scholarly journals TOR signaling modulates Cdk8-dependent GAL gene expression in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Nicole Hawe ◽  
Konstantin Mestnikov ◽  
Riley Horvath ◽  
Mariam Eji-Lasisi ◽  
Cindy Lam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Mediator Complex ◽  
Tor Signaling ◽  
A Subunit ◽  
Insight Into

AbstractCdk8 of the RNA Polymerase II mediator complex regulates genes by phosphorylating sequence specific transcription factors. Despite conserved importance for eukaryotic transcriptional regulation, the signals regulating Cdk8 are unknown. Full induction of the yeast GAL genes requires phosphorylation of Gal4 by Cdk8, and we exploited this requirement for growth of gal3 yeast on galactose to identify mutants affecting Cdk8 activity. Several mutants from the screen produced defects in TOR signaling. A mutant designated gal four throttle (gft) 1, gft1, was identified as an allele of hom3, encoding aspartokinase. Defects in gft1/ hom3 caused hypersensitivity to rapamycin, and constitutive nuclear localization of Gat1. Furthermore, mutations of tor1 or tco89, encoding TORC1 components, also prevented GAL expression in gal3 yeast, and tco89 was determined to be allelic to gft7. Disruption of cdc55, encoding a subunit of PP2A regulated by TOR signaling, suppressed the effect of gft1/ hom3, gft7/ tco89, and tor1 mutations on GAL expression in gal3 yeast, but not of cdk8/ srb10 disruptions or Gal4 S699A mutation. Mutations of gft1/ hom3 and tor1 did not affect kinase activity of Cdk8 in vitro, but caused loss of Gal4 phosphorylation in vivo. These observations demonstrate that TOR signaling regulates GAL induction through the activity of PP2A/ Cdc55, and are consistent with the contention that Cdk8-dependent phosphorylation of Gal4 S699 is opposed by PP2A/ Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.

scholarly journals TORC1 signaling modulates Cdk8-dependent GAL gene expression in Saccharomyces cerevisiae

Genetics ◽  
2021 ◽  
Author(s):  
Riley Horvath ◽  
Nicole Hawe ◽  
Cindy Lam ◽  
Konstantin Mestnikov ◽  
Mariam Eji-Lasisi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Nuclear Localization ◽  
Protein Phosphatase ◽  
Regulatory Subunit ◽  
Gal Genes ◽  
Dependent Site ◽  
Insight Into

Abstract Cdk8 of the RNA Polymerase II mediator kinase complex regulates gene expression by phosphorylating sequence-specific transcription factors. This function is conserved amongst eukaryotes, but the signals and mechanisms regulating Cdk8 activity and phosphorylation of its substrates are unknown. Full induction of the GAL genes in yeast requires phosphorylation of the transcriptional activator Gal4 by Cdk8. We used a screen to identify regulators of the Cdk8-dependent phosphorylation on Gal4, from which we identified multiple mutants with defects in TORC1 signaling. One mutant, designated gal four throttle 1 (gft1) was identified as a recessive allele of hom3, encoding aspartokinase, and mutations in hom3 caused effects typical of inhibition of TORC1, including rapamycin sensitivity and enhanced nuclear localization of the TORC1-responsive transcription factor Gat1. Mutations in hom3 also inhibit phosphorylation of Gal4 in vivo at the Cdk8-dependent site on Gal4, as did mutations of tor1, but these mutations did not affect activity of Cdk8 assayed in vitro. Disruption of cdc55, encoding a regulatory subunit of the TORC1-regulated protein phosphatase PP2A, suppressed the effect of hom3 and tor1 mutations on GAL expression, and also restored phosphorylation of Gal4 at the Cdk8-dependent site in vivo. These observations demonstrate that TORC1 signaling regulates GAL induction through the activity of PP2A/Cdc55, and suggest that Cdk8-dependent phosphorylation of Gal4 is opposed by PP2A/Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.

scholarly journals Inhibition of Transcription Induces Phosphorylation of YB-1 at Ser102 and Its Accumulation in the Nucleus

Cells ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 104 ◽  
Author(s):  
Dmitry A. Kretov ◽  
Daria A. Mordovkina ◽  
Irina A. Eliseeva ◽  
Dmitry N. Lyabin ◽  
Dmitry N. Polyakov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Regulatory Mechanisms ◽  
Nuclear Accumulation ◽  
Nuclear Retention ◽  
Post Translational Modifications

The Y-box binding protein 1 (YB-1) is an RNA/DNA-binding protein regulating gene expression in the cytoplasm and the nucleus. Although mostly cytoplasmic, YB-1 accumulates in the nucleus under stress conditions. Its nuclear localization is associated with aggressiveness and multidrug resistance of cancer cells, which makes the understanding of the regulatory mechanisms of YB-1 subcellular distribution essential. Here, we report that inhibition of RNA polymerase II (RNAPII) activity results in the nuclear accumulation of YB-1 accompanied by its phosphorylation at Ser102. The inhibition of kinase activity reduces YB-1 phosphorylation and its accumulation in the nucleus. The presence of RNA in the nucleus is shown to be required for the nuclear retention of YB-1. Thus, the subcellular localization of YB-1 depends on its post-translational modifications (PTMs) and intracellular RNA distribution.

scholarly journals c-Jun Homodimers Can Function as a Context-Specific Coactivator

2007 ◽  
Vol 27 (8) ◽  
pp. 2919-2933 ◽  
Author(s):  
Benoit Grondin ◽  
Martin Lefrancois ◽  
Mathieu Tremblay ◽  
Marianne Saint-Denis ◽  
André Haman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Protein Interactions ◽  
Expression Profiles ◽  
Basic Domain ◽  
Interaction Mode

ABSTRACT Transcription factors can function as DNA-binding-specific activators or as coactivators. c-Jun drives gene expression via binding to AP-1 sequences or as a cofactor for PU.1 in macrophages. c-Jun heterodimers bind AP-1 sequences with higher affinity than homodimers, but how c-Jun works as a coactivator is unknown. Here, we provide in vitro and in vivo evidence that c-Jun homodimers are recruited to the interleukin-1β (IL-1β) promoter in the absence of direct DNA binding via protein-protein interactions with DNA-anchored PU.1 and CCAAT/enhancer-binding protein β (C/EBPβ). Unexpectedly, the interaction interface with PU.1 and C/EBPβ involves four of the residues within the basic domain of c-Jun that contact DNA, indicating that the capacities of c-Jun to function as a coactivator or as a DNA-bound transcription factor are mutually exclusive. Our observations indicate that the IL-1β locus is occupied by PU.1 and C/EBPβ and poised for expression and that c-Jun enhances transcription by facilitating a rate-limiting step, the assembly of the RNA polymerase II preinitiation complex, with minimal effect on the local chromatin status. We propose that the basic domain of other transcription factors may also be redirected from a DNA interaction mode to a protein-protein interaction mode and that this switch represents a novel mechanism regulating gene expression profiles.

scholarly journals Gene expression and thioguanine nucleotide disposition in acute lymphoblastic leukemia after in vivo mercaptopurine treatment

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1778-1785 ◽  
Author(s):  
Gianluigi Zaza ◽  
Meyling Cheok ◽  
Wenjian Yang ◽  
John C. Panetta ◽  
Ching-Hon Pui ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Gene Probes ◽  
Thioguanine Nucleotide ◽  
Genomic Basis ◽  
Insight Into ◽  
In Vitro Data

Abstract To elucidate interpatient variability in thioguanine nucleotide (TGN) concentrations in acute lymphoblastic leukemia (ALL) cells, we determined the TGN concentrations in leukemic blasts from 82 children with newly diagnosed ALL after intravenous administration of mercaptopurine (MP). Patients treated with MP alone achieved higher TGN concentrations than those treated with the combination of methotrexate plus mercaptopurine (MTX + MP). Analysis of the expression of approximately 9600 genes in ALL cells obtained at diagnosis identified 60 gene probes significantly associated with TGN accumulation in patients treated with MP alone and 75 gene probes in patients treated with MTX + MP, with no overlap between the 2 sets of genes. Genes significantly associated with intracellular TGN accumulation after MP alone included those encoding MP metabolic enzymes and transporters (eg, SLC29A1). Inhibition of SLC29A1 by nitrobenzylmercaptopurine ribonucleoside (NBMPR) caused a 33% to 45% reduction of TGN in ALL cells in vitro (P < .006), consistent with the gene expression findings. Genes associated with TGN concentration after combination therapy included those involved in protein and adenosine triphosphate (ATP)-biosynthesis. Together, these in vivo and in vitro data provide new insight into the genomic basis of interpatient differences in intracellular TGN accumulation and reveal significant differences between treatment with MP alone and treatment with MP and MTX. (Blood. 2005;106:1778-1785)

scholarly journals Signal-Induced Transcriptional Activation by Dif Requires the dTRAP80 Mediator Module

2003 ◽  
Vol 23 (4) ◽  
pp. 1358-1367 ◽  
Author(s):  
Jin Mo Park ◽  
Jung Mo Kim ◽  
Lark Kyun Kim ◽  
Se Nyun Kim ◽  
Jeongsil Kim-Ha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Specific Binding ◽  
Regulation Of Gene Expression ◽  
Transcriptional Activator ◽  
Mediator Complex ◽  
Activator Proteins ◽  
In Vitro Interactions

ABSTRACT The Mediator complex is the major multiprotein transcriptional coactivator complex in Drosophila melanogaster. Mediator components interact with diverse sets of transcriptional activator proteins to elicit the sophisticated regulation of gene expression. The distinct phenotypes associated with certain mutations in some of the Mediator genes and the specific in vitro interactions of Mediator gene products with transcriptional activator proteins suggest the presence of activator-specific binding subunits within the Mediator complex. However, the physiological relevance of these selective in vitro interactions has not been addressed. Therefore, we analyzed dTRAP80, one of the putative activator-binding subunits of the Mediator, for specificity of binding to a number of natural transcriptional activators from Drosophila. Among the group of activator proteins that requires the Mediator complex for transcriptional activation, only a subset of these proteins interacted with dTRAP80 in vitro and only these dTRAP80-interacting activators were defective for activation under dTRAP80-deficient in vivo conditions. In particular, activation of Drosophila antimicrobial peptide drosomycin gene expression by the NF-κB-like transcription factor Dif during induction of the Toll signaling pathway was dependent on the dTRAP80 module. These results, and the indirect support from the dTRAP80 artificial recruitment assay, indicate that dTRAP80 serves as a genuine activator-binding target responsible for a distinct group of activators.

scholarly journals Yeast GMP Kinase Mutants Constitutively Express AMP Biosynthesis Genes by Phenocopying a Hypoxanthine-Guanine Phosphoribosyltransferase Defect

Genetics ◽  
2000 ◽  
Vol 156 (3) ◽  
pp. 953-961
Author(s):  
Karine Lecoq ◽  
Manfred Konrad ◽  
Bertrand Daignan-Fornier
Keyword(s):  
Gene Expression ◽  
Vegetative Growth ◽  
Kinase Activity ◽  
Feedback Inhibition ◽  
Purine Base ◽  
Growth Defects ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Lines Of Evidence

Abstract We have characterized a new locus, BRA3, leading to deregulation of the yeast purine synthesis genes (ADE genes). We show that bra3 mutations are alleles of the GUK1 gene, which encodes GMP kinase. The bra3 mutants have a low GMP kinase activity, excrete purines in the medium, and show vegetative growth defects and resistance to purine base analogs. The bra3 locus also corresponds to the previously described pur5 locus. Several lines of evidence indicate that the decrease in GMP kinase activity in the bra3 mutants results in GMP accumulation and feedback inhibition of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), encoded by the HPT1 gene. First, guk1 and hpt1 mutants share several phenotypes, such as adenine derepression, purine excretion, and 8-azaguanine resistance. Second, overexpression of HPT1 allows suppression of the deregulated phenotype of the guk1 mutants. Third, we show that purified yeast HGPRT is inhibited by GMP in vitro. Finally, incorporation of hypoxanthine into nucleotides is similarly diminished in hpt1 and guk1 mutants in vivo. We conclude that the decrease in GMP kinase activity in the guk1 mutants results in deregulation of the ADE gene expression by phenocopying a defect in HGPRT. The possible occurrence of a similar phenomenon in humans is discussed.

scholarly journals Mediator Complex Subunit 19 Promotes the Development of Hepatocellular Carcinoma by Regulating the AKT/mTOR Signaling Pathway

2022 ◽  
Vol 11 ◽  
Author(s):  
Yuting Zhang ◽  
Peifang Qin ◽  
Xingfeng Xu ◽  
Mao Li ◽  
Haitao Huang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Rna Polymerase Ii ◽  
Malignant Tumors ◽  
Mediator Complex ◽  
Clinical Prognosis ◽  
Immune Infiltration ◽  
Downstream Effectors ◽  
A Subunit

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors, the pathogenesis of which remains unclear. Mediator complex subunit 19 (MED19), a subunit of the Mediator complex, is a multi-protein co-activator necessary for DNA transcription factors to induce RNA polymerase II transcription. In the current study, we aimed to study the role of MED19 in HCC and elucidate its mechanism.MethodsMED19 expression in HCC tissues was determined. The relationship between MED19 and the clinical prognosis was explored. The influence of MED19 on HCC cell viability, migration, invasion, and apoptosis was studied. The expression of AKT/mTOR pathway genes and proteins was detected by qRT-PCR and western blot. The correlation between MED19 and immune infiltration was investigated.ResultsMED19 was upregulated in HCC tissues compared with tumor-adjacent tissues, and was associated with a poor prognosis. Furthermore, high MED19 expression was correlated with race, gender, etc. Knockdown of MED19 inhibited cell proliferation, migration, invasion, and promoted apoptosis. Knockdown of MED19 decreased p-AKT and p-mTOR protein expression. Additionally, the downstream effectors of the AKT/mTOR pathway, p70S6K1 and 4EBP1, were affected by MED19. Notably, MED19 expression was positively correlated with the infiltration levels of B cells, CD4+ T cells, CD8+ T cells, macrophages, etc.ConclusionMED19 is significantly upregulated in HCC tissues and cells. MED19 may promote the progression of HCC in vitro and may be related to immune infiltration. Together, our data show that MED19 could be considered as a new possible biomarker as well as a novel therapeutic target for HCC.

scholarly journals Mediator complex subunit Med19 binds directly GATA DNA-binding zinc finger and functions with Med1 in GATA-driven gene regulation in vivo

2020 ◽  
Author(s):  
Clément Immarigeon ◽  
Sandra Bernat-Fabre ◽  
Emmanuelle Guillou ◽  
Alexis Verger ◽  
Elodie Prince ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Zinc Finger ◽  
Target Gene ◽  
Target Genes ◽  
Mediator Complex ◽  
Transcriptional Responses ◽  
Evolutionarily Conserved

AbstractThe evolutionarily-conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Polymerase II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. However, binary MED subunit - TF partnerships are probably oversimplified models. Using Drosophila TFs of the GATA family - Pannier (Pnr) and Serpent (Srp) - as a model, we have previously established GATA cofactor evolutionarily-conserved function for the Med1 Mediator subunit. Here, we show that another subunit, Med19, is required for GATA-dependent gene expression and interacts physically with Pnr and Srp in cellulo, in vivo and in vitro through their conserved C-zinc finger (ZF), indicating general GATA co-activator functions. Interestingly, Med19 is critical for the regulation of all tested GATA target genes which is not the case for Med1, suggesting differential use of MED subunits by GATAs depending on the target gene. Lastly, despite their presumed distant position within the MED middle module, both subunits interact physically. In conclusion, our data shed new light first on the MED complex, engaging several subunits to mediate TF-driven transcriptional responses and second, on GATA TFs, showing that ZF DNA-binding domain also serves for transactivation.

scholarly journals A transcriptional mediator protein that is required for activation of many RNA polymerase II promoters and is conserved from yeast to humans.

1997 ◽  
Vol 17 (8) ◽  
pp. 4622-4632 ◽  
Author(s):  
Y C Lee ◽  
S Min ◽  
B S Gim ◽  
Y J Kim
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Mediator Complex ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Inducible Promoters ◽  
Activation Of Transcription

A temperature-sensitive mutation was obtained in Med6p, a component of the mediator complex from the yeast Saccharomyces cerevisiae. The mediator complex has been shown to enable transcriptional activation in vitro. This mutation in Med6p abolished activation of transcription from four of five inducible promoters tested in vivo. There was no effect, however, on uninduced transcription, transcription of constitutively expressed genes, or transcription by RNA polymerases I and III. Mediator-RNA polymerase II complex isolated from the mutant yeast strain was temperature sensitive for transcriptional activation in a reconstituted in vitro system due to a defect in initiation complex formation. A database search revealed the existence of MED6-related genes in humans and Caenorhabditis elegans, suggesting that the role of mediator in transcriptional activation is conserved throughout the evolution.

PIP4Kβ interacts with and modulates nuclear localization of the high-activity PtdIns5P-4-kinase isoform PIP4Kα

2010 ◽  
Vol 430 (2) ◽  
pp. 223-235 ◽  
Author(s):  
Yvette Bultsma ◽  
Willem-Jan Keune ◽  
Nullin Divecha
Keyword(s):  
Crystal Structure ◽  
Rna Interference ◽  
High Activity ◽  
Nuclear Localization ◽  
Functional Interaction ◽  
Specific Antibodies ◽  
Tumour Suppressor P53 ◽  
Insight Into

The β-isoform of PIP4K (PtdIns5P-4-kinase) regulates the levels of nuclear PtdIns5P, which in turn modulates the acetylation of the tumour suppressor p53. The crystal structure of PIP4Kβ demonstrated that it can form a homodimer with the two subunits arranged in opposite orientations. Using MS, isoform-specific antibodies against PIP4Ks, RNAi (RNA interference) suppression and overexpression studies, we show that PIP4Kβ interacts in vitro and in vivo with the PIP4Kα isoform. As the two isoforms phosphorylate the same substrate to generate the same product, the interaction could be considered to be functionally redundant. However, contrary to expectation, we find that PIP4Kβ has 2000-fold less activity towards PtdIns5P compared with PIP4Kα, and that the majority of PIP4K activity associated with PIP4Kβ comes from its interaction with PIP4Kα. Furthermore, PIP4Kβ can modulate the nuclear localization of PIP4Kα, and PIP4Kα has a role in regulating PIP4Kβ functions. The results of the present study suggest a rationale for the functional interaction between PIP4Kα and PIP4Kβ and provide insight into how the relative levels of the two enzymes may be important in their physiological and pathological roles.

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