Role of the RAM Network in Cell Polarity and Hyphal Morphogenesis inCandida albicans

RAM (regulation of Ace2p transcription factor and polarized morphogenesis) is a conserved signaling network that regulates polarized morphogenesis in yeast, worms, flies, and humans. To investigate the role of the RAM network in cell polarity and hyphal morphogenesis of Candida albicans, each of the C. albicans RAM genes (CaCBK1, CaMOB2, CaKIC1, CaPAG1, CaHYM1, and CaSOG2) was deleted. All C. albicans RAM mutants exhibited hypersensitivity to cell-wall- or membrane-perturbing agents, exhibiting cell-separation defects, a multinucleate phenotype and loss of cell polarity. Yeast two-hybrid and in vivo functional analyses of CaCbk1p and its activator, CaMob2p, the key factors in the RAM network, demonstrated that the direct interaction between the SMA domain of CaCbk1p and the Mob1/phocein domain of CaMob2p was necessary for hyphal growth of C. albicans. Genome-wide transcription profiling of a Camob2 mutant suggested that the RAM network played a role in serum- and antifungal azoles–induced activation of ergosterol biosynthesis genes, especially those involved in the late steps of ergosterol biosynthesis, and might be associated, at least indirectly, with the Tup1p-Nrg1p pathway. Collectively, these results demonstrate that the RAM network is critically required for hyphal growth as well as normal vegetative growth in C. albicans.

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Genome-wide CRISPR-Cas9 screen identified KLF11 as a druggable suppressor for sarcoma cancer stem cells

Science Advances ◽

10.1126/sciadv.abe3445 ◽

2021 ◽

Vol 7 (5) ◽

pp. eabe3445

Author(s):

Yicun Wang ◽

Jinhui Wu ◽

Hui Chen ◽

Yang Yang ◽

Chengwu Xiao ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Negative Feedback ◽

Direct Interaction ◽

Therapy Resistance ◽

Negative Regulator ◽

Chemotherapy Response ◽

Genome Wide

Cancer stem cells (CSCs) are involved in tumorigenesis, recurrence, and therapy resistance. To identify critical regulators of sarcoma CSCs, we performed a reporter-based genome-wide CRISPR-Cas9 screen and uncovered Kruppel-like factor 11 (KLF11) as top candidate. In vitro and in vivo functional annotation defined a negative role of KLF11 in CSCs. Mechanistically, KLF11 and YAP/TEAD bound to adjacent DNA sites along with direct interaction. KLF11 recruited SIN3A/HDAC to suppress the transcriptional output of YAP/TEAD, which, in turn, promoted KLF11 transcription, forming a negative feedback loop. However, in CSCs, this negative feedback was lost because of epigenetic silence of KLF11, causing sustained YAP activation. Low KLF11 was associated with poor prognosis and chemotherapy response in patients with sarcoma. Pharmacological activation of KLF11 by thiazolidinedione effectively restored chemotherapy response. Collectively, our study identifies KLF11 as a negative regulator in sarcoma CSCs and potential therapeutic target.

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Positive Regulation of fur Gene Expression via Direct Interaction of Fur in a Pathogenic Bacterium, Vibrio vulnificus

Journal of Bacteriology ◽

10.1128/jb.01791-06 ◽

2007 ◽

Vol 189 (7) ◽

pp. 2629-2636 ◽

Cited By ~ 35

Author(s):

Hyun-Jung Lee ◽

So Hyun Bang ◽

Kyu-Ho Lee ◽

Soon-Jung Park

Keyword(s):

Gene Expression ◽

Pathogenic Bacteria ◽

Vibrio Vulnificus ◽

Iron Concentration ◽

Direct Interaction ◽

Repeated Sequence ◽

Upstream Region ◽

Fur Protein

ABSTRACT In pathogenic bacteria, the ability to acquire iron, which is mainly regulated by the ferric uptake regulator (Fur), is essential to maintain growth as well as its virulence. In Vibrio vulnificus, a human pathogen causing gastroenteritis and septicemia, fur gene expression is positively regulated by Fur when the iron concentration is limited (H.-J. Lee et al., J. Bacteriol. 185:5891-5896, 2003). Footprinting analysis revealed that an upstream region of the fur gene was protected by the Fur protein from DNase I under iron-depleted conditions. The protected region, from −142 to −106 relative to the transcription start site of the fur gene, contains distinct AT-rich repeats. Mutagenesis of this repeated sequence resulted in abolishment of binding by Fur. To confirm the role of this cis-acting element in Fur-mediated control of its own gene in vivo, fur expression was monitored in V. vulnificus strains using a transcriptional fusion containing the mutagenized Fur-binding site (fur mt::luxAB). Expression of fur mt::luxAB showed that it was not regulated by Fur and was not influenced by iron concentration. Therefore, this study demonstrates that V. vulnificus Fur acts as a positive regulator under iron-limited conditions by direct interaction with the fur upstream region.

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DEFINING CANDIDATE PARKINSON’S DISEASE GENES THROUGH THE ANALYSIS OF GENOME-WIDE HOMOZYGOSITY

10.1101/2020.11.23.20235671 ◽

2020 ◽

Author(s):

Steven J Lubbe ◽

Yvette C. Wong ◽

Bernabe Bustos ◽

Soojin Kim ◽

Jana Vandrovcova ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Autosomal Recessive Inheritance ◽

Homozygosity Mapping ◽

Disease Genes ◽

Compound Heterozygous ◽

Genome Wide ◽

Biallelic Mutations ◽

Functional Analyses

ABSTRACTEarly-onset Parkinson’s disease (EOPD) can be caused by biallelic mutations in PRKN, DJ1 and PINK1. However, while the identification of novel genes is becoming increasingly challenging, new insights into EOPD genetics have important relevance for understanding the pathways driving disease pathogenesis. Here, using extended runs of homozygosity (ROH) >8Mb as a marker for possible autosomal recessive inheritance, we identified 90 EOPD patients with extended ROH. Investigating rare, damaging homozygous variants to identify candidate genes for EOPD, 81 genes were prioritised. Through the assessment of biallelic (homozygous and compound heterozygous) variant frequencies in cases and controls from three independent cohorts totalling 3,381 PD patients and 2,463 controls, we identified two biallelic MIEF1 variant carriers among EOPD patients. We further investigated the role of disease-associated variants in MIEF1 which encodes for MID51, an outer mitochondrial membrane protein, and found that putative EOPD-associated variants in MID51 preferentially disrupted its oligomerization state. These findings provide further support for the role of mitochondrial dysfunction in the development of PD. Together, we have used genome-wide homozygosity mapping to identify potential EOPD genes, and future studies incorporating expanded datasets and further functional analyses will help to determine their roles in disease aetiology.

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Allelic variation contributes to bacterial host specificity

Nature Communications ◽

10.1038/ncomms9754 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 46

Author(s):

Min Yue ◽

Xiangan Han ◽

Leon De Masi ◽

Chunhong Zhu ◽

Xun Ma ◽

...

Keyword(s):

Allelic Variation ◽

Multinomial Logistic Regression ◽

Gene Gain ◽

Nucleotide Polymorphisms ◽

Genome Wide ◽

Serovar Typhimurium ◽

Functional Analyses ◽

High Degree

Abstract Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population and functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. Together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts.

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A Genome-Wide RNA Interference Screen Identifies a Differential Role of the Mediator CDK8 Module Subunits for GATA/ RUNX-Activated Transcription in Drosophila

Molecular and Cellular Biology ◽

10.1128/mcb.01625-09 ◽

2010 ◽

Vol 30 (11) ◽

pp. 2837-2848 ◽

Cited By ~ 25

Author(s):

Vanessa Gobert ◽

Dani Osman ◽

Stéphanie Bras ◽

Benoit Augé ◽

Muriel Boube ◽

...

Keyword(s):

Cell Differentiation ◽

Rna Interference ◽

Cell Fate ◽

Gata Factor ◽

Hematopoietic System ◽

Crystal Cell ◽

Genome Wide ◽

A Genome

ABSTRACT Transcription factors of the RUNX and GATA families play key roles in the control of cell fate choice and differentiation, notably in the hematopoietic system. During Drosophila hematopoiesis, the RUNX factor Lozenge and the GATA factor Serpent cooperate to induce crystal cell differentiation. We used Serpent/Lozenge-activated transcription as a paradigm to identify modulators of GATA/RUNX activity by a genome-wide RNA interference screen in cultured Drosophila blood cells. Among the 129 factors identified, several belong to the Mediator complex. Mediator is organized in three modules plus a regulatory “CDK8 module,” composed of Med12, Med13, CycC, and Cdk8, which has long been thought to behave as a single functional entity. Interestingly, our data demonstrate that Med12 and Med13 but not CycC or Cdk8 are essential for Serpent/Lozenge-induced transactivation in cell culture. Furthermore, our in vivo analysis of crystal cell development show that, while the four CDK8 module subunits control the emergence and the proliferation of this lineage, only Med12 and Med13 regulate its differentiation. We thus propose that Med12/Med13 acts as a coactivator for Serpent/Lozenge during crystal cell differentiation independently of CycC/Cdk8. More generally, we suggest that the set of conserved factors identified herein may regulate GATA/RUNX activity in mammals.

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Identification of Genes That Contribute to the Pathogenesis of Invasive Pneumococcal Disease byIn VivoTranscriptomic Analysis

Infection and Immunity ◽

10.1128/iai.00295-12 ◽

2012 ◽

Vol 80 (9) ◽

pp. 3268-3278 ◽

Cited By ~ 44

Author(s):

Abiodun D. Ogunniyi ◽

Layla K. Mahdi ◽

Claudia Trappetti ◽

Nadine Verhoeven ◽

Daphne Mermans ◽

...

Keyword(s):

Invasive Pneumococcal Disease ◽

Pneumococcal Disease ◽

Targeted Mutagenesis ◽

Content Type ◽

Genome Wide ◽

Blood Relative ◽

High Level ◽

Upregulated Genes

ABSTRACTStreptococcus pneumoniae(the pneumococcus) continues to be responsible for a high level of global morbidity and mortality resulting from pneumonia, bacteremia, meningitis, and otitis media. Here we have used a novel technique involving niche-specific, genome-widein vivotranscriptomic analyses to identify genes upregulated in distinct niches during pathogenesis after intranasal infection of mice with serotype 4 or 6A pneumococci. The analyses yielded 28 common, significantly upregulated genes in the lungs relative to those in the nasopharynx and 25 significantly upregulated genes in the blood relative to those in the lungs in both strains, some of which were previously unrecognized. The role of five upregulated genes from either the lungs or the blood in pneumococcal pathogenesis and virulence was then evaluated by targeted mutagenesis. One of the mutants (ΔmalX) was significantly attenuated for virulence in the lungs, two (ΔaliAand ΔilvH) were significantly attenuated for virulence in the blood relative to the wild type, and two others (ΔcbiOand ΔpiuA) were completely avirulent in a mouse intranasal challenge model. We also show that the products ofaliA,malX, andpiuAare promising candidates for incorporation into multicomponent protein-based pneumococcal vaccines currently under development. Importantly, we suggest that this new approach is a viable complement to existing strategies for the discovery of genes critical to the distinct stages of invasive pneumococcal disease and potentially has broad application for novel protein antigen discovery in other pathogens such asS. pyogenes,Haemophilus influenzaetype b, andNeisseria meningitidis.

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Role of Ndt80p in Sterol Metabolism Regulation and Azole Resistance in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00074-09 ◽

2009 ◽

Vol 8 (8) ◽

pp. 1174-1183 ◽

Cited By ~ 64

Author(s):

Adnane Sellam ◽

Faïza Tebbji ◽

André Nantel

Keyword(s):

Candida Albicans ◽

Stress Responses ◽

Efflux Pump ◽

Transcriptional Profiling ◽

Ergosterol Biosynthesis ◽

Gene Promoters ◽

Sterol Metabolism ◽

Metabolism Regulation ◽

Genome Wide

ABSTRACT The Ndt80p transcription factor modulates azole tolerance in Candida albicans by controlling the expression of the gene for the drug efflux pump Cdr1p. To date, the contribution of this transcriptional modulator to drug tolerance is not yet well understood. Here, we investigate the role of Ndt80p in mediating fluconazole tolerance by determining its genome-wide occupancy using chromatin immunoprecipitation coupled to high-density tiling arrays. Ndt80p was found to bind a large number of gene promoters with diverse biological functions. Gene ontology analysis of these Ndt80p targets revealed a significant enrichment in gene products related to the cell wall, carbohydrate metabolism, stress responses, hyphal development, multidrug transport, and the cell cycle. Ndt80p was found on the promoters of ergosterol biosynthesis genes, including on the azole target Erg11p. Additionally, expression profiling was used to identify fluconazole-responsive genes that require Ndt80p for their proper expression. We found that Ndt80p is crucial for the expression of numerous fluconazole-responsive genes, especially genes involved in ergosterol metabolism. Therefore, by combining genome-wide location and transcriptional profiling, we have characterized the Ndt80p fluconazole-dependent regulon and demonstrated the key role of this global transcriptional regulator in modulating sterol metabolism and drug resistance in C. albicans.

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Requirement of splicing factor hnRNP A2B1 of hnRNPs for tumorigenesis of melanoma stem cells

10.21203/rs.3.rs-92870/v1 ◽

2020 ◽

Author(s):

Mengqi Chu ◽

Haitao Wan ◽

Xiaobo Zhang

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Tumor Therapy ◽

Cycle Arrest ◽

Genome Wide ◽

A Genome ◽

Rna Immunoprecipitation ◽

Wide Scale

Abstract Background: Cancer stem cells play essential roles in tumorigenesis, thus being the important targets for tumor therapy. The hnRNP family proteins, the important splicing factors, are found to be associated with tumor progression. However, the influence of hnRNPs on cancer stem cells has not been extensively explored.Methods: Quantitative real-time PCR and Western blot were used to examine the gene expression level. RNA immunoprecipitation assay and RNA sequencing were conducted to identify the RNAs interacted with hnRNP A2B1 on a genome-wide scale. The in vivo assays were performed in nude mice.Results: In this study, the results showed that hnRNP A2B1 of 19 hnRNPs was significantly upregulated in melanoma stem cells compared with non-stem cells, suggesting the important role of hnRNP A2B1 in cancer stem cells. The hnRNP A2B1 silencing triggered the cell cycle arrest in G2 phase, leading to apoptosis of melanoma stem cells. The results revealed that hnRNP A2B1 could bind to the precursor mRNAs of pro-apoptosis genes (DAPK1, SYT7 and RNF128) and anti-apoptosis genes (EIF3H, TPPP3 and DOCK2) to regulate the splicing of these 6 genes, thus promoting the expressions of anti-apoptosis genes and suppressing the expressions of pro-apoptosis genes. The in vivo data indicated that hnRNP A2B1 was required for tumorigenesis of melanoma stem cells in vivo by affecting the splicing of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7, thus suppressing apoptosis of melanoma stem cells.Conclusions: HnRNP A2B1 was required for tumorigenesis of melanoma stem cells. Therefore our findings presented novel molecular insights into the roles of hnRNPs in cancer stem cells.

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The RHO GTPASE CDC42 Regulator CDC42GAP Plays a Critical Role in Neutrophil Migration Via Podosome-Like Formation.

Blood ◽

10.1182/blood.v104.11.651.651 ◽

2004 ◽

Vol 104 (11) ◽

pp. 651-651 ◽

Cited By ~ 1

Author(s):

Marie-Dominique Filippi ◽

Haiming Xu ◽

Jason Towe ◽

Chad E. Harris ◽

Kathleen Szczur ◽

...

Keyword(s):

Cell Polarity ◽

Rho Gtpase ◽

Critical Role ◽

Neutrophil Migration ◽

Cell Movement ◽

Directed Migration ◽

Mutant Cells

Abstract Neutrophils (PMN) are a critical cell in inflammatory processes. In response to environmental stimuli, they activate various signal transduction pathways allowing them to move rapidly to a site of microbial invasion and to perform phagocytosis, cytokine and oxygen substrate release. Rho GTPase proteins, Rac1, Rac2, CDC42 and Rho, are central regulators of cell movement via actin rearrangement. We have demonstrated the specific role of Rac1 and Rac2 in PMN functions (Gu and Filippi et al, Science 2003; Filippi et al. Nat Immuol., 2004) which raises the question of the specificity of the other Rho GTPases. CDC42 primarily regulates the formation of filopodia. CDC42 controls cell polarity and migration in hematopoietic cell lines. Most of previous studies have utilized dominant active or negative mutants which lack specificity and cannot be easily used to define in vivo cell biology. Here, we used mice genetically deficient in the CDC42 negative regulator CDC42 GTPase Activating Protein (GAP) to study the role of CDC42 in neutrophil functions in vitro and in vivo. Heterozygote (CDC42GAP+/−) or homozygote (CDC42GAP−/−) mutant mice displayed normal neutrophil differentiation in vitro and in vivo. PMN deficient in CDC42GAP displayed 2-fold increased in CDC42 activity. In vivo recruitment of PMN in peritoneal cavities after thioglycollate exposure was significantly impaired in CDC42GAP+/− mice compared with wild type (WT) mice (25.5±0.76 x 105 vs 35.7±0.38 x 105, p<0.05). Both CDC42GAP+/− and CDC42GAP−/− PMN demonstrated defective directed migration in vitro in response to fMLP in a Boyden chamber assay compared with WT (248±31 and 199±20 versus 314±29 migrated cells, p<0.05), suggesting that CDC42 plays a critical role in neutrophil migration in vitro and in vivo. To further understand the role of CDC42GAP in neutrophil migration, single-cell analysis by time-lapse videomicroscopy was performed. Surprisingly, CDC42GAP+/− PMN demonstrated higher migration velocity compared with WT cells in response to fMLP, but this increased speed was associated with an abnormal shape. Upon beta-2 integrin ligation, CDC42GAP+/− PMN demonstrated abnormal elongated trailing tail associated with increased tail filopodia. Importantly, the podosome-like structures identified by a vinculin ring surrounding F-actin at the ventral plasma membrane that are present in the leading edge of WT PMN was absent in the mutant cells. CDC42GAP−/− PMN demonstrated more dramatic F-actin impairment upon integrin ligation compared with CDC42GAP+/− and WT cells and remarkably showed complete loss of cell polarity, consistent with the known role of CDC42 in cell polarity. We hypothesize that the lack of podosome formation in mutant cells could account for the increased speed observed in CDC42GAP+/− cells and therefore result in ineffective directed migration in vivo. Altogether, this suggests that regulation of CDC42 activity plays a pivotal role in neutrophil migration likely via integrin-dependent podosome-like formation. This reinforces the importance of turnover of attachment structures during cell movement and suggests a new role for CDC42 in actin-based attachment structure in neutrophils.

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Functional Network Analysis Reveals Versatile MicroRNAs in Human Heart

Cellular Physiology and Biochemistry ◽

10.1159/000430324 ◽

2015 ◽

Vol 36 (4) ◽

pp. 1628-1643 ◽

Cited By ~ 5

Author(s):

Yingqi Xu ◽

Wenliang Zhu ◽

Yong Sun ◽

Zhe Wang ◽

Wei Yuan ◽

...

Keyword(s):

Protein Interactions ◽

Human Heart ◽

Myocardial Injury ◽

Dynamic Balance ◽

Mirna Gene ◽

Vital Role ◽

Association Data ◽

Functional Analyses

Background: Acting on many mRNAs allows the power of a single miRNA to modulate multiple pathophysiological phenotypes. One question is whether versatile miRNAs exist in the pathological scenarios of myocardial infarction (MI) and heart failure (HF). Methods: A hypergeometric analysis, in combination with network-based functional analyses, was performed on the available human protein interaction and miRNA-gene association data to highlight versatile miRNAs among the significantly dysregulated miRNAs in MI and HF. In vivo, mice models of MI and HF were then established to investigate whether dysregulated expression be undertaken by versatile miRNA identified here. Results: Systematic analyses really identified the previously validated miRNAs that have been verified of multiple important roles in MI and HF, demonstrating method effectiveness. By using this means, we innovatively revealed the vital role of miR-7 in maintaining the dynamic balance of protein interactions and its obvious overexpression in MI and HF that implies pathological involvement. Functional experiments are definitely needed for further revealing its potential influences on MI- or HF-led myocardial injury. Conclusion: Our results have implications not only for the coming miRNA-based strategy in treating MI and HF but also for further understanding on gene regulation by miRNAs in human heart.

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