Phase separation of TAZ compartmentalizes the transcription machinery to promote gene expression

AbstractTAZ promotes cell proliferation, development, and tumorigenesis by regulating target gene transcription. However, how TAZ orchestrates the transcriptional responses remains poorly defined. Here we demonstrate that TAZ forms nuclear condensates via liquid-liquid phase separation to compartmentalize its DNA binding co-factor TEAD4, the transcription co-activators BRD4 and MED1 and the transcription elongation factor CDK9 for activation of gene expression. TAZ, but not its paralog YAP, forms phase-separated droplets in vitro and liquid-like nuclear condensates in vivo, and this ability is negatively regulated by Hippo signaling via LATS-mediated phosphorylation and mediated by the coiled-coil domain. Deletion of the TAZ coiled-coil domain or substitution with the YAP coiled-coil domain does not affect the interaction of TAZ with its partners, but prevents its phase separation and more importantly, its ability to induce target gene expression. Thus, our study identifies a novel mechanism for the transcriptional activation by TAZ and demonstrates for the first time that pathway-specific transcription factors also engage the phase separation mechanism for efficient transcription activation.

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Phase separation of the LINE-1 ORF1 protein is mediated by the N-terminus and coiled-coil domain

10.1101/2020.10.29.360719 ◽

2020 ◽

Author(s):

JC Newton ◽

GY Li ◽

MT Naik ◽

NL Fawzi ◽

JM Sedivy ◽

...

Keyword(s):

Dna Damage ◽

Phase Separation ◽

Human Genome ◽

Genomic Instability ◽

Coiled Coil ◽

Retrotransposable Element ◽

Coiled Coil Domain ◽

N Terminus ◽

Long Interspersed Nuclear Element

AbstractLong Interspersed Nuclear Element-1 (LINE-1 or L1) is a retrotransposable element that autonomously replicates in the human genome, resulting in DNA damage and genomic instability. Activation of L1 in senescent cells triggers a type I interferon response and age-associated inflammation. Two open reading frames encode an ORF1 protein functioning as mRNA chaperone and an ORF2 protein providing catalytic activities necessary for retrotransposition. No function has been identified for the conserved, disordered N-terminal region of ORF1. Using microscopy and NMR spectroscopy, we demonstrate that ORF1 forms liquid droplets in vitro in a salt-dependent manner and that interactions between its N-terminal region and coiled-coil domain are necessary for phase separation. Mutations disrupting blocks of charged residues within the N-terminus impair phase separation while some mutations within the coiled-coil domain enhance phase separation. Demixing of the L1 particle from the cytosol may provide a mechanism to protect the L1 transcript from degradation.Statement of significanceOver half of the human genome is comprised of repetitive sequences. The Long Interspersed Nuclear Element-1 (L1) is an autonomous mobile DNA element that can alter its genomic location, resulting in genomic instability and DNA damage. L1 encodes two proteins that are required for this function: the ORF1 RNA chaperone and the enzymatic ORF2. Here, we demonstrate that ORF1 forms liquid-liquid phase separated states in vitro, which is mediated by electrostatic interactions between the conserved, disordered N-terminus and coiled-coil domain. This work provides a framework to explore how L1 phase separation may enhance the ability of the retrotransposable element to colonize the genome by preventing degradation of the L1 transcript and evasion of host immune responses.

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2049-P: The Chd4 Helicase of the Nucleosome Remodeling and Deacetylase Complex Modulates Pdx1 Target Gene Expression In Vitro

Diabetes ◽

10.2337/db20-2049-p ◽

2020 ◽

Vol 69 (Supplement 1) ◽

pp. 2049-P

Author(s):

REBECCA K. DAVIDSON ◽

NOLAN CASEY ◽

JASON SPAETH

Keyword(s):

Gene Expression ◽

Target Gene ◽

Nucleosome Remodeling ◽

Target Gene Expression

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Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

Biochemistry and Cell Biology ◽

10.1139/o05-062 ◽

2005 ◽

Vol 83 (4) ◽

pp. 535-547 ◽

Cited By ~ 7

Author(s):

Gareth N Corry ◽

D Alan Underhill

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcription Factors ◽

Transcriptional Activation ◽

Current Knowledge ◽

Nuclear Architecture ◽

Subnuclear Localization ◽

Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

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Fos and jun cooperate in transcriptional regulation via heterologous activation domains.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5532 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5532-5535 ◽

Cited By ~ 45

Author(s):

C Abate ◽

D Luk ◽

E Gagne ◽

R G Roeder ◽

T Curran

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Regulation Of Gene Expression ◽

Negative Influence ◽

Activation Domain ◽

Activation Domains ◽

Transcriptional Activation Domain ◽

Transcriptional Stimulation

The products of c-fos and c-jun (Fos and Jun) function in gene regulation by interacting with the AP-1 binding site. Here we have examined the contribution of Fos and Jun toward transcriptional activity by using Fos and Jun polypeptides purified from Escherichia coli. Fos contained a transcriptional activation domain as well as a region which exerted a negative influence on transcriptional activity in vitro. Moreover, distinct activation domains in both Fos and Jun functioned cooperatively in transcriptional stimulation. Thus, regulation of gene expression by Fos and Jun results from an integration of several functional domains in a bimolecular complex.

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The Caenorhabditis elegans unc-64 Locus Encodes a Syntaxin That Interacts Genetically with Synaptobrevin

Molecular Biology of the Cell ◽

10.1091/mbc.9.6.1235 ◽

1998 ◽

Vol 9 (6) ◽

pp. 1235-1252 ◽

Cited By ~ 151

Author(s):

Owais Saifee ◽

Liping Wei ◽

Michael L. Nonet

Keyword(s):

Caenorhabditis Elegans ◽

Synaptic Vesicle ◽

Acetylcholinesterase Inhibitor ◽

Coiled Coil ◽

Vesicle Fusion ◽

Loss Of Function ◽

Hydrophobic Membrane ◽

Coiled Coil Domain ◽

Synaptic Vesicle Fusion

We describe the molecular cloning and characterization of theunc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63–64% identity to human syntaxin 1A, a membrane- anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their C-terminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those ofDrosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion.

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Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

The Journal of Cell Biology ◽

10.1083/jcb.200206113 ◽

2002 ◽

Vol 159 (6) ◽

pp. 993-1004 ◽

Cited By ~ 126

Author(s):

Christine L. Humphries ◽

Heath I. Balcer ◽

Jessica L. D'Agostino ◽

Barbara Winsor ◽

David G. Drubin ◽

...

Keyword(s):

Binding Protein ◽

Coiled Coil ◽

Actin Binding ◽

Complex Activity ◽

Actin Binding Protein ◽

Coiled Coil Domain ◽

Allele Specific ◽

And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

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Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex

Biochemistry Research International ◽

10.1155/2018/6406372 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9

Author(s):

Gwang Sik Kim ◽

Young Chul Lee

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Transcriptional Activation ◽

Reporter Gene Expression ◽

Temperature Sensitive ◽

Internal Deletion ◽

Cell Extracts ◽

Evolutionarily Conserved

Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis ofSaccharomyces cerevisiaeMed6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitivemed6mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to thelexAoperator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 tolexAoperator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator.

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In vitro chemosensitivity of freshly explanted tumor cells to pemetrexed is correlated with target gene expression

Investigational New Drugs ◽

10.1007/s10637-007-9060-9 ◽

2007 ◽

Vol 25 (5) ◽

pp. 417-423 ◽

Cited By ~ 60

Author(s):

Axel-Rainer Hanauske ◽

Ulrike Eismann ◽

Olaf Oberschmidt ◽

Heike Pospisil ◽

Steve Hoffmann ◽

...

Keyword(s):

Gene Expression ◽

Tumor Cells ◽

Target Gene ◽

Target Gene Expression ◽

In Vitro Chemosensitivity

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Multimerization and tubulin binding are required for the SPIRAL2 protein to localize to and stabilize microtubule minus ends

10.1101/2021.10.05.463238 ◽

2021 ◽

Author(s):

YUANWEI FAN ◽

Natasha Bilkey ◽

Ram Dixit

Keyword(s):

Coiled Coil ◽

Spatial Arrangement ◽

In Planta ◽

Structural Determinants ◽

Coiled Coil Domain ◽

Tubulin Binding ◽

And Function ◽

Necessary And Sufficient ◽

Basic Region

Accruing evidence points to the control of microtubule minus-end dynamics as being crucial for the spatial arrangement and function of the microtubule cytoskeleton. In plants, the SPIRAL2 (SPR2) protein has emerged as a microtubule minus-end regulator that is structurally distinct from the animal minus-end regulators. Previously, SPR2 was shown to autonomously localize to microtubule minus ends and decrease their depolymerization rate. Here, we used in vitro and in planta experiments to identify the structural determinants required for SPR2 to recognize and stabilize microtubule minus ends. We show that SPR2 contains a single N-terminal TOG domain that binds to soluble tubulin. The TOG domain, a basic region, and coiled-coil domain are necessary and sufficient to target and stabilize microtubule minus ends. We demonstrate that the coiled-coil domain mediates multimerization of SPR2 that provides avidity for microtubule binding and is essential for binding to soluble tubulin. While TOG domain-containing proteins are traditionally thought to function as microtubule plus-end regulators, our results reveal that nature has repurposed the TOG domain of SPR2 to regulate microtubule minus ends.

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Mutants of cubitus interruptus that are independent of PKA regulation are independent of hedgehog signaling

Development ◽

10.1242/dev.126.16.3607 ◽

1999 ◽

Vol 126 (16) ◽

pp. 3607-3616 ◽

Cited By ~ 3

Author(s):

Y. Chen ◽

J.R. Cardinaux ◽

R.H. Goodman ◽

S.M. Smolik

Keyword(s):

Gene Expression ◽

Target Gene ◽

Ectopic Expression ◽

Proteolytic Processing ◽

Dominant Negative ◽

Cubitus Interruptus ◽

Target Gene Expression ◽

Exogenous Expression

Hedgehog (HH) is an important morphogen involved in pattern formation during Drosophila embryogenesis and disc development. cubitus interruptus (ci) encodes a transcription factor responsible for transducing the hh signal in the nucleus and activating hh target gene expression. Previous studies have shown that CI exists in two forms: a 75 kDa proteolytic repressor form and a 155 kDa activator form. The ratio of these forms, which is regulated positively by hh signaling and negatively by PKA activity, determines the on/off status of hh target gene expression. In this paper, we demonstrate that the exogenous expression of CI that is mutant for four consensus PKA sites [CI(m1-4)], causes ectopic expression of wingless (wg) in vivo and a phenotype consistent with wg overexpression. Expression of CI(m1-4), but not CI(wt), can rescue the hh mutant phenotype and restore wg expression in hh mutant embryos. When PKA activity is suppressed by expressing a dominant negative PKA mutant, the exogenous expression of CI(wt) results in overexpression of wg and lethality in embryogenesis, defects that are similar to those caused by the exogenous expression of CI(m1-4). In addition, we demonstrate that, in cell culture, the mutation of any one of the three serine-containing PKA sites abolishes the proteolytic processing of CI. We also show that PKA directly phosphorylates the four consensus phosphorylation sites in vitro. Taken together, our results suggest that positive hh and negative PKA regulation of wg gene expression converge on the regulation of CI phosphorylation.

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