scholarly journals Multiple Nuclear Localization Signals Mediate Nuclear Localization of the GATA Transcription Factor AreA

2014 ◽  
Vol 13 (4) ◽  
pp. 527-538 ◽  
Author(s):  
Cameron C. Hunter ◽  
Kendra S. Siebert ◽  
Damien J. Downes ◽  
Koon Ho Wong ◽  
Sara D. Kreutzberger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Nitrogen Sources ◽  
Nuclear Accumulation ◽  
Content Type ◽  
Nuclear Localization Signals ◽  
Gata Transcription Factor

ABSTRACTTheAspergillus nidulansGATA transcription factor AreA activates transcription of nitrogen metabolic genes in response to nitrogen limitation and is known to accumulate in the nucleus during nitrogen starvation. Sequence analysis of AreA revealed multiple nuclear localization signals (NLSs), five putative classical NLSs conserved in fungal AreA orthologs but not in theSaccharomyces cerevisiaefunctional orthologs Gln3p and Gat1p, and one putative noncanonical RRX33RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructedareAmutants with mutations in individual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes. Deletion of all five classical NLSs individually or collectively did not affect utilization of nitrogen sources or AreA-dependent gene expression and did not prevent AreA nuclear localization. Mutation of the bipartite NLS conferred the inability to utilize alternative nitrogen sources and abolished AreA-dependent gene expression likely due to effects on DNA binding but did not prevent AreA nuclear localization. Mutation of all six NLSs simultaneously prevented AreA nuclear accumulation. The bipartite NLS alone strongly directed GFP to the nucleus, whereas the classical NLSs collaborated to direct GFP to the nucleus. Therefore, AreA contains multiple conserved NLSs, which show redundancy and together function to mediate nuclear import. The noncanonical bipartite NLS is conserved in GATA factors fromAspergillus, yeast, and mammals, indicating an ancient origin.

scholarly journals Novel Antifungal Drug Discovery Based on Targeting Pathways Regulating the Fungus-Conserved Upc2 Transcription Factor

2013 ◽  
Vol 58 (1) ◽  
pp. 258-266 ◽  
Author(s):  
Christina Gallo-Ebert ◽  
Melissa Donigan ◽  
Ilana L. Stroke ◽  
Robert N. Swanson ◽  
Melissa T. Manners ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Fungal Pathogens ◽  
Direct Interaction ◽  
Antifungal Drugs ◽  
Sterol Biosynthesis ◽  
Content Type ◽  
Stressed Cells

ABSTRACTInfections byCandida albicansand related fungal pathogens pose a serious health problem for immunocompromised patients. Azole drugs, the most common agents used to combat infections, target the sterol biosynthetic pathway. Adaptation to azole therapy develops as drug-stressed cells compensate by upregulating several genes in the pathway, a process mediated in part by the Upc2 transcription factor. We have implemented a cell-based high-throughput screen to identify small-molecule inhibitors of Upc2-dependent induction of sterol gene expression in response to azole drug treatment. The assay is designed to identify not only Upc2 DNA binding inhibitors but also compounds impeding the activation of gene expression by Upc2. An AlphaScreen assay was developed to determine whether the compounds identified interact directly with Upc2 and inhibit DNA binding. Three compounds identified by the cell-based assay inhibited Upc2 protein level andUPC2-LacZgene expression in response to a block in sterol biosynthesis. The compounds were growth inhibitory and attenuated antifungal-induced sterol gene expressionin vivo. They did so by reducing the level of Upc2 protein and Upc2 DNA binding in the presence of drug. The mechanism by which the compounds restrict Upc2 DNA binding is not through a direct interaction, as demonstrated by a lack of DNA binding inhibitory activity using the AlphaScreen assay. Rather, they likely inhibit a novel pathway activating Upc2 in response to a block in sterol biosynthesis. We suggest that the compounds identified represent potential precursors for the synthesis of novel antifungal drugs.

scholarly journals Cottontail Rabbit Papillomavirus E1 and E2 Proteins Mutually Influence Their Subcellular Localizations

2018 ◽  
Vol 92 (21) ◽  
Author(s):  
Markus Schneider ◽  
Aylin Yigitliler ◽  
Frank Stubenrauch ◽  
Thomas Iftner
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Direct Interaction ◽  
Genome Replication ◽  
Content Type ◽  
Mutant Proteins ◽  
Nuclear Localization Signals ◽  
Cottontail Rabbit ◽  
Direct Binding

ABSTRACTThe papillomavirus (PV) E2 protein is a nuclear, sequence-specific DNA-binding protein that regulates transcription and nuclear retention of viral genomes. E2 also interacts with the viral E1 protein to replicate the viral genome. E2 residue K111 is highly conserved among PV and has been implicated in contributing to nuclear transport, transcription, and replication. Cottontail rabbit (Sylvilagus floridanus) PV (CRPV or SfPV1) E2 K111R, A, or Q mutations are transcription deficient and localized to the cytoplasm, comparable to other PV types. The addition of a nuclear localization signal (NLS) resulted in nuclear E2 K111 mutant proteins but did not restore transcriptional activation, and this is most likely due to an impaired binding to the cellular Brd4 protein. Surprisingly, coexpression of E1 with E2 K111 mutations resulted in their nuclear localization and, for K111A and R mutations, the activation of an E1/E2-dependent reporter construct. Interestingly, the nuclear localization of E2 K111Q mutant protein was independent from the presence of the conserved bipartite NLS in E1 and the direct interaction between E1 and E2. On the other hand, the cytoplasmic E1 NLS mutation could be targeted to the nucleus by wild-type E2, and this was dependent upon an interaction between E1 and E2. In summary, our studies have uncovered that E1 and E2 control each other's subcellular localization: direct binding of E2 to E1 can direct E1 to the nucleus independently from the E1 NLS, and E1 can direct E2 to the nucleus without an intact NLS or direct binding to E2.IMPORTANCEPapillomaviruses encode the DNA-binding E1 and E2 proteins, which form a complex and are essential for genome replication. Both proteins are targeted to the nucleus via nuclear localization signals. Our studies have uncovered that cytoplasmic mutant E1 or E2 proteins can be localized to the nucleus when E1 or E2 is also present. An interaction between E1 and E2 is necessary to target cytoplasmic E1 mutant proteins to the nucleus, but cytoplasmic E2 mutant proteins can be targeted to the nucleus without a direct interaction, which points to a novel function of E1.

Nuclear accumulation of the E2F heterodimer regulated by subunit composition and alternative splicing of a nuclear localization signal

1996 ◽  
Vol 109 (10) ◽  
pp. 2443-2452 ◽  
Author(s):  
S. de la Luna ◽  
M.J. Burden ◽  
C.W. Lee ◽  
N.B. La Thangue
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Alternative Splicing ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Binding Activity ◽  
Nuclear Accumulation ◽  
Dna Binding Activity ◽  
Localization Signal

The cellular transcription factor E2F plays a critical role in integrating cell cycle progression with the transcription apparatus by virtue of a physical interaction and control by key regulators of the cell cycle, such as pRb, cyclins and cyclin-dependent kinases. Generic E2F DNA binding activity arises when a member of two families of proteins, E2F and DP, form heterodimeric complexes, an interaction which results in co-operative transcriptional and DNA binding activity. Here, we characterise a new and hitherto unexpected mechanism of control influencing the activity of E2F which is mediated at the level of intracellular location through a dependence on heterodimer formation for nuclear translocation. Nuclear accumulation is dramatically influenced by two distinct processes: alternative splicing of a nuclear localization signal and subunit composition of the E2F heterodimer. These data define a new level of control in the E2F transcription factor whereby interplay between subunits dictates the levels of nuclear DNA binding activity.

scholarly journals Identification of nuclear localization signals in the human homeoprotein MSX1

2018 ◽  
Vol 96 (4) ◽  
pp. 483-489 ◽  
Author(s):  
Akio Shibata ◽  
Junichiro Machida ◽  
Seishi Yamaguchi ◽  
Masashi Kimura ◽  
Tadashi Tatematsu ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Fluorescent Protein ◽  
Nuclear Accumulation ◽  
Web Based ◽  
Core Motif ◽  
Nuclear Localization Signals ◽  
Proliferation And Differentiation ◽  
Localization Signal ◽  
Green Fluorescent

MSX1 is one of the homeoproteins with the homeodomain (HD) sequence, which regulates proliferation and differentiation of mesenchymal cells. In this study, we investigated the nuclear localization signal (NLS) in the MSX1 HD by deletion and amino acid substitution analyses. The web-based tool NLStradamus predicted 2 putative basic motifs in the N- and C-termini of the MSX1 HD. Green fluorescent protein (GFP) chimera studies revealed that NLS1 (161RKHKTNRKPR170) and NLS2 (216NRRAKAKR223) were independently insufficient for robust nuclear localization. However, they can work cooperatively to promote nuclear localization of MSX1, as was shown by the 2 tandem NLS motifs partially restoring functional NLS, leading to a significant nuclear accumulation of the GFP chimera. These results demonstrate a unique NLS motif in MSX1, which consists of an essential single core motif in helix-I, with weak potency, and an auxiliary subdomain in helix-III, which alone does not have nuclear localization potency. Additionally, other peptide sequences, other than predicted 2 motifs in the spacer, may be necessary for complete nuclear localization in MSX1 HD.

scholarly journals Shade-induced nuclear localization of PIF7 is regulated by phosphorylation and 14-3-3 proteins in Arabidopsis

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Xu Huang ◽  
Qian Zhang ◽  
Yupei Jiang ◽  
Chuanwei Yang ◽  
Qianyue Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Nuclear Localization ◽  
Stem Elongation ◽  
Hypocotyl Elongation ◽  
Nuclear Accumulation ◽  
Shade Avoidance ◽  
Light Perception ◽  
Shade Avoidance Syndrome

Shade avoidance syndrome enables shaded plants to grow and compete effectively against their neighbors. In Arabidopsis, the shade-induced de-phosphorylation of the transcription factor PIF7 (PHYTOCHROME-INTERACTING FACTOR 7) is the key event linking light perception to stem elongation. However, the mechanism through which phosphorylation regulates the activity of PIF7 is unclear. Here, we show that shade light induces the de-phosphorylation and nuclear accumulation of PIF7. Phosphorylation-resistant site mutations in PIF7 result in increased nuclear localization and shade-induced gene expression, and consequently augment hypocotyl elongation. PIF7 interacts with 14-3-3 proteins. Blocking the interaction between PIF7 and 14-3-3 proteins or reducing the expression of 14-3-3 proteins accelerates shade-induced nuclear localization and de-phosphorylation of PIF7, and enhances the shade phenotype. By contrast, the 14-3-3 overexpressing line displays an attenuated shade phenotype. These studies demonstrate a phosphorylation-dependent translocation of PIF7 when plants are in shade and a novel mechanism involving 14-3-3 proteins, mediated by the retention of PIF7 in the cytoplasm that suppresses the shade response.

scholarly journals The High Mobility Group Box Transcription Factor Nhp6Ap Enters the Nucleus by a Calmodulin-dependent, Ran-independent Pathway

2007 ◽  
Vol 282 (46) ◽  
pp. 33743-33751 ◽  
Author(s):  
John A. Hanover ◽  
Dona C. Love ◽  
Nikki DeAngelis ◽  
Meghan E. O'Kane ◽  
Raquel Lima-Miranda ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Nuclear Localization ◽  
High Mobility ◽  
High Mobility Group ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Nuclear Entry ◽  
Nuclear Localization Signals ◽  
Calmodulin Binding ◽  
Evolutionarily Conserved

A gradient of Ran·GTP typically regulates traffic through the nuclear pore by modulating association of receptors with cargo. However, here we demonstrate that the yeast high mobility group box transcription factor Nhp6Ap enters the nucleus via a novel nuclear localization signal recognized by calcium calmodulin in a process that does not require Ran. Calmodulin is strictly required for the nondiffusional nuclear entry of Nhp6Ap. Calmodulin and DNA exhibit mutually exclusive binding to NHP6A, indicating that the directionality of Nhp6Ap nuclear accumulation may be driven by DNA-dependent dissociation of calmodulin. Our findings demonstrate that calmodulin can serve as a molecular switch triggering nuclear entry with subsequent dissociation of calmodulin binding upon interaction of cargo with chromatin. This pathway appears to be evolutionarily conserved; mammalian high mobility group box transcription factors often have two nuclear localization signals: one a classical Ran-dependent signal and a second that binds calmodulin. The finding that Nhp6Ap nuclear entry requires calmodulin but not Ran indicates that Nhp6Ap is a good model for studying this poorly understood but evolutionarily conserved calmodulin-dependent nuclear import pathway.

scholarly journals EspM Is a Conserved Transcription Factor That Regulates Gene Expression in Response to the ESX-1 System

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kevin G. Sanchez ◽  
Micah J. Ferrell ◽  
Alexandra E. Chirakos ◽  
Kathleen R. Nicholson ◽  
Robert B. Abramovitch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Protein Transport ◽  
Transcriptional Response ◽  
Secretion System ◽  
Pathogenic Species ◽  
Macrophage Infection ◽  
Content Type ◽  
Link Type ◽  
Phagosomal Membrane

ABSTRACT Pathogenic mycobacteria encounter multiple environments during macrophage infection. Temporally, the bacteria are engulfed into the phagosome, lyse the phagosomal membrane, and interact with the cytosol before spreading to another cell. Virulence factors secreted by the mycobacterial ESX-1 (ESAT-6-system-1) secretion system mediate the essential transition from the phagosome to the cytosol. It was recently discovered that the ESX-1 system also regulates mycobacterial gene expression in Mycobacterium marinum (R. E. Bosserman, T. T. Nguyen, K. G. Sanchez, A. E. Chirakos, et al., Proc Natl Acad Sci U S A 114:E10772–E10781, 2017, https://doi.org/10.1073/pnas.1710167114), a nontuberculous mycobacterial pathogen, and in the human-pathogenic species M. tuberculosis (A. M. Abdallah, E. M. Weerdenburg, Q. Guan, R. Ummels, et al., PLoS One 14:e0211003, 2019, https://doi.org/10.1371/journal.pone.0211003). It is not known how the ESX-1 system regulates gene expression. Here, we identify the first transcription factor required for the ESX-1-dependent transcriptional response in pathogenic mycobacteria. We demonstrate that the gene divergently transcribed from the whiB6 gene and adjacent to the ESX-1 locus in mycobacterial pathogens encodes a conserved transcription factor (MMAR_5438, Rv3863, now espM). We prove that EspM from both M. marinum and M. tuberculosis directly and specifically binds the whiB6-espM intergenic region. We show that EspM is required for ESX-1-dependent repression of whiB6 expression and for the regulation of ESX-1-associated gene expression. Finally, we demonstrate that EspM functions to fine-tune ESX-1 activity in M. marinum. Taking the data together, this report extends the esx-1 locus, defines a conserved regulator of the ESX-1 virulence pathway, and begins to elucidate how the ESX-1 system regulates gene expression. IMPORTANCE Mycobacterial pathogens use the ESX-1 system to transport protein substrates that mediate essential interactions with the host during infection. We previously demonstrated that in addition to transporting proteins, the ESX-1 secretion system regulates gene expression. Here, we identify a conserved transcription factor that regulates gene expression in response to the ESX-1 system. We demonstrate that this transcription factor is functionally conserved in M. marinum, a pathogen of ectothermic animals; M. tuberculosis, the human-pathogenic species that causes tuberculosis; and M. smegmatis, a nonpathogenic mycobacterial species. These findings provide the first mechanistic insight into how the ESX-1 system elicits a transcriptional response, a function of this protein transport system that was previously unknown.

scholarly journals Evolutionary Analysis of Transcriptional Regulation Mediated by Cdx2 in Rodents

2021 ◽  
Author(s):  
Weizheng Liang ◽  
Guipeng Li ◽  
Huanhuan Cui ◽  
Yukai Wang ◽  
Wencheng Wei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Phenotypic Diversity ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Evolutionary Analysis ◽  
Protein Sequence Analysis ◽  
Systematic Analysis

AbstractDifferences in gene expression, which can arise from divergence in cis-regulatory elements or alterations in transcription factors binding specificity, are one of the most important causes of phenotypic diversity during evolution. By protein sequence analysis, we observed high sequence conservation in the DNA binding domain (DBD) of the transcription factor Cdx2 across many vertebrates, whereas three amino acid changes were exclusively found in mouse Cdx2 (mCdx2), suggesting potential positive selection in the mouse lineage. Multi-omics analyses were then carried out to investigate the effects of these changes. Surprisingly, there were no significant functional differences between mCdx2 and its rat homologue (rCdx2), and none of the three amino acid changes had any impact on its function. Finally, we used rat-mouse allodiploid embryonic stem cells (RMES) to study the cis effects of Cdx2-mediated gene regulation between the two rodents. Interestingly, whereas Cdx2 binding is largely divergent between mouse and rat, the transcriptional effect induced by Cdx2 is conserved to a much larger extent.Author summaryOur study 1) represented a first systematic analysis of species-specific adaptation in DNA binding pattern of transcription factor. Although the mouse-specific amino acid changes did not manifest functional impact in our system, several explanations may account for it (See Discussion part for the detail); 2) represented a first study of cis-regulation between two reproductively isolated species by using a novel allodiploid system; 3) demonstrated a higher conservation of transcriptional output than that of DNA binding, suggesting the evolvability/plasticity of the latter; 4) finally provided a rich data resource for Cdx2 mediated regulation, including gene expression, chromatin accessibility and DNA binding etc.

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