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Project description:Topoisomerase 1 Nicking-dependent HP1gamma Engagement Mediates Acute Activation of Phase-separated Enhancers
| PRJNA560174 | ENA
Project description:3D chromatin organization plays an important role in transcription regulation and gene expression. The 3D genome is highly maintained by several architectural proteins, such as CTCF, Yin Yang 1, and cohesin complex. This structural organization brings regulatory DNA elements in close proximity to their target promoters. In this review, we discuss the 3D chromatin organization of super-enhancers and their relationship to phase-separated condensates. Super-enhancers are large clusters of DNA elements. They can physically contact with their target promoters by chromatin looping during transcription. Multiple transcription factors can bind to enhancer and promoter sequences and recruit a complex array of transcriptional co-activators and RNA polymerase II to effect transcriptional activation. Phase-separated condensates of transcription factors and transcriptional co-activators have been implicated in assembling the transcription machinery at particular enhancers. Cancer cells can hijack super-enhancers to drive oncogenic transcription to promote cell survival and proliferation. These dysregulated transcriptional programs can cause cancer cells to become highly dependent on transcriptional regulators, such as Mediator and BRD4. Moreover, the expression of oncogenes that are driven by super-enhancers is sensitive to transcriptional perturbation and often occurs in phase-separated condensates, supporting therapeutic rationales of targeting SE components, 3D genome organization, or dysregulated condensates in cancer.
| S-EPMC9221043 | biostudies-literature
Project description:Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell biology and pathogenesis. BRD4 is a nuclear protein instrumental to the establishment of phase-separated super-enhancers (SEs) to direct the transcription of important genes. We previously observed that protein droplets of BRD4 became hydrophobic as their size increase, implying an ability of SEs to limit the ionization of water molecules by irradiation. Here, we aim to establish if SEs confer radiation resistance in cancer cells. We established an in vitro DNA damage assay that measures the effect of radicals provoked by the Fenton reaction on DNA integrity. This revealed that DNA damage was markedly reduced when BRD4 underwent phase separation with DNA. Accordingly, co-focal imaging analyses revealed that SE foci and DNA damage foci are mutually exclusive in irradiated cells. Lastly, we observed that the radioresistance of cancer cells was significantly reduced when irradiation was combined with ARV-771, a BRD4 de-stabilizer. Our data revealed the existence of innately radioresistant genomic regions driven by phase separation in cancer cells. The disruption of these phase-separated components enfolding genomic DNA may represent a novel strategy to augment the effects of radiotherapy.
| S-EPMC11262858 | biostudies-literature
Project description: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 571 DNA sequence motifs across the human genome and 6650 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. Compared to nonclustered TF binding, densely clustered TF binding sites are more enriched at cell-type-specific super-enhancers with higher chromatin accessibility, elevated chromatin interaction and stronger association with cancer outcomes. Our results indicate that the clustered genomic binding patterns and the phase separation properties of TFs collectively contribute to the formation of transcriptional condensates.
| S-EPMC11760973 | biostudies-literature
Project description:Phase separation of intracellular components has been recently realized as a mechanism by which cells achieve membraneless organization. Here, we study the associative liquid-liquid phase separation (LLPS) of DNA upon complexation with cationic polypeptides. Comparing the phase behavior of different single-stranded DNA as well as double-stranded DNA (dsDNA) sequences that differ in persistence lengths, we find that DNA local flexibility, not simply charge density, determines the LLPS. Furthermore, in a nucleotide- and DNA-dependent manner, free nucleotide triphosphates promote LLPS of polypeptide-dsDNA complexes that are otherwise prone to precipitation. Under these conditions, dsDNA undergoes a secondary phase separation forming liquid-crystalline subcompartments inside the droplets. These results point toward a role of local DNA flexibility, encoded in the sequence, in the regulation and selectivity of multicomponent LLPS in membraneless intracellular organization.
| S-EPMC6303412 | biostudies-literature
Project description:Aberrant T-cell factor (TCF) transcription is implicated in the majority of colorectal cancers (CRCs). TCF transcription induces epithelial-mesenchymal transition (EMT), promoting a tumor-initiating cell (TIC) phenotype characterized by increased proliferation, multidrug resistance (MDR), invasion and metastasis. The data presented herein characterize topoisomerase IIα (TopoIIα) as a required component of TCF transcription promoting EMT. Using chromatin immunoprecipitation (ChIP) and protein co-immunoprecipitation (co-IP) studies, we show that TopoIIα forms protein-protein interactions with β-catentin and TCF4 and interacts with Wnt response elements (WREs) and promoters of direct target genes of TCF transcription, including: MYC, vimentin, AXIN2 and LEF1. Moreover, both TopoIIα and TCF4 ChIP with the N-cadherin promoter, which is a new discovery indicating that TCF transcription may directly regulate N-cadherin expression. TopoIIα N-terminal ATP-competitive inhibitors, exemplified by the marine alkaloid neoamphimedine (neo), block TCF activity in vitro and in vivo. Neo effectively inhibits TopoIIα and TCF4 from binding WREs/promoter sites, whereas protein-protein interactions remain intact. Neo inhibition of TopoIIα-dependent TCF transcription also correlates with significant antitumor effects in vitro and in vivo, including the reversion of EMT, the loss of TIC-mediated clonogenic colony formation, and the loss of cell motility and invasion. Interestingly, non-ATP-competitive inhibitors of TopoIIα, etoposide and merbarone, were ineffective at preventing TopoIIα-dependent TCF transcription. Thus, we propose that TopoIIα participation in TCF transcription may convey a mechanism of MDR to conventional TopoIIα inhibitors. However, our results indicate that TopoIIα N-terminal ATP-binding sites remain conserved and available for drug targeting. This article defines a new strategy for targeted inhibition of TCF transcription that may lead to effective therapies for the treatment of CRC and potentially other Wnt-dependent cancers.
| S-EPMC5036162 | biostudies-literature
Project description:The discovery that enhancers are regulated transcription units, encoding eRNAs, has raised new questions about the mechanisms of their activation. Here, we report an unexpected molecular mechanism that underlies ligand-dependent enhancer activation, based on DNA nicking to relieve torsional stress from eRNA synthesis. Using dihydrotestosterone (DHT)-induced binding of androgen receptor (AR) to prostate cancer cell enhancers as a model, we show rapid recruitment, within minutes, of DNA topoisomerase I (TOP1) to a large cohort of AR-regulated enhancers. Furthermore, we show that the DNA nicking activity of TOP1 is a prerequisite for robust eRNA synthesis and enhancer activation and is kinetically accompanied by the recruitment of ATR and the MRN complex, followed by additional components of DNA damage repair machinery to the AR-regulated enhancers. Together, our studies reveal a linkage between eRNA synthesis and ligand-dependent TOP1-mediated nicking-a strategy exerting quantitative effects on eRNA expression in regulating AR-bound enhancer-dependent transcriptional programs.
| S-EPMC4422651 | biostudies-literature
Project description:Liquid-liquid phase separation (LLPS) plays a critical role in regulating gene transcription via the formation of transcriptional condensates. However, LLPS has not been reported to be engineered as a tool to activate endogenous gene expression in mammalian cells or in vivo. Here, we developed a droplet-forming CRISPR (clustered regularly interspaced short palindromic repeats) gene activation system (DropCRISPRa) to activate transcription with high efficiency via combining the CRISPR-SunTag system with FETIDR-AD fusion proteins, which contain an N-terminal intrinsically disordered region (IDR) of a FET protein (FUS or TAF15) and a transcription activation domain (AD, VP64/P65/VPR). In this system, the FETIDR-AD fusion protein formed phase separation condensates at the target sites, which could recruit endogenous BRD4 and RNA polymerase II with an S2 phosphorylated C-terminal domain (CTD) to enhance transcription elongation. IDR-FUS9Y>S and IDR-FUSG156E, two mutants with deficient and aberrant phase separation respectively, confirmed that appropriate phase separation was required for efficient gene activation. Further, the DropCRISPRa system was compatible with a broad set of CRISPR-associated (Cas) proteins and ADs, including dLbCas12a, dAsCas12a, dSpCas9 and the miniature dUnCas12f1, and VP64, P65 and VPR. Finally, the DropCRISPRa system could activate target genes in mice. Therefore, this study provides a robust tool to activate gene expression for foundational research and potential therapeutics.
| S-EPMC10250237 | biostudies-literature
Project description:Transcription factors (TFs) bind specifically to discrete regions of mammalian genomes called cis-regulatory elements. Among those are enhancers, which play key roles in regulation of gene expression during development and differentiation. Despite the recognized central regulatory role exerted by chromatin in control of TF functions, much remains to be learned regarding the chromatin structure of enhancers and how it is established. Here, we have analyzed on a genomic-scale enhancers that recruit FOXA1, a pioneer transcription factor that triggers transcriptional competency of these cis-regulatory sites. Importantly, we found that FOXA1 binds to genomic regions showing local DNA hypomethylation and that its cell-type-specific recruitment to chromatin is linked to differential DNA methylation levels of its binding sites. Using neural differentiation as a model, we showed that induction of FOXA1 expression and its subsequent recruitment to enhancers is associated with DNA demethylation. Concomitantly, histone H3 lysine 4 methylation is induced at these enhancers. These epigenetic changes may both stabilize FOXA1 binding and allow for subsequent recruitment of transcriptional regulatory effectors. Interestingly, when cloned into reporter constructs, FOXA1-dependent enhancers were able to recapitulate their cell type specificity. However, their activities were inhibited by DNA methylation. Hence, these enhancers are intrinsic cell-type-specific regulatory regions of which activities have to be potentiated by FOXA1 through induction of an epigenetic switch that includes notably DNA demethylation.
| S-EPMC3065703 | biostudies-literature
Project description:The discovery that enhancers are regulated transcription units, encoding eRNAs, has raised new questions about the mechanisms of their activation. Here, we report an unexpected molecular mechanism that underlies ligand-dependent enhancer activation, based on DNA nicking to relieve torsional stress from eRNA synthesis. Using dihydrotestosterone (DHT)-induced binding of androgen receptor (AR) to prostate cancer cell enhancers as a model, we show rapid recruitment, within minutes, of DNA topoisomerase I (TOP1) to a large cohort of AR-regulated enhancers. Furthermore, we show that the DNA nicking activity of TOP1 is a prerequisite for robust eRNA synthesis and enhancer activation and is kinetically accompanied by the recruitment of ATR and the MRN complex, followed by additional components of DNA damage repair machinery to the AR-regulated enhancers. Together, our studies reveal a linkage between eRNA synthesis and ligand-dependent TOP1-mediated nicking - a strategy exerting quantitative effects on eRNA expression in regulating AR-bound enhancer-dependent transcriptional programs. Genome-wide binding analysis of AR, TOP1, MRE11 in prostate cancer cell line LNCaP with or without 5alpha-dihydrotestosterone (DHT) treatment. Nascent RNA analysis by global nuclear run-on (GRO-seq) in LNCaP cells transfected with siRNA with or without DHT treatment. Distribution of transcriptionally engaged RNA Pol II in LNCaP cells with or without DHT treatment by precision nuclear run-on and sequencing (PRO-seq).
2015-01-22 | E-GEOD-63202 | biostudies-arrayexpress