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Coactivator condensation drives cell lineage specification

ABSTRACT: During development, cells make switch-like decisions to activate new gene programs specifying cell lineage. The mechanisms underlying these decisive choices remain unclear. Here we show that the cardiovascular transcriptional coactivator, Myocardin (MYOCD), activates cell identity genes by concentration-dependent and switch-like formation of transcriptional condensates. MYOCD forms such condensates and activates cell identity genes at critical concentration thresholds achieved during smooth muscle cell and cardiomyocyte differentiation. The C-terminal disordered region of MYOCD is necessary and sufficient for condensate formation. Disrupting this region’s ability to form condensates disrupts gene activation. Rescuing condensate formation by replacing this region with disordered regions from functionally unrelated proteins rescues gene activation. Our findings demonstrate that MYOCD condensate formation is required for gene activation during differentiation. We propose that the formation of transcriptional condensates at critical concentrations of cell type-specific regulators provides a molecular switch underlying the activation of key cell identity genes during cell lineage specification.

ORGANISM(S): Mus musculus

PROVIDER: GSE250206 | GEO | 2024/02/08

REPOSITORIES: GEO

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Coactivator condensation drives lineage specification

Project description:During development, cells make switch-like decisions to activate the expression of new gene programs leading to lineage specification1. The mechanisms underlying these decisive choices remain unclear2. Here we find that Myocardin (MYOCD), a cardiomyocyte and smooth muscle cell-specific transcriptional coactivator3-6, activates lineage-specific gene programs by concentration-dependent and switch-like formation of nuclear condensates. While compartmentalization of the transcriptional machinery by condensates has been associated with gene activation7,8, directly linking the two processes has been a major challenge for the field9,10. By modeling the natural changes in MYOCD concentration during development, coupled with quantitative fluorescence microscopy, single-cell resolution reporter assays, and cellular differentiation assays, we demonstrate that condensate formation is directly linked to transcriptional activation and lineage specification. During cardiomyocyte and smooth muscle cell differentiation, the formation of MYOCD condensates precedes activation of cell identity genes and these condensates are present at sites of cell identity gene transcription. MYOCD condensates form, activate gene expression, and specify cell state at critical concentration thresholds, dependent upon the C-terminal disordered region of MYOCD. Disrupting condensate formation by manipulating the sequence of this region impairs gene activation which can be rescued by replacing this region with condensate-forming disordered regions from functionally unrelated proteins. These results demonstrate that coactivator condensation at critical concentrations enables switch-like changes in gene expression programs crucial for lineage specification.

2024-03-20 | GSE235928 | GEO

Molecular features driving condensate formation and gene expression by the BRD4-NUT fusion oncoprotein are overlapping but distinct [RNA-seq]

Project description:Aberrant formation of biomolecular condensates has been proposed to play a role in several cancers. The oncogenic fusion protein BRD4-NUT forms condensates and drives changes in gene expression in Nut Carcinoma (NC). Here we sought to understand the molecular elements of BRD4-NUT and its associated histone acetyltransferase (HAT), p300, that promote these activities. We determined that a minimal fragment of NUT (MIN) in fusion with BRD4 is necessary and sufficient to bind p300 and form condensates. Furthermore, a BRD4-p300 fusion protein also forms condensates and drives gene expression similarly to BRD4-NUT(MIN), suggesting the p300 fusion may mimic certain features of BRD4-NUT. The intrinsically disordered regions, transcription factor-binding domains, and HAT activity of p300 all collectively contribute to condensate formation by BRD4-p300, suggesting that these elements might contribute to condensate formation by BRD4-NUT. Conversely, only the HAT activity of BRD4-p300 appears necessary to mimic the transcriptional profile of cells expressing BRD4-NUT. Our results suggest a model for condensate formation by the BRD4-NUT:p300 complex involving a combination of positive feedback and phase separation, and show that multiple overlapping, yet distinct, regions of p300 contribute to condensate formation and transcriptional regulation.

2023-05-24 | GSE233302 | GEO

Molecular features driving condensate formation and gene expression by the BRD4-NUT fusion oncoprotein are overlapping but distinct [ChIP-seq]

2023-05-24 | GSE233301 | GEO

Stress-induced nuclear condensation of NELF drives global transcriptional downregulation

Project description:In response to stress, human cells actively downregulate ongoing gene expression programs. Translational downregulation is accompanied by the assembly of stress granules which are cytosolic condensates containing ribosomes and mRNA. Transcriptional downregulation is caused by an increased recruitment of Negative Elongation Factors (NELF) to promoters, inhibiting RNA Pol II elongation. We report here that NELF forms nuclear condensates upon stress which can be recapitulated by liquid-liquid phase separation of recombinant NELF in vitro. Stress leads to rapid dephosphorylation and SUMOylation of NELF in cells facilitating condensate formation driven by intrinsically disordered domains of NELFA and NELFE subunits. Using NELF mutants that do not form condensates we provide evidence that condensation is required for increased recruitment of NELF to downregulated promoters and cellular survival in stress. Thus, our study has uncovered a novel nuclear condensate that is conceptually similar to cytosolic stress granule in downregulating gene expression during stressful conditions.

2021-02-05 | GSE140053 | GEO

Formation of biomolecular condensates in bacteria by tuning protein electrostatics

Project description:While eukaryotic cells have a myriad of membrane-bound organelles enabling the isolation of different chemical environments, prokaryotic cells lack these defined reaction vessels. Biomolecular condensates—organelles that lack a membrane—provide a strategy for cellular organization without a physical barrier while allowing for the dynamic, responsive organization of the cell. It is well established that intrinsically disordered protein domains drive condensate formation via liquid–liquid phase separation; however, the role of globular protein domains on intracellular phase separation remains poorly understood. We hypothesized that the overall charge of globular proteins would dictate the formation and concentration of condensates and systematically probed this hypothesis with supercharged proteins and nucleic acids in E. coli. Within this study, we demonstrated that condensates form via electrostatic interactions between engineered proteins and RNA and that these condensates are dynamic and only enrich specific nucleic acid and protein components. Herein, we propose a simple model for the phase separation based on protein charge that can be used to predict intracellular condensate formation. With these guidelines, we have paved the way to designer functional synthetic membraneless organelles with tunable control over globular protein function.

2020-04-24 | MSV000085333 | MassIVE

MeCP2 links heterochromatin condensates and neurodevelopmental disease

Project description:MeCP2 (methyl CpG binding protein 2) is a key component of constitutive heterochromatin, which plays important roles in chromosome maintenance and transcriptional silencing. Mutations in MeCP2 cause Rett syndrome (RTT), a postnatal progressive neurodevelopmental disorder associated with severe mental disability and autism-like symptoms that manifests in girls during early childhood. Heterochromatin, long considered a dense and relatively static structure, is now understood to exhibit properties consistent with a liquid-like condensate. Here we report that MeCP2 is a dynamic component of heterochromatin condensates in cells, is stimulated by DNA to form liquid-like condensates, contains multiple domains that contribute to condensate formation, manifests physicochemical properties that selectively concentrate heterochromatin cofactors compared to components of transcriptionally active condensates, and when altered by RTT-causing mutations is disrupted in its ability to form condensates. We propose that MeCP2 enhances heterochromatin/euchromatin separation through its condensate partitioning properties and that condensate disruption may be a common consequence of RTT patient mutations.

2020-06-23 | GSE139033 | GEO

Engineering Material Properties of Transcription Factor Condensates to Control Gene Expression in HEK-293T Cells

Project description:Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. We systematically engineered light-inducible transcription factor condensates with different material properties and analyzed their influence on transcription activation. When transcription factor condensates were transformed into solid-like gels, we observed a reduced activation of gene expression. We wanted to evaluate the impact that the condensate formation of the transcription factor RelA had on its endogenous promoters using expression data. To this aim, HEK-293T cells were transfected either with empty vector (1-3) or eGFP-RelA (4-6) or eGFP-RelA, Cry2olig-mCh-FUSN-NLS-NbGFP and Cry2olig-mCh-FUSN-NLS, along with an NF-κB-responsive SEAP reporter (7-9). In each of the three groups of transfected cells, 8 h after transfection, 3 samples were kept in the dark (D) and 3 under blue light illumination (BL, 5 μmol m-² s-1) for 24 h prior to RNA extraction. RNA-seq libraries from the 18 samples were sequenced by BGI on a DNBSEQ platform using paired-end chemistry with a read length of 100 base pairs each. Each strand was sequenced across two separate lanes (L03, L04), generating a total of 4 FASTQ files per sample.

2024-04-01 | E-MTAB-13905 | biostudies-arrayexpress

Systematic investigation on the effect of O-GlcNAcylation on condensate formation under heat stress and recovery

Project description:O-GlcNAc is known to regulate the aggregation and condensate formation of several proteins, but how O-GlcNAc regulates protein solubilities globally has yet to be systematically investigated. Here, we employed a chemoproteomic workflow integrating selective enrichment and multiplex proteomics to quantify the solubilities of O-GlcNAcylated and non-modified proteins, as well as their role in RNA condensate formation. The solubilities were also quantified after heat stress and recovery to investigate the condensate formation during heat stress response. Surprisingly. we found O-GlcNAc leads to dramatic solubility decrease of the modified proteins, and the degree of solubility drop is related to the protein localization and functions. Site-specific analysis found the adjacent residues and secondary structures of the glycosites are pivotal for the effect of O-GlcNAc for solubility change, and the number of acidic residues is related to the different effect of O-GlcNAc in heat stress and recovery. Additionally, it was found that O-GlcNAc promotes the dissociation of RNA condensates during heat stress, while it enhances the formation of RNA condensates in the recovery phase. The glycosites that facilitate the dissociation or the formation of the RNA condensates are distinct, and the physiological properties of their surroundings residues are diverse. This work advances our understanding of the role of O-GlcNAc in regulating biomolecular condensates and will be a useful resource for future research in biomolecular condensates.

2023-08-18 | MSV000092690 | MassIVE

Phosphorylation regulates viral biomolecular condensates to promote infectious progeny production

Project description:Biomolecular condensates play important roles in diverse biological processes. Many viruses form biomolecular condensates which have been implicated in various functions critical for productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral biomolecular condensates that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquid-like properties over condensate hardening. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and maintained in a state conducive to their function in viral progeny production. In addition, our findings have implications for antiviral strategies aimed at targeting the regulation of viral biomolecular condensates to limit viral multiplication.

2023-12-06 | PXD046971 | Pride

Localized condensation of the cBAF remodeler is governed by ARID1A/B and is required for transcriptional activation [CUT&RUN]

Project description:Specifically, we find that the N-terminal disordered region of ARID1A/B and the DNA binding ARID domain are required for condensate formation and chromatin targeting.

2023-10-01 | GSE235698 | GEO

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