Transcription activation is enhanced by multivalent interactions independent of phase separation.

Transcription factors (TFs) consist of a DNA-binding domain and an activation domain (AD) that are frequently considered to be independent and exchangeable modules. However, recent studies report that the physicochemical properties of the AD can control TF assembly at chromatin by driving phase separation into transcriptional condensates. Here, we dissected transcription activation by comparing different synthetic TFs at a reporter gene array with real-time single-cell fluorescence microscopy. In these experiments, binding site occupancy, residence time, and coactivator recruitment in relation to multivalent TF interactions were compared. While phase separation propensity and activation strength of the AD were linked, the actual formation of liquid-like TF droplets had a neutral or inhibitory effect on transcription activation. We conclude that multivalent AD-mediated interactions enhance the transcription activation capacity of a TF by increasing its residence time in the chromatin-bound state and facilitating the recruitment of coactivators independent of phase separation.

Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation.

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.

ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats.

Alternative lengthening of telomeres (ALT) occurs in ∼10% of cancer entities. However, little is known about the heterogeneity of ALT activity since robust ALT detection assays with high-throughput in situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate ALT activity in single cells from the accumulation of single-stranded telomeric DNA and RNA. It involves a one-step fluorescent in situ hybridization approach followed by fluorescence microscopy imaging. Our method reliably identified ALT in cancer cell lines from different tumor entities and was validated in three established models of ALT induction and suppression. Furthermore, we successfully applied ALT-FISH to spatially resolve ALT activity in primary tissue sections from leiomyosarcoma and neuroblastoma tumors. Thus, our assay provides insights into the heterogeneity of ALT tumors and is suited for high-throughput applications, which will facilitate screening for ALT-specific drugs.

Systems in Flames: Dynamic Coproduction of Social-Ecological Processes.

Ecologists who study human-dominated places have adopted a social-ecological systems framework to recognize the coproduced links between ecological and social processes. However, many social scientists are wary of the way ecologists use the systems concept to represent such links. This wariness is sometimes due to a misunderstanding of the contemporary use of the systems concept in ecology. We aim to overcome this misunderstanding by discussing the contemporary systems concept using refinements from biophysical ecology. These refinements allow the systems concept to be used as a bridge rather than a barrier to social-ecological interaction. We then use recent examples of extraordinary fire to illustrate the usefulness and flexibility of the concept for understanding the dynamism of fire as a social-ecological interaction. The systems idea is a useful interdisciplinary abstraction that can be contextualized to account for societally important problems and dynamics.

Real-time observation of light-controlled transcription in living cells.

Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed blue light-induced chromatin recruitment (BLInCR) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a cluster of reporter genes in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ∼2 min after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models of transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.This article has an associated First Person interview with the first author of the paper.

Specificity, propagation, and memory of pericentric heterochromatin.

The cell establishes heritable patterns of active and silenced chromatin via interacting factors that set, remove, and read epigenetic marks. To understand how the underlying networks operate, we have dissected transcriptional silencing in pericentric heterochromatin (PCH) of mouse fibroblasts. We assembled a quantitative map for the abundance and interactions of 16 factors related to PCH in living cells and found that stably bound complexes of the histone methyltransferase SUV39H1/2 demarcate the PCH state. From the experimental data, we developed a predictive mathematical model that explains how chromatin-bound SUV39H1/2 complexes act as nucleation sites and propagate a spatially confined PCH domain with elevated histone H3 lysine 9 trimethylation levels via chromatin dynamics. This "nucleation and looping" mechanism is particularly robust toward transient perturbations and stably maintains the PCH state. These features make it an attractive model for establishing functional epigenetic domains throughout the genome based on the localized immobilization of chromatin-modifying enzymes.

Compartments in medulloblastoma with extensive nodularity are connected through differentiation along the granular precursor lineage.

Medulloblastomas with extensive nodularity are cerebellar tumors characterized by two distinct compartments and variable disease progression. The mechanisms governing the balance between proliferation and differentiation in MBEN remain poorly understood. Here, we employ a multi-modal single cell transcriptome analysis to dissect this process. In the internodular compartment, we identify proliferating cerebellar granular neuronal precursor-like malignant cells, along with stromal, vascular, and immune cells. In contrast, the nodular compartment comprises postmitotic, neuronally differentiated malignant cells. Both compartments are connected through an intermediate cell stage resembling actively migrating CGNPs. Notably, we also discover astrocytic-like malignant cells, found in proximity to migrating and differentiated cells at the transition zone between the two compartments. Our study sheds light on the spatial tissue organization and its link to the developmental trajectory, resulting in a more benign tumor phenotype. This integrative approach holds promise to explore intercompartmental interactions in other cancers with varying histology.

Assessing the Phase Separation Propensity of Proteins in Living Cells Through Optodroplet Formation.

Phase separation is emerging as a key mechanism to describe the formation of membraneless organelles in the cell. It depends on the multivalent (self-) interaction properties of the macromolecules involved and can be observed in aqueous solutions under controlled conditions in vitro with purified components. However, to experimentally demonstrate that this process indeed occurs in the complex environment of living cells remains difficult. Here, we describe an assay based on light-induced association of proteins into complexes termed optodroplets that are in the hundred nm to µm size range. The formation and dissociation of these optodroplets can be followed over time in living cells by fluorescence microscopy to evaluate the propensity of proteins to demix and to form phase-separated subcompartments. The optodroplet assay is based on the fusion of a protein of interest with the photolyase homology region (PHR) protein domain from Arabidopsis thaliana, which can undergo reversible homo-oligomerization upon illumination with blue light. Using this approach, candidate proteins and their interaction-deficient or interaction-enhanced variants can be compared to each other or to reference proteins with known phase separation features. By quantifying the resulting microscopy images, the propensity of a given protein construct to assemble into a phase-separated subcompartment can be assessed.

DNA sequence-dependent formation of heterochromatin nanodomains.

The mammalian epigenome contains thousands of heterochromatin nanodomains (HNDs) marked by di- and trimethylation of histone H3 at lysine 9 (H3K9me2/3), which have a typical size of 3-10 nucleosomes. However, what governs HND location and extension is only partly understood. Here, we address this issue by introducing the chromatin hierarchical lattice framework (ChromHL) that predicts chromatin state patterns with single-nucleotide resolution. ChromHL is applied to analyse four HND types in mouse embryonic stem cells that are defined by histone methylases SUV39H1/2 or GLP, transcription factor ADNP or chromatin remodeller ATRX. We find that HND patterns can be computed from PAX3/9, ADNP and LINE1 sequence motifs as nucleation sites and boundaries that are determined by DNA sequence (e.g. CTCF binding sites), cooperative interactions between nucleosomes as well as nucleosome-HP1 interactions. Thus, ChromHL rationalizes how patterns of H3K9me2/3 are established and changed via the activity of protein factors in processes like cell differentiation.

Tracing Reversible Light-Induced Chromatin Binding with Near-infrared Fluorescent Proteins.

Blue light-induced chromatin recruitment (BLInCR) is a versatile optogenetic tool to enrich effector proteins at specific loci within the nucleus using illumination in the 400-500 nm range. The resulting chromatin binding reaction is reversible on the time scale of minutes. BLInCR is advantageous over ligand-binding induced methods since it does not require a change of growth medium for the relatively slow depletion of the inducer from the nucleus. However, applying BLInCR for reversibility experiments is challenging because of the need to spectrally separate light-induced activation from visualization of the chromatin locus and effector and/or reader proteins by light microscopy. Here, we describe an improved BLInCR protocol for light-dependent association and dissociation of effectors using the near-infrared fluorescent protein iRFP713. Due to its spectral properties, iRFP713 can be detected separately from the red fluorescent protein mCherry. Thus, it becomes possible to trace two proteins labeled with iRFP713 and mCherry independently of the light activation reaction. This approach largely facilitates applications of the BLInCR system for experiments that test the reversibility, persistence, and memory of chromatin states.

Light-Induced Transcription Activation for Time-Lapse Microscopy Experiments in Living Cells.

Gene expression can be monitored in living cells via the binding of fluorescently tagged proteins to RNA repeats engineered into a reporter transcript. This approach makes it possible to trace temporal changes of RNA production in real time in living cells to dissect transcription regulation. For a mechanistic analysis of the underlying activation process, it is essential to induce gene expression with high accuracy. Here, we describe how this can be accomplished with an optogenetic approach termed blue light-induced chromatin recruitment (BLInCR). It employs the recruitment of an activator protein to a target promoter via the interaction between the PHR and CIBN plant protein domains. This process occurs within seconds after setting the light trigger and is reversible. Protocols for continuous activation as well as pulsed activation and reactivation with imaging either by laser scanning confocal microscopy or automated widefield microscopy are provided. For the semiautomated quantification of the resulting image series, an approach has been implemented in a set of scripts in the R programming language. Thus, the complete workflow of the BLInCR method is described for mechanistic studies of the transcription activation process as well as the persistence and memory of the activated state.

Applying microdroplets as sensors for label-free detection of chemical reactions.

Despite its tremendous high-throughput screening capabilities, widespread applications of droplet-based microfluidics are still limited by the poor availability of appropriate analytical assays. Here we report on a novel sensor method that exploits the osmosis-driven change in droplet size as a quantitative and label-free marker for reactions inside the droplets. We present an analysis of the underlying mechanism and apply the method for monitoring metabolic activity at a single-cell level.