Mediator Condensates Localize Signaling Factors to Key Cell Identity Genes

Zamudio, Alicia V.; Dall’Agnese, Alessandra; Henninger, Jonathan E.; Manteiga, John C.; Afeyan, Lena K.; Hannett, Nancy M.; Coffey, Eliot L.; Li, Charles; Oksuz, Ozgur; Sabari, Benjamin R.; Boija, Ann; Klein, I.; Hawken, Susana Wilson; Spille, Jan-Hendrik; Decker, Tim-Michael; Cissé, Ibrahima; Abraham, Brian J.; Lee, Tong I.; Taatjes, Dylan J.; Schuijers, Jurian; Young, Richard A.

doi:10.1016/j.molcel.2019.08.016

Cited by 185 publications

(170 citation statements)

References 68 publications

(102 reference statements)

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“…Recent work has shown that many enhancer-associated factors, such as Mediator (e.g. MED1) and BRD4, assemble into phase-separated activation complexes, and these interactions appear to be integral to their ability to activate transcription 2,3,[34][35][36][37] . Further, several studies have linked the phase separation-mediated assembly of activation complexes at enhancers (particularly SEs) and promoters to the initiation and maintenance of interactions between enhancers and promoters 2,3,[35][36][37] .…”

Section: Next Generationmentioning

confidence: 99%

BET inhibition disrupts transcription but retains enhancer-promoter contact

Crump

Ballabio

Godfrey

et al. 2019

Preprint

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In higher eukaryotes, enhancers are DNA sequences that enable complex temporal and tissue-specific regulation of genes. Although it is not entirely clear how enhancer-promoter interactions can increase gene expression, this proximity has been observed in multiple systems at multiple loci and is thought to be essential for the maintenance of gene expression. The formation of phase condensates is thought to be an essential component of enhancer function. Here, we show that pharmacological targeting of cells with inhibitors of BET (Bromodomain and Extra-Terminal domain) proteins can have a strong impact on transcription but very little impact on enhancer-promoter interactions. Treatment with 1,6-hexanediol, which dissolves phase condensate structures and reduces BET and Mediator protein binding at enhancers, can also have a strong effect on gene transcription, without disrupting enhancer-promoter interactions. These results suggest that activation of transcription and maintenance of enhancerpromoter interactions are separable events. Our findings further suggest that enhancer-promoter interactions are not dependent on high levels of BRD4 (Bromodomain-containing protein 4) and Mediator, and are likely maintained by a complex set of factors including additional activator complexes and loop extrusion by CTCF/cohesin.

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Section: Next Generationmentioning

confidence: 99%

BET inhibition disrupts transcription but retains enhancer-promoter contact

Crump

Ballabio

Godfrey

et al. 2019

Preprint

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“…Recent evidence hints at how this may be achieved. Mediator subunits in its tail sub-module have been shown to engage in a high number of low specificity and weak interactions via phase separation, thereby achieving a “fuzzy” interaction between Mediator and the activation domains of many different transcription factors [ [24] , [25] , [26] , [27] ]. The second arm of the Mediator bridge extends to interact with the pre-initiation complex, inducing the recruitment of RNA Pol II at target promoters [ 20 ] ( Fig.…”

Section: Mediator: a Bridge Between Enhancers And Promotersmentioning

confidence: 99%

“…RNA Pol II is known to participate in discrete membrane-less protein aggregates previously described as transcription factories [ [50] , [51] , [52] ]. More recently, studies have elucidated how these factors efficiently co-segregate from the nuclear milieu based on a shared propensity for phase separation, leading to the term transcriptional condensates [ 24 , 25 , [50] , [51] , [52] , [53] ]. Mediator and several other protein elements of the transcriptional machinery contain intrinsically-disordered regions (IDRs) that confer their phase separation [ 24 , 25 , 50 , [52] , [53] , [54] ].…”

Section: Mediator Intrinsically Disordered Regions and Transcriptiomentioning

confidence: 99%

Manipulating the Mediator complex to induce naïve pluripotency

Lynch

Bernad

Calvo

et al. 2020

Experimental Cell Research

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Human naïve pluripotent stem cells (PSCs) represent an optimal homogenous starting point for molecular interventions and differentiation strategies. This is in contrast to the standard primed PSCs which fluctuate in identity and are transcriptionally heterogeneous. However, despite many efforts, the maintenance and expansion of human naïve PSCs remains a challenge. Here, we discuss our recent strategy for the stabilization of human PSC in the naïve state based on the use of a single chemical inhibitor of the related kinases CDK8 and CDK19. These kinases phosphorylate and negatively regulate the multiprotein Mediator complex, which is critical for enhancer-driven recruitment of RNA Pol II. The net effect of CDK8/19 inhibition is a global stimulation of enhancers, which in turn reinforces transcriptional programs including those related to cellular identity. In the case of pluripotent cells, the presence of CDK8/19i efficiently stabilizes the naïve state. Importantly, in contrast to previous chemical methods to induced the naïve state based on the inhibition of the FGF-MEK-ERK pathway, CDK8/19i-naïve human PSCs are chromosomally stable and retain developmental potential after long-term expansion. We suggest this could be related to the fact that CDK8/19 inhibition does not induce DNA demethylation. These principles may apply to other fate decisions.

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“…Embryonic development depends on the precise spatial and temporal regulation of gene expression. Enhancers and other cis-regulatory elements embody the logic of the regulatory genome via their specific sets of binding motifs for different sequence-specific transcriptional activators and inhibitors (Zamudio et al, 2019). It is now clear that 'shadow, ' 'distributed,' or 'redundant' enhancers are ubiquitous features of many genomes (Cannavò et al, 2016, Osterwalder et al, 2018, Madgwick et al, 2019.…”

Section: Introductionmentioning

confidence: 99%

Ciona Brachyuryproximal and distal enhancers have different FGF dose-response relationships

Harder

Hix

Reeves

et al. 2020

Preprint

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Many genes are regulated by two or more enhancers that drive similar expression patterns. Evolutionary theory suggests that these seemingly redundant enhancers must have functionally important differences. In the simple ascidian chordate Ciona, the transcription factor Brachyury is induced exclusively in the presumptive notochord downstream of lineage specific regulators and FGF-responsive Ets family transcription factors. Here we exploit the ability to finely titrate FGF signaling activity via the MAPK pathway using the MEK inhibitor U0126 to quantify the dependence of transcription driven by different Brachyury reporter constructs on this direct upstream regulator. We find that the more powerful promoter-adjacent proximal enhancer and a weaker distal enhancer have fundamentally different dose-response relationships to MAPK inhibition. The Distal enhancer is more sensitive to MAPK inhibition but shows a less cooperative response, whereas the Proximal enhancer is less sensitive and more cooperative. A longer construct containing both enhancers has a complex dose-response curve that supports the idea that the proximal and distal enhancers are moderately super-additive. We show that the overall expression loss from intermediate doses of U0126 is not only a function of the fraction of cells expressing these reporters, but also involves graded decreases in expression at the single-cell level. Expression of the endogenous gene shows a comparable dose-response relationship to the full length reporter, and we find that different notochord founder cells are differentially sensitive to MAPK inhibition. Together, these results indicate that although the two Brachyury enhancers have qualitatively similar expression patterns, they respond to FGF in quantitatively different ways and act together to drive high levels of Brachyury expression with a characteristic input/output relationship. This indicates that they are fundamentally not redundant genetic elements.

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Mediator Condensates Localize Signaling Factors to Key Cell Identity Genes

Abstract: Highlights d Signaling factors incorporate into Mediator condensates at super-enhancers d b-Catenin IDRs are required for both phase separation and target gene activation d Both condensate interactions and TF interactions contribute to b-catenin localization

Cited by 185 publications

References 68 publications

BET inhibition disrupts transcription but retains enhancer-promoter contact

BET inhibition disrupts transcription but retains enhancer-promoter contact

Manipulating the Mediator complex to induce naïve pluripotency

Ciona Brachyuryproximal and distal enhancers have different FGF dose-response relationships

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