RTG-dependent Mitochondria-to-Nucleus Signaling Is Regulated byMKS1and Is Linked to Formation of Yeast Prion [URE3]

2002 ◽  
Vol 13 (3) ◽  
pp. 795-804 ◽  
Author(s):  
Takayuki Sekito ◽  
Zhengchang Liu ◽  
Janet Thornton ◽  
Ronald A. Butow
Keyword(s):  
Gene Expression ◽  
Catabolite Repression ◽  
Target Gene ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Nitrogen Catabolite Repression ◽  
Yeast Prion ◽  
Phosphorylation Pattern ◽  
Bhlh Transcription Factors ◽  
Do So

An important function of the RTG signaling pathway is maintenance of intracellular glutamate supplies in yeast cells with dysfunctional mitochondria. Herein, we report that MKS1is a negative regulator of the RTG pathway, acting between Rtg2p, a proximal sensor of mitochondrial function, and the bHLH transcription factors Rtg1p and Rtg3p. In mks1Δcells, RTG target gene expression is constitutive, bypassing the requirement for Rtg2p, and is no longer repressible by glutamate. We show further that Mks1p is a phosphoprotein whose phosphorylation pattern parallels that of Rtg3p in response to activation of the RTG pathway, and that Mks1p is in a complex with Rtg2p. MKS1 was previously implicated in the formation of [URE3], an inactive prion form of a negative regulator of the nitrogen catabolite repression pathway, Ure2p.rtgΔ mutations induce [URE3] and can do so independently of MKS1. We find that glutamate suppresses [URE3] formation, suggesting that the Mks1p effect on the formation of [URE3] can occur indirectly via regulation of theRTG pathway.

scholarly journals The Yeast GATA Factor Gat1 Occupies a Central Position in Nitrogen Catabolite Repression-Sensitive Gene Activation

2009 ◽  
Vol 29 (13) ◽  
pp. 3803-3815 ◽  
Author(s):  
Isabelle Georis ◽  
André Feller ◽  
Fabienne Vierendeels ◽  
Evelyne Dubois
Keyword(s):  
Gene Expression ◽  
Catabolite Repression ◽  
Nitrogen Availability ◽  
Gene Activation ◽  
Nitrogen Sources ◽  
Central Position ◽  
Limiting Factor ◽  
Nitrogen Catabolite Repression ◽  
Gata Factor ◽  
Gata Factors

ABSTRACT Saccharomyces cerevisiae cells are able to adapt their metabolism according to the quality of the nitrogen sources available in the environment. Nitrogen catabolite repression (NCR) restrains the yeast's capacity to use poor nitrogen sources when rich ones are available. NCR-sensitive expression is modulated by the synchronized action of four DNA-binding GATA factors. Although the first identified GATA factor, Gln3, was considered the major activator of NCR-sensitive gene expression, our work positions Gat1 as a key factor for the integrated control of NCR in yeast for the following reasons: (i) Gat1 appeared to be the limiting factor for NCR gene expression, (ii) GAT1 expression was regulated by the four GATA factors in response to nitrogen availability, (iii) the two negative GATA factors Dal80 and Gzf3 interfered with Gat1 binding to DNA, and (iv) Gln3 binding to some NCR promoters required Gat1. Our study also provides mechanistic insights into the mode of action of the two negative GATA factors. Gzf3 interfered with Gat1 by nuclear sequestration and by competition at its own promoter. Dal80-dependent repression of NCR-sensitive gene expression occurred at three possible levels: Dal80 represses GAT1 expression, it competes with Gat1 for binding, and it directly represses NCR gene transcription.

scholarly journals cis-Dominant mutations which dramatically enhance DUR1,2 gene expression without affecting its normal regulation.

1984 ◽  
Vol 4 (5) ◽  
pp. 947-955 ◽  
Author(s):  
G Chisholm ◽  
T Cooper
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Control System ◽  
Normal Control ◽  
Chromatin Structure ◽  
Catabolite Repression ◽  
Mutant Phenotype ◽  
Nitrogen Catabolite Repression ◽  
Upstream Control ◽  
Affect Gene Expression

We have isolated three cis-dominant mutations which dramatically enhance DUR1 ,2 gene expression in Saccharomyces cerevisiae. The mutant phenotype, which is expressed both in haploid and MATa/MAT alpha diploid strains, does not appear to be an alteration of the normal control system for this gene because its expression remained fully inducible and sensitive to nitrogen catabolite repression. Instead, we found much higher levels of DUR1 ,2-specific RNA under both uninduced and induced conditions, i.e., the overproduction trait was superimposed on normal regulation of the gene. The mutations seemed to affect gene expression in a unidirectional manner or to be specific for DUR1 ,2 gene expression, because other genes in proximity to the mutations were not affected. We feel that these mutations may alter the chromatin structure in the vicinity of the DUR1 ,2 upstream control sequences or, alternatively, may be Ty insertions which no longer possess the ROAM characteristics reported by others and ourselves.

scholarly journals NOTCH1 Signaling Is Activated in CLL By Mutations of FBXW7 and Low Expression of USP28 at 11q23

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 946-946 ◽  
Author(s):  
Viola Close ◽  
William Close ◽  
Sabrina Julia Kugler ◽  
Michaela Reichenzeller ◽  
Deyan Yordanov Yosifov ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Silico ◽  
Research Funding ◽  
Target Gene ◽  
Negative Regulator ◽  
Novel Mutations ◽  
Target Gene Expression ◽  
In Silico Modeling ◽  
Wd40 Domain ◽  
Support Research

Abstract Background Oncogenic proteins can be stabilized either via genetic mutations or via defects in the ubiquitin ligating- and degradation machinery. NOTCH1 protein stability is affected by genetic mutations in approximately 10% of chronic lymphocytic leukemia (CLL) patients and mutations are associated with a worse prognosis. Yet, even in the absence of NOTCH1 mutations, NOTCH1 is activated in almost half of all CLL patients. Aims In order to shed light on NOTCH1 activation in CLL, we analyzed the impact of its modulator, the E3-ubiquitin ligase FBXW7 and the deubiquitinase USP28 that is a negative regulator of FBXW7 and is located in chromosomal band 11q23. Methods FBXW7 mutation analysis was performed via targeted next generation sequencing from a total of 905 patients. In silico modeling of potential substrate binding to the mutated FBXW7 was performed by the use of PolyPhen-2 and validated via co-immunoprecipitation of overexpressed mutated FBXW7 and NOTCH1 wild type proteins. Accumulation of FBXW7 substrates was tested via Western blot in CRISPR/Cas9 induced FBXW7 mutated HG-3 CLL cell lines and in FBXW7 mutated primary CLL cells, identifying the transcription factor NOTCH1 as FBXW7 target. Expression of NOTCH1 target genes were analyzed in FBXW7 mutated CLL cases via gene expression profiling (n=4) and RT-qPCR (n=19). In an independent cohort, USP28 and NOTCH1 target gene expression was analyzed in a total of 285 patients via gene expression profiling. Results Heterozygous FBXW7 mutations were identified in 41/905 (4.5%) CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain, while an additional 10% of mutations were located on the first exon of the abundantly expressed FBXW7 α-isoform. We identified substrate targets of FBXW7 in CLL via the generation of a truncation of the WD40 domain of FBXW7 in the CLL cell line HG-3 using CRISPR/Cas9, identifying NOTCH1 as an FBXW7 target. In silico modeling of FBXW7 mutations on protein binding predicted that novel mutations within the WD40 domain affected substrate recognition capacity of FBXW7. Interestingly, modeling predicted W425C and a new hotspot mutation G423V (found in 3/905 of CLL cases) to ablate NOTCH1 binding while the mutation A503V was predicted not to impair binding of NOTCH1 to FBXW7. The in silico modeling was confirmed by co-immunoprecipitation experiments of overexpressed NOTCH1 and FBXW7 and further revealed that mutations within the α-isoform specific N-terminus T15VR and V154I still enabled NOTCH1 binding. Intriguingly, in primary CLL cells FBXW7 mutations correlated with an increase in NOTCH1 levels that remained stable even upon inhibition of translation, underlining the enhanced protein stabilization by FBXW7 mutations. Furthermore, FBXW7 mutations in CLL resulted in an increased NOTCH1 target gene expression. FBXW7 activity is not only modulated by gene mutations, but also by the deubiquitinase USP28 which is localized in the recurrently deleted region 11q22-q23 close to the ATM tumor suppressor gene. We hypothesized that USP28 would impact on FBXW7 activity and thus on NOTCH1 stability and activity. In line with this concept we found that in primary cells from a cohort of 285 CLL patients, low USP28 expression significantly correlated with increased NOTCH1 target gene expression, independent of the 11q deletion status of the patients where USP28 is localized. Discussion In CLL patient cells we have identified novel mutations within the WD40 binding domain of FBXW7 in addition to the common hot spot mutations (R465, R479, R505). Amongst these novel mutations, G423V and W425C are recurrent and result in decreased binding of the FBXW7 substrate NOTCH1 and hence in an accumulation and induction of NOTCH1 activity. Furthermore, expression of USP28, the negative regulator of FBXW7, significantly correlated with increased NOTCH1 target gene expression. Hence, our findings uncover modulation of NOTCH1 in CLL via the FBXW7-USP28-NOTCH1 axis (Figure 1) in addition to genomic NOTCH1 modification, thus explaining the high proportion of CLL cases that harbor an activation of NOTCH1 leading to more aggressive disease. Disclosures Tausch: AbbVie: Consultancy, Other: Travel grants; Celgene: Consultancy, Other: Travel grants; Gilead: Consultancy, Other: Travel grants. Döhner:AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Agios: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding. Stilgenbauer:Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding.

scholarly journals Expression of the DAL80 gene, whose product is homologous to the GATA factors and is a negative regulator of multiple nitrogen catabolic genes in Saccharomyces cerevisiae, is sensitive to nitrogen catabolite repression.

1991 ◽  
Vol 11 (12) ◽  
pp. 6205-6215 ◽  
Author(s):  
T S Cunningham ◽  
T G Cooper
Keyword(s):  
Zinc Finger ◽  
Catabolite Repression ◽  
Regulatory Gene ◽  
Coiled Coil ◽  
Negative Regulator ◽  
Nitrogen Catabolite Repression ◽  
Activator Proteins ◽  
Gata Factors ◽  
Catabolic Genes ◽  
Mammalian Tissues

We have cloned the negative regulatory gene (DAL80) of the allantoin catabolic pathway, characterized its structure, and determined the physiological conditions that control DAL80 expression and its influence on the expression of nitrogen catabolic genes. Disruption of the DAL80 gene demonstrated that it regulates multiple nitrogen catabolic pathways. Inducer-independent expression was observed for the allantoin pathway genes DAL7 and DUR1,2, as well as the UGA1 gene required for gamma-aminobutyrate catabolism in the disruption mutant. DAL80 transcription was itself highly sensitive to nitrogen catabolite repression (NCR), and its promoter contained 12 sequences homologous to the NCR-sensitive UASNTR. The deduced DAL80 protein structure contains zinc finger and coiled-coil motifs. The DAL80 zinc finger motif possessed high homology to the transcriptional activator proteins required for expression of NCR-sensitive genes in fungi and the yeast GLN3 gene product required for functioning of the NCR-sensitive DAL UASNTR. It was also homologous to the three GATAA-binding proteins reported to be transcriptional activators in avian and mammalian tissues. The latter correlations raise the possibility that both positive and negative regulators of allantoin pathway transcription may bind to similar sequences.

scholarly journals Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification

Gut ◽  
2019 ◽  
Vol 69 (6) ◽  
pp. 1092-1103 ◽  
Author(s):  
Sam O Kleeman ◽  
Viktor H Koelzer ◽  
Helen JS Jones ◽  
Ester Gil Vazquez ◽  
Hayley Davis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colorectal Cancer ◽  
Target Gene ◽  
Area Under The Curve ◽  
Ground Truth ◽  
Negative Regulator ◽  
Molecular Biomarker ◽  
Target Gene Expression ◽  
Pathway Activation ◽  
Signal Transduction Proteins

ObjectivePathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal transduction proteins (Adenomatous polyposis coli, β-catenin), causing constitutive pathway activation and ii) ligand-dependent (LD) mutations affecting the synergistic R-Spondin axis (RNF43, RSPO-fusions) acting through amplification of endogenous Wnt signal transmembrane transduction. Our aim was to exploit differential Wnt target gene expression to generate a mutation-agnostic biomarker for LD tumours.DesignWe undertook harmonised multi-omic analysis of discovery (n=684) and validation cohorts (n=578) of colorectal tumours collated from publicly available data and the Stratification in Colorectal Cancer Consortium. We used mutation data to establish molecular ground truth and subdivide lesions into LI/LD tumour subsets. We contrasted transcriptional, methylation, morphological and clinical characteristics between groups.ResultsWnt disrupting mutations were mutually exclusive. Desmoplastic stromal upregulation of RSPO may compensate for absence of epithelial mutation in a subset of stromal-rich tumours. Key Wnt negative regulator genes were differentially expressed between LD/LI tumours, with targeted hypermethylation of some genes (AXIN2, NKD1) occurring even in CIMP-negative LD cancers. AXIN2 mRNA expression was used as a discriminatory molecular biomarker to distinguish LD/LI tumours (area under the curve >0.93).ConclusionsEpigenetic suppression of appropriate Wnt negative feedback loops is selectively advantageous in LD tumours and differential AXIN2 expression in LD/LI lesions can be exploited as a molecular biomarker. Distinguishing between LD/LI tumour types is important; patients with LD tumours retain sensitivity to Wnt ligand inhibition and may be stratified at diagnosis to clinical trials of Porcupine inhibitors.

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