDEs, where ROBHandHLYP was found to be the most accurate. By using this method, we calculated O-H BDEs of over 140 oximes in a systematic fashion. All of the calculated O-H BDEs fell in the range from 76.8 to 89.8 kcal/mol. An amino group on the azomethine carbon was found to strengthen the O-H bond, whereas bulky alkyl substituents on oximes decreased O-H BDEs due to their large steric-strain-relieving effects in the process of O-H bond cleavage. Para substituents had little effect on the BDEs of benzaldoximes and phenyl methyl ketoximes. Finally, on the basis of a spin distribution calculation, aryl-, alkyl-, and carbonyl-substituted iminoxyl radicals were found to be sigma-radicals, whereas amino-substituted iminoxyl radicals were of pi-structure.