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.

Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences.

The idea that liquid-liquid phase separation (LLPS) may be a general mechanism by which molecules in the complex cellular milieu may self-organize has generated much excitement and fervor in the cell biology community. While this concept is not new, its rise to preeminence has resulted in renewed interest in the mechanisms that shape and drive diverse cellular self-assembly processes from gene expression to cell division to stress responses. In vitro biochemical data have been instrumental in deriving some of the fundamental principles and molecular grammar by which biological molecules may phase separate, and the molecular basis of these interactions. Definitive evidence is lacking as to whether the same principles apply in the physiological environment inside living cells. In this Perspective, we analyze the evidence supporting phase separation in vivo across multiple cellular processes. We find that the evidence for in vivo LLPS is often phenomenological and inadequate to discriminate between phase separation and other possible mechanisms. Moreover, the causal relationship and functional consequences of LLPS in vivo are even more elusive. We underscore the importance of performing quantitative measurements on proteins in their endogenous state and physiological abundance, as well as make recommendations for experiments that may yield more conclusive results.

Dense Bicoid hubs accentuate binding along the morphogen gradient.

Morphogen gradients direct the spatial patterning of developing embryos; however, the mechanisms by which these gradients are interpreted remain elusive. Here we used lattice light-sheet microscopy to perform in vivo single-molecule imaging in early Drosophila melanogaster embryos of the transcription factor Bicoid that forms a gradient and initiates patterning along the anteroposterior axis. In contrast to canonical models, we observed that Bicoid binds to DNA with a rapid off rate throughout the embryo such that its average occupancy at target loci is on-rate-dependent. We further observed Bicoid forming transient "hubs" of locally high density that facilitate binding as factor levels drop, including in the posterior, where we observed Bicoid binding despite vanishingly low protein levels. We propose that localized modulation of transcription factor on rates via clustering provides a general mechanism to facilitate binding to low-affinity targets and that this may be a prevalent feature of other developmental transcription factors.

Single-molecule tracking technologies for quantifying the dynamics of gene regulation in cells, tissue and embryos.

For decades, we have relied on population and time-averaged snapshots of dynamic molecular scale events to understand how genes are regulated during development and beyond. The advent of techniques to observe single-molecule kinetics in increasingly endogenous contexts, progressing from in vitro studies to living embryos, has revealed how much we have missed. Here, we provide an accessible overview of the rapidly expanding family of technologies for single-molecule tracking (SMT), with the goal of enabling the reader to critically analyse single-molecule studies, as well as to inspire the application of SMT to their own work. We start by overviewing the basics of and motivation for SMT experiments, and the trade-offs involved when optimizing parameters. We then cover key technologies, including fluorescent labelling, excitation and detection optics, localization and tracking algorithms, and data analysis. Finally, we provide a summary of selected recent applications of SMT to study the dynamics of gene regulation.

Phase separation drives heterochromatin domain formation.

Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability, and silencing of transposon and gene expression. Heterochromatin is highly enriched for repetitive sequences, and is defined epigenetically by methylation of histone H3 at lysine 9 and recruitment of its binding partner heterochromatin protein 1 (HP1). A prevalent view of heterochromatic silencing is that these and associated factors lead to chromatin compaction, resulting in steric exclusion of regulatory proteins such as RNA polymerase from the underlying DNA. However, compaction alone does not account for the formation of distinct, multi-chromosomal, membrane-less heterochromatin domains within the nucleus, fast diffusion of proteins inside the domain, and other dynamic features of heterochromatin. Here we present data that support an alternative hypothesis: that the formation of heterochromatin domains is mediated by phase separation, a phenomenon that gives rise to diverse non-membrane-bound nuclear, cytoplasmic and extracellular compartments. We show that Drosophila HP1a protein undergoes liquid-liquid demixing in vitro, and nucleates into foci that display liquid properties during the first stages of heterochromatin domain formation in early Drosophila embryos. Furthermore, in both Drosophila and mammalian cells, heterochromatin domains exhibit dynamics that are characteristic of liquid phase-separation, including sensitivity to the disruption of weak hydrophobic interactions, and reduced diffusion, increased coordinated movement and inert probe exclusion at the domain boundary. We conclude that heterochromatic domains form via phase separation, and mature into a structure that includes liquid and stable compartments. We propose that emergent biophysical properties associated with phase-separated systems are critical to understanding the unusual behaviours of heterochromatin, and how chromatin domains in general regulate essential nuclear functions.

Chromatin topology, condensates and gene regulation: shifting paradigms or just a phase?

In the past decade, two major advances in our understanding of nuclear organization have taken the field of gene regulation by storm. First, technologies that can analyze the three-dimensional conformation of chromatin have revealed how the genome is organized and have provided novel insights into how regulatory regions in the genome interact. Second, the recognition that many proteins can form membraneless compartments through liquid-liquid phase separation (LLPS) has challenged long-standing notions of how proteins within the nucleus are organized and has offered a tantalizing general mechanism by which many aspects of nuclear function may be regulated. However, the functional roles of chromatin topology and LLPS in regulating gene expression remain poorly understood. These topics were discussed with great fervor during an open discussion held at a recent workshop titled 'Chromatin-based regulation of development' organized by The Company of Biologists. Here, we summarize the major points covered during this debate and discuss how they tie into current thinking in the field of gene regulation.

Faster and less phototoxic 3D fluorescence microscopy using a versatile compressed sensing scheme.

Three-dimensional fluorescence microscopy based on Nyquist sampling of focal planes faces harsh trade-offs between acquisition time, light exposure, and signal-to-noise. We propose a 3D compressed sensing approach that uses temporal modulation of the excitation intensity during axial stage sweeping and can be adapted to fluorescence microscopes without hardware modification. We describe implementations on a lattice light sheet microscope and an epifluorescence microscope, and show that images of beads and biological samples can be reconstructed with a 5-10 fold reduction of light exposure and acquisition time. Our scheme opens a new door towards faster and less damaging 3D fluorescence microscopy.

Dysregulation in microRNA expression in peripheral blood mononuclear cells of sepsis patients is associated with immunopathology.

Sepsis is a major cause of death worldwide. It triggers systemic inflammation, the role of which remains unclear. In the current study, we investigated the induction of microRNA (miRNA) during sepsis and their role in the regulation of inflammation. Patients, on days 1 and 5 following sepsis diagnosis, had reduced T cells but elevated monocytes. Plasma levels of IL-6, IL-8, IL-10 and MCP-1 dramatically increased in sepsis patients on day 1. T cells from sepsis patients differentiated primarily into Th2 cells, whereas regulatory T cells decreased. Analysis of 1163 miRNAs from PBMCs revealed that miR-182, miR-143, miR-145, miR-146a, miR-150, and miR-155 were dysregulated in sepsis patients. miR-146a downregulation correlated with increased IL-6 expression and monocyte proliferation. Bioinformatics analysis uncovered the immunological associations of dysregulated miRNAs with clinical disease. The current study demonstrates that miRNA dysregulation correlates with clinical manifestations and inflammation, and therefore remains a potential therapeutic target against sepsis.

Optical measurement of cycle-dependent cell growth.

Determining the growth patterns of single cells offers answers to some of the most elusive questions in contemporary cell biology: how cell growth is regulated and how cell size distributions are maintained. For example, a linear growth in time implies that there is no regulation required to maintain homeostasis; an exponential pattern indicates the opposite. Recently, there has been great effort to measure single cells using microelectromechanical systems technology, and several important questions have been explored. However, a unified, easy-to-use methodology to measure the growth rate of individual adherent cells of various sizes has been lacking. Here we demonstrate that a newly developed optical interferometric technique, known as spatial light interference microscopy, can measure the cell dry mass of many individual adherent cells in various conditions, over spatial scales from micrometers to millimeters, temporal scales ranging from seconds to days, and cell types ranging from bacteria to mammalian cells. We found evidence of exponential growth in Escherichia coli, which agrees very well with other recent reports. Perhaps most importantly, combining spatial light interference microscopy with fluorescence imaging provides a unique method for studying cell cycle-dependent growth. Thus, by using a fluorescent reporter for the S phase, we measured single cell growth over each phase of the cell cycle in human osteosarcoma U2OS cells and found that the G2 phase exhibits the highest growth rate, which is mass-dependent and can be approximated by an exponential.

Dynamic microenvironments shape nuclear organization and gene expression.

Live imaging has revealed that the regulation of gene expression is largely driven by transient interactions. For example, many regulatory proteins bind chromatin for just seconds, and loop-like genomic contacts are rare and last only minutes. These discoveries have been difficult to reconcile with our canonical models that are predicated on stable and hierarchical interactions. Proteomic microenvironments that concentrate nuclear factors may explain how brief interactions can still mediate gene regulation by creating conditions where reactions occur more frequently. Here, we summarize new imaging technologies and recent discoveries implicating microenvironments as a potential driver of nuclear function. Finally, we propose that key properties of proteomic microenvironments, such as their size, enrichment, and lifetimes, are directly linked to regulatory function.

A fine kinetic balance of interactions directs transcription factor hubs to genes.

Eukaryotic gene regulation relies on the binding of sequence-specific transcription factors (TFs). TFs bind chromatin transiently yet occupy their target sites by forming high-local concentration microenvironments (hubs and condensates) that increase the frequency of binding events. Despite their ubiquity, such microenvironments have been difficult to study in endogenous contexts due to technical limitations. Here, we overcome these limitations and investigate how hubs drive TF occupancy at their targets. Using a DNA binding perturbation to a hub-forming TF, Zelda, in Drosophila embryos, we find that hub properties, including the stability and frequencies of associations to targets, are key determinants of TF occupancy. Our data suggest that the targeting of these hubs is driven not just by specific DNA motif recognition, but also by a fine-tuned kinetic balance of interactions between TFs and their co-binding partners.

Imipridones inhibit tumor growth and improve survival in an orthotopic liver metastasis mouse model of human uveal melanoma.

Purpose: Uveal melanoma (UM) is a highly aggressive disease with very few treatment options. We previously demonstrated that mUM is characterized by high oxidative phosphorylation (OXPHOS). Here we tested the anti-tumor, signaling and metabolic effects of imipridones, CLPP activators which reduce OXPHOS indirectly and have demonstrated safety in patients. Experimental Design: We assessed CLPP expression in UM patient samples. We tested the effects of imipridones (ONC201, ONC212) on the growth, survival, signaling and metabolism of UM cell lines in vitro, and for therapeutic effects in vivo in UM liver metastasis models. Results: CLPP expression was confirmed in primary and mUM patient samples. ONC201/212 treatment of UM cell lines in vitro decreased OXPHOS effectors, inhibited cell growth and migration, and induced apoptosis. ONC212 increased metabolic stress and apoptotic pathways, inhibited amino acid metabolism, and induced cell death-related lipids. ONC212 also decreased tumor burden and increased survival in vivo in two UM liver metastasis models. Conclusion: Imipridones are a promising strategy for further testing and development in mUM.

Nanoscale fluctuations and surface tension measurements in droplets using phase-resolved low-coherence interferometry.

A common-path interferometer was designed with rapidly tunable broadband swept laser source, which provides quantitative phase measurements of nanometer scale motions with very high sensitivity. With this setup, we are able to detect the thermal fluctuations in liquid droplets hanging at the end of an optical fiber. The measured nanoscale displacement fluctuations of various droplet surfaces were used to extract the surface tension. This newly developed technique proved the feasibility of noninvasive, fast, phase-resolved dynamic light scattering measurement of fluid mechanical properties.

Localization of the Drosophila pioneer factor GAF to subnuclear foci is driven by DNA binding and required to silence satellite repeat expression.

The eukaryotic genome is organized to enable the precise regulation of gene expression. This organization is established as the embryo transitions from a fertilized gamete to a totipotent zygote. To understand the factors and processes that drive genomic organization, we focused on the pioneer factor GAGA factor (GAF) that is required for early development in Drosophila. GAF transcriptionally activates the zygotic genome and is localized to subnuclear foci. This non-uniform distribution is driven by binding to highly abundant GA repeats. At GA repeats, GAF is necessary to form heterochromatin and silence transcription. Thus, GAF is required to establish both active and silent regions. We propose that foci formation enables GAF to have opposing transcriptional roles within a single nucleus. Our data support a model in which the subnuclear concentration of transcription factors acts to organize the nucleus into functionally distinct domains essential for the robust regulation of gene expression.

Diffraction phase microscopy with white light.

We present white light diffraction phase microscopy (wDPM) as a quantitative phase imaging method that combines the single shot measurement benefit associated with off-axis methods, high temporal phase stability associated with common path geometries, and high spatial phase sensitivity due to the white light illumination. We propose a spatiotemporal filtering method that pushes the limit of the pathlength sensitivity to the subangstrom level at practical spatial and temporal bandwidths. We illustrate the utility of wDPM with measurements on red blood cell morphology and HeLa cell growth over 18 hours.

Spatial light interference microscopy (SLIM).

We present spatial light interference microscopy (SLIM) as a new optical microscopy technique, capable of measuring nanoscale structures and dynamics in live cells via interferometry. SLIM combines two classic ideas in light imaging: Zernike's phase contrast microscopy, which renders high contrast intensity images of transparent specimens, and Gabor's holography, where the phase information from the object is recorded. Thus, SLIM reveals the intrinsic contrast of cell structures and, in addition, renders quantitative optical path-length maps across the sample. The resulting topographic accuracy is comparable to that of atomic force microscopy, while the acquisition speed is 1,000 times higher. We illustrate the novel insight into cell dynamics via SLIM by experiments on primary cell cultures from the rat brain. SLIM is implemented as an add-on module to an existing phase contrast microscope, which may prove instrumental in impacting the light microscopy field at a large scale.

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.

Diffraction Phase Cytometry: blood on a CD-ROM.

We demonstrate Diffraction Phase Cytometry (DPC) as a single shot, full-field, high throughput quantitative phase imaging modality, dedicated to analyzing whole blood smears. Utilizing a commercial CD as a sample substrate, along with dynamic spatial filtering via a liquid crystal spatial light modulator, we have developed a compact instrument capable of making quantitative, physiologically relevant measurements. To illustrate the ability of the system to function as a highly sensitive cytometer we imaged a large number (N=1,537) of live human erythrocytes in whole blood without preparation. We retrieved a comprehensive set of geometrical parameters including cell volume and surface area, which are not directly available using existing cytometers. Furthermore, we retrieved the minimum cylindrical diameter, through which red blood cells can pass, and deliver oxygen. These initial results prove the concept for an inexpensive lab-on-a-chip blood screening device.

A new class of disordered elements controls DNA replication through initiator self-assembly.

The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.