Coactivator condensation at super-enhancers links phase separation and gene control (ChIP-Seq)
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE112806 | GEO | 2018/04/07
REPOSITORIES: GEO
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Project description:Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. We recently proposed that a phase-separated multi-molecular assembly underlies the formation and function of SEs. Here, we demonstrate that the SE-enriched factors BRD4 and MED1 form nuclear puncta that occur at SEs and exhibit properties of liquid-like condensates. Disruption of BRD4 and MED1 puncta by 1,6-hexanediol is accompanied by a loss of BRD4 and MED1 at SEs and a loss of RNAPII from SE-driven genes. We find that the intrinsically disordered regions (IDRs) of BRD4 and MED1 are sufficient to form phase-separated droplets in vitro and the MED1 IDR promotes phase separation in living cells. The MED1 IDR droplets are capable of compartmentalizing BRD4 and other transcriptional machinery in nuclear extracts. These results support the idea that SEs form phase-separated condensates that compartmentalize the transcription apparatus at key genes, provide insights into the role of cofactor IDRs in this process, and offer new insights into mechanisms involved in control of key cell identity genes.
2018-04-07 | GSE112807 | GEO
Project description:HIPPO-YAP/TAZ signaling has been implicated in supratentorial ependymoma formation from neural progenitor cells (NPC) in the brain, however, the underlying mechanisms to trigger the neural progenitor cell transformation remains elusive. Here, we uncover that patient-derived tumorigenic YAP-fusion proteins (YAP-MAMLD1 and C11ORF95-YAP) promote ependymoma tumorigenesis through forming liquid-liquid phase-separated condensates. Intrinsically disordered regions (IDR) in the fusion proteins promote oligomerization of YAP-transcriptional co-activators and self-assembly of nuclear puncta-like membrane-less organelles. Phase separation of YAP-fusion proteins further facilitates the compartmentalization of transcriptional coactivators, BRD4 and MED1, resulting in pervasive enhancer landscape changes and exclusion of transcriptional repressors such as PRC2 complexes. YAP-fusion proteins-induced nuclear puncta recruit RNA polymerase II to promote transcriptional bursting of multiple oncogenic pathways. Moreover, we show that IDR-mediated phase separation is necessary for YAP-fusion protein-induced tumor formation. Distinct YAP fusion-proteins identified in other human tumors also encompass IDR features. Together, our data suggest that IDR-mediated phase separation is an integral component of YAP-fusion protein-induced tumorigenesis and might serve as a therapeutic target in supratentorial ependymoma.
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Project description:HIPPO-YAP/TAZ signaling has been implicated in supratentorial ependymoma formation from neural progenitor cells (NPC) in the brain, however, the underlying mechanisms to trigger the neural progenitor cell transformation remains elusive. Here, we uncover that patient-derived tumorigenic YAP-fusion proteins (YAP-MAMLD1 and C11ORF95-YAP) promote ependymoma tumorigenesis through forming liquid-liquid phase-separated condensates. Intrinsically disordered regions (IDR) in the fusion proteins promote oligomerization of YAP-transcriptional co-activators and self-assembly of nuclear puncta-like membrane-less organelles. Phase separation of YAP-fusion proteins further facilitates the compartmentalization of transcriptional coactivators, BRD4 and MED1, resulting in pervasive enhancer landscape changes and exclusion of transcriptional repressors such as PRC2 complexes. YAP-fusion proteins-induced nuclear puncta recruit RNA polymerase II to promote transcriptional bursting of multiple oncogenic pathways. Moreover, we show that IDR-mediated phase separation is necessary for YAP-fusion protein-induced tumor formation. Distinct YAP fusion-proteins identified in other human tumors also encompass IDR features. Together, our data suggest that IDR-mediated phase separation is an integral component of YAP-fusion protein-induced tumorigenesis and might serve as a therapeutic target in supratentorial ependymoma.
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Project description:RNA-binding proteins with intrinsically disordered regions (IDRs) such as Rbm14 can phase separate in vitro. To what extent the phase separation contributes to their physiological functions is however unclear. Here we show that zebrafish rbm14 regulates embryonic dorsoventral patterning through phase separation. Zebrafish rbm14 morphants displayed dorsalized phenotypes associated with attenuated BMP signaling. Consistently, depletion of mammalian Rbm14 downregulated BMP regulators and effectors Nanog, Smad4/5, and Id1/2, whereas overexpression of the BMP-related proteins in the morphants significantly restored the developmental defects. Importantly, rbm14's IDR demixed into liquid droplets in vitro despite poor sequence conservation with its mammalian counterpart. While its phase separation mutants or IDR failed to rescue the morphants, its chimeric proteins containing an IDR from other phase-separation proteins were effective. Rbm14 complexed with proteins involved in RNA metabolism and phase separated into cellular ribonucleoprotein compartments. Consistently, RNA deep sequencing analysis on the morphant embryos revealed increased alternative splicing events as well as largescale transcriptomic downregulations. Our results suggest that Rbm14 functions in ribonucleoprotein compartments through phase separation to modulate multiple aspects of RNA metabolism. Furthermore, IDRs conserve in phase separation ability but not primary sequence and can be functionally interchangeable.
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Project description:Development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human hematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains nucleoporin?s IDR, tandemly dispersed phenylalanine-and-glycine (FG) repeats. However, it remains elusive how unstructured IDRs contribute to oncogenesis. We show that IDR harbored within NUP98-HOXA9, a homeodomain-containing transcription factor (TF) chimera recurrently detected in leukemias, is essential for establishing liquid-liquid phase separation (LLPS) puncta of chimera and for inducing leukemic transformation. Strikingly, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera TFs but is also required for formation of a broad, ?super-enhancer?-like binding pattern, typically seen at a battery of leukemogenic genes, potentiating their transcriptional activation. Artificial HOX chimera (FUS-HOXA9), created by replacing NUP98?s FG repeats with an unrelated LLPS-forming IDR of FUS, had similar enhancement effects on chimera?s genome-wide binding and target gene activation. Hi-C mapping further demonstrated that phase-separated NUP98-HOXA9 induces CTCF-independent chromatin looping enriched at proto-oncogenes. Together, this report describes a proof-of-principle example wherein cancer acquires mutation to establish oncogenic TF condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumorous transformation. As LLPS-competent molecules are frequently implicated in diseases, this mechanism can potentially be generalized to many malignant and pathological settings.
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Project description:Development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human hematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains nucleoporin?s IDR, tandemly dispersed phenylalanine-and-glycine (FG) repeats. However, it remains elusive how unstructured IDRs contribute to oncogenesis. We show that IDR harbored within NUP98-HOXA9, a homeodomain-containing transcription factor (TF) chimera recurrently detected in leukemias, is essential for establishing liquid-liquid phase separation (LLPS) puncta of chimera and for inducing leukemic transformation. Strikingly, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera TFs but is also required for formation of a broad, ?super-enhancer?-like binding pattern, typically seen at a battery of leukemogenic genes, potentiating their transcriptional activation. Artificial HOX chimera (FUS-HOXA9), created by replacing NUP98?s FG repeats with an unrelated LLPS-forming IDR of FUS, had similar enhancement effects on chimera?s genome-wide binding and target gene activation. Hi-C mapping further demonstrated that phase-separated NUP98-HOXA9 induces CTCF-independent chromatin looping enriched at proto-oncogenes. Together, this report describes a proof-of-principle example wherein cancer acquires mutation to establish oncogenic TF condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumorous transformation. As LLPS-competent molecules are frequently implicated in diseases, this mechanism can potentially be generalized to many malignant and pathological settings.
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Project description:Some transcription factors (TFs) can form liquid-liquid phase separated (LLPS) condensates. However, the function of these TF condensates in 3D genome organization and gene regulation remains elusive. In response to methionine (met) starvation in budding yeast, Met4 and a few sequence-specific co-activators, including Met32, induce a set of genes involved in met biosynthesis. Here, we show that the endogenous Met4 and Met32 form puncta-like structures that significantly overlap in yeast nuclei upon met depletion. Recombinant Met4 and Met32 form mixed droplets with LLPS properties in vitro. In relation to chromatin, Met4 puncta co-localize with target genes, and at least a subset of these target genes are clustered in 3D in a Met4-dependent manner. A MET3pr-GFP reporter inserted near several native Met4 binding sites becomes co-localized with Met4 puncta and displays enhanced transcriptional activity. A Met4 variant with a partial truncation of an intrinsically disordered region (IDR) shows less puncta formation, and this mutant selectively reduces the reporter activity near Met4 binding sites to the basal level. Overall, these results support a model where Met4 and co-activators form condensates to bring multiple target genes into a vicinity with higher local TF concentrations, which facilitates a strong response to met depletion (-met).
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Project description:Some transcription factors (TFs) can form liquid-liquid phase separated (LLPS) condensates. However, the function of these TF condensates in 3D genome organization and gene regulation remains elusive. In response to methionine (met) starvation in budding yeast, Met4 and a few sequence-specific co-activators, including Met32, induce a set of genes involved in met biosynthesis. Here, we show that the endogenous Met4 and Met32 form puncta-like structures that significantly overlap in yeast nuclei upon met depletion. Recombinant Met4 and Met32 form mixed droplets with LLPS properties in vitro. In relation to chromatin, Met4 puncta co-localize with target genes, and at least a subset of these target genes are clustered in 3D in a Met4-dependent manner. A MET3pr-GFP reporter inserted near several native Met4 binding sites becomes co-localized with Met4 puncta and displays enhanced transcriptional activity. A Met4 variant with a partial truncation of an intrinsically disordered region (IDR) shows less puncta formation, and this mutant selectively reduces the reporter activity near Met4 binding sites to the basal level. Overall, these results support a model where Met4 and co-activators form condensates to bring multiple target genes into a vicinity with higher local TF concentrations, which facilitates a strong response to met depletion (-met).
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Project description:Development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human hematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains nucleoporin’s IDR, tandemly dispersed phenylalanine-and-glycine (FG) repeats. However, it remains largely elusive how unstructured IDRs contribute to oncogenesis. We here show that IDR or FG repeats harbored within NUP98-HOXA9, a HOX transcription factor (TF) chimera recurrently detected in acute leukemia patients, is essential for establishing nuclear liquid-liquid phase separation (LLPS) puncta and for inducing leukemic transformation of primary hematopoietic cells in vitro and in vivo. Strikingly, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera TF oncoproteins but is also required for formation of a broad, ‘super-enhancer’-like binding pattern, typically seen at a battery of leukemia-related loci exemplified by HOX, MEIS and PBX genes, potentiating their transcriptional activation. An artificial HOX chimera, created by replacing NUP98’s FG repeats with an unrelated LLPS-forming IDR of FUS, had similar enhancement effects on chimera’s chromatin binding and target gene activation. Via Hi-C mapping, we further demonstrated that the phase-separated NUP98-HOXA9 protein assembly is able to induce de novo formation of CTCF-independent chromatin looping enriched at leukemic oncogenes. Together, this report describes a proof-of-principle example wherein cancer acquires mutation to establish multi-molecule assemblies of oncogenic TFs via a phase separation mechanism, which simultaneously enhances their chromatin targeting and induces organization of aberrant three-dimensional chromatin structure during tumorous transformation. As a range of LLPS-competent molecules are implicated in various human cancers, this mechanism can potentially be generalized to many malignant and diseased settings.
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