Session N38: Biomolecular Condensates in the Cell Nucleus

Condensates in Chromatin Organization and Cancer

Biomolecular condensates assemble in living cells through phase separation and related phase transitions. An underappreciated feature of these dynamic molecular assemblies is that they form interfaces with cellular structures, including membranes, cytoskeleton, DNA and RNA, and other membraneless compartments. These interfaces are expected to give rise to capillary forces, but there are few ways of quantifying and harnessing these forces in living cells. Here, we introduce VECTOR (ViscoElastic Chromatin Tethering and ORganization), which uses light-inducible biomolecular condensates to generate capillary forces at targeted DNA loci. VECTOR can be utilized to programmably reposition genomic loci on a timescale of seconds to minutes, quantitatively revealing local heterogeneity in the viscoelastic material properties of chromatin. These synthetic condensates are built from components that naturally form liquid-like structures in living cells, highlighting the potential role for native condensates to generate forces and do work to reorganize the genome and impact chromatin architecture.   

Imaging transcription condensates in three dimensional genomes

Imaging transcription condensates in three dimensional genome

YAP/TEAD signaling is essential for organismal development, cell proliferation, and cancer progression. As a transcriptional coactivator, how YAP activates its downstream target genes is incompletely understood. YAP forms biomolecular condensates in response to hyperosmotic stress, concentrating transcription-related factors to activate downstream target genes. However, whether YAP forms condensates under other signals, how YAP condensates organize and function, and how YAP condensates activate transcription in general are unknown. Here, we report that endogenous YAP forms sub-micron scale condensates in response to Hippo pathway regulation and actin cytoskeletal tension. The transcription factor TEAD1 actively stabilizes YAP condensates, which also recruit BRD4, a coactivator that is enriched at active enhancers. Using single molecule tracking, we found that YAP condensates slowed YAP diffusion within condensate boundaries, a possible mechanism for promoting YAP target search. These results reveal that YAP condensate formation is a highly regulated process that is critical for YAP/TEAD target gene expression.   

The ripening dynamics of nuclear biomolecular condensates

Bio-molecular condensates are implicated in many cellular processes. Condensate growth dynamics within the complex cellular environment are therefore of great importance. Chromatin mechanics have been shown to restrict condensate coarsening in the nucleus but the role of condensate properties on these growth dynamics is not well understood. Here, we show that condensate surface tension and the interplay with chromatin mechanics govern condensate growth in the nucleus. By using a chemical dimerization approach to induce condensates with different proteins in live cell nuclei, we found that one grew mainly through diffusion while the other grew mainly by ripening. To explain these distinct growth patterns, we developed a quantitative physical model that assessed nuclear condensate growth as diffusion-based growth in an elastic mesh network, where the condensates experience size dependent pressure from the chromatin. Our model reveals that alongside local chromatin stiffness gradients, the condensate surface tension influences the ripening propensity of condensates and can explain the experimental growth patterns. By matching the experimentally observed ripening patterns with model predictions, we reveal that the condensate with greater surface tension undergoes Ostwald ripening while the condensate with lower surface tension experiences suppressed Ostwald ripening and occasionally, Elastic ripening. The model can also explain the difference in the growth exponent for the two condensates and predicts that the growth of high surface tension condensate would be less sensitive to changes in chromatin environment, which is confirmed by experiment. Through combination of theory and experiments, our work reveals that nuclear condensate surface tension can be high enough to compete with chromatin stiffness to govern condensate growth dynamics, which sheds light on how cells can regulate condensate properties and chromatin stiffness together to control condensate stability and sizes in the nucleus.

    

Condensate-mediated chromatin organization through elastocapillary interactions

Biomolecular condensates in the nucleus play a pivotal role in the organization and functionality of chromatin. Here, we propose a novel computational model for the complex elastocapillary interactions between condensates and fiber networks, augmented experimentally by a set of light-inducible proteinaceous condensates with a wide range of binding affinities to chromatin. In both simulations and experiments, we found distinct regimes where condensates cavitate or engulf the chromatin network. In addition, the size and morphology of condensates as well as the compaction of chromatin are dictated by the interplay between wetting affinity and chromatin stiffness. More broadly, our computational model is suitable for studying a wide range of capillary phenomena in biology and materials science and, together with our experimental platform, paves the way for further investigation of condensate-mediated nuclear organization and functionality.   

Transcription factor clusters as information transfer agents

Precise regulation of gene expression requires accurate interpretation of nuclear transcription factor (TF) concentrations at DNA binding sites. While TF molecule clusters have been found to be associated with gene loci, the extent to which these clusters convey the nuclear concentration information to the gene locus and their functional implications remain unclear. In this work, we present evidence that the nuclear concentration gradient of Bicoid, a central transcription factor driving the anterior-posterior patterning genes during early fly development, is accurately reflected by the sub-nuclear clusters. This sub-nuclear clustering phenomenon yields a two-fold concentration amplification, significantly enhancing the accuracy of TF readout, and thereby facilitating precise nuclear positioning within the fly embryo. Furthermore, the characteristics of these clusters were found to be distinct from liquid-liquid phase separation. Notably, their physical sizes remain consistent throughout the embryo, while their concentrations change along the embryo's axis. We propose that TF clustering reduces the time required for the accurate interpretation of concentration. Thus TF clustering emerges as a mechanism enabling the rapid and precise assessment of cellular protein concentrations by gene enhancers. This mechanism offers an essential means for the transfer of positional information, which is fundamental to various biological processes.   

Genomic clustered pattern of transcription factor binding reflects phase-separated transcriptional condensates

Many transcription factors (TFs) have been shown to bind to super-enhancers, forming transcriptional condensates to activate transcription in various cellular systems. However, the genomic and epigenomic determinants of phase-separated transcriptional condensate formation remain poorly understood. Questions regarding which TFs tend to associate with transcriptional condensates and what factors influence their association are largely unanswered. Here we systematically analyzed 528 DNA sequence motifs across the human genome and 6,650 TF binding profiles across different cell types to identify the molecular features contributing to the formation of transcriptional condensates. We found that the genomic distributions of sequence motifs for different TFs exhibit distinct clustering tendencies. Notably, TF motifs with a high genomic clustering tendency are significantly associated with super-enhancers. TF binding profiles showing a high genomic clustering tendency are further enriched at cell-type-specific super-enhancers. TFs with a high binding clustering tendency also possess high liquid-liquid phase separation abilities. Our results indicate that the clustered genomic binding patterns and the phase separation properties of TFs collectively contribute to the formation of transcriptional condensates.   

The transcription factor TEAD1 forms repressive biomolecular condensates in renal cell carcinomas

The chromatin adaptor protein YAP1 is an important regulator of transcriptional activation and chromatin architecture in response to Hippo pathway signaling. YAP1 binds TEAD-family transcription factors and forms phase-separated biomolecular condensates, specifically transcriptional hubs, in response to hyperosmolarity and other cellular and environmental cues. In Renal Cell Carcinoma (RCC), a particularly lethal form of kidney cancer with poor treatment options, YAP1 and its transcription factor partner TEAD1 are often up-regulated and indicate poor prognosis. We have found that in certain patient-derived RCC cell lines with high levels of YAP1 and TEAD1 expression, TEAD1 forms atypical large nuclear foci, distinct from normal kidney cells and other subtypes of RCC. These few large TEAD1 foci are co-occupant with the repressive histone mark H3K9me3 and are generally located near the nuclear periphery or nucleolus. This is different from TEAD1 patterns both in normal cells and within other regions of the same nuclei, where TEAD1 is distributed into many sub-resolution foci and associates with markers of active chromatin. We have used a combination of high-resolution microscopy, RNA-seq, ChIP-seq, and biochemical methods to identify the unique chromatin architecture of these cancer-associated large TEAD1 foci and propose that they represent a novel subtype of biomolecular condensate where normally activating transcription factors are recruited into repressive heterochromatin. As YAP1-TEAD complexes are important mediators of chromatin architecture and genome organization, these findings provide insight into how those mechanisms can be hijacked in cancer.   

Chromatin Heterogeneity Modulates Nuclear Phase Behavior and Condensates Dynamics

The cell nucleus serves as a dynamic substrate responsible for storing and processing genetic material. This genetic material is intricately organized into dense, thread-like structures known as chromatin fibers, which, in turn, form a complex network with varying spatial densities. While extensive research has been conducted on the structural aspects of the chromatin network, the impact of its spatially heterogeneous mechanical properties on liquid-liquid phase separation within the nucleus remains enigmatic. In this study, we employ an optogenetic oligomerization system to investigate the role of the chromatin network in the phase separation dynamics within living cell nuclei. By modulating the spatial heterogeneity of the chromatin network through the use of a histone deacetylase inhibitor (HDACi) and inducing phase separation via blue light exposure to oligomerize proteins with different intrinsically disordered regions (IDRs), we reveal that the phase separation process is markedly inhibited when the chromatin network becomes globally homogeneous. Furthermore, we observe a reduction in the size of the phase-separated droplets and deviations from the typical coalescence-driven growth mechanism upon chromatin homogenization. Our study suggests alterations in the intranuclear mechanical properties concomitant with chromatin homogenization, which correlates with the change in the phase behavior in the nucleus. These findings emphasize the critical importance of the material state of the chromatin network in influencing liquid-liquid phase separation within the nucleus, and bear implications for the biophysical regulation of biomolecular condensates.

    

Spatial Organization of Regulatory Chromatin At Transcription Condensates

Many biomolecular condensates associate with specific chromatin loci in the cell nucleus. Transcription condensates in mouse embryonic stem cells are thought to form at super-enhancer chromatin domains that are characterized by extended accumulations of transcription factor and coactivator binding sites. Bulk assays suggest that active chromatin marks are required for transcription condensate homeostasis. We uncover the epigenetic fingerprints of chromatin at transcription condensates below the diffraction limit with 3D multicolor single molecule super-resolution microscopy. Our results show general enrichment of nucleosomal chromatin inside transcription condensates and divergent spatial profiles of histone modifications associated with distinct types of chromatin regulatory elements. We link these findings to sequential stages of the transcription process. In particular, condensates appear to be foci of transcription initiation. Our findings begin to elucidate structure-function relationships of condensates in transcription regulation.   

Mechanisms of sensitive and selective nucleation of chromatin-associated biomolecular condensates

In order to perform useful biological functions, phase-separated biomolecular condensates comprising proteins and RNAs must assemble at specific locations within a living cell. However, the biophysical mechanisms by which such spatial control is achieved remain poorly understood. To address this question, we present an experimentally motivated coarse-grained model of a class of transcriptional condensates that are believed to play a role in initiating DNA transcription. Specifically, we consider transcriptional condensates composed of the bromodomain protein BRD4, which selectively associates with chromatin via specific interactions with acetylated histone tails. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we elucidate how BRD4–chromatin interactions tune both the partitioning of chromatin into BRD4 condensates and the nucleation pathway by which BRD4 condensates assemble. We show that both the patterning of histone acetylation marks and the oligomerization state of BRD4 molecules govern the sensitivity and specificity of chromatin-seeded heterogeneous nucleation, whereas disruption of BRD4–chromatin interactions suppresses the chromatin-associated nucleation pathway. Our findings provide a molecularly detailed view of the biophysical mechanisms governing BRD4 condensate formation and suggest potential strategies for regulating transcription via spatiotemporal control of transcriptional condensate nucleation.   

Role of electrostatics and phase separation in regulation of gene expression in Escherichia Coli

Intracellular physical organization and transport of biomolecules have been shown to play a key role in a wide range of cellular functions of biological cells. The spatial organization and temporal regulation of biomolecules are essential for regulating gene expression, cellular signaling, cell division, biochemical reactions, stress response, etc. One of the predominant modes of spatial organization of biomolecules inside biological cells is liquid-liquid phase separation. Though we have knowledge of many mechanisms that regulate the phase separation of biomolecules inside the cells such as multivalent interactions, hydrogen bonding, and electrostatic interactions, the exact function of many of the biomolecular condensates remains largely unknown. In this study, we explore the role of nucleoprotein phase separation on the dynamics of mRNA translation by the ribosomal machinery. We first probe the effect of cell growth-mediated stoichiometry on the nucleoprotein phase separation in Escherichia Coli. We further investigate the temporal evolution of the phase properties of these biomolecular condensates. Finally, we investigate the effect of this phase separation on the elongation dynamics of mRNA translation where we observe the competing effects of transport and sequestration regulate the elongation rate.