Fur4 mediated uracil-scavenging to screen for surface protein regulators

Cell surface membrane proteins perform diverse and critical functions and are spatially and temporally regulated by membrane trafficking pathways. Although perturbations in these pathways underlie many pathologies, our understanding of these pathways at a mechanistic level remains incomplete. Using yeast as a model, we have developed an assay that reports on the surface activity of the Fur4 uracil permease in uracil auxotroph strains grown in the presence of limited uracil. This assay was used to screen a haploid deletion library that identified mutants with both diminished and enhanced comparative growth in restricted uracil media. Factors identified, including various multi-subunit complexes, were enriched for membrane trafficking and transcriptional functions, in addition to various uncharacterised genes. Bioinformatic analysis of expression profiles from many strains lacking identified transcription factors required for efficient uracil-scavenging revealed they control expression of other uracil-scavenging factors, in addition to membrane trafficking genes essential for viability, and therefore not represented in the screen. Finally, we performed a secondary mating factor secretion screen to functionally categorise factors implicated in uracil-scavenging, most of which are conserved throughout evolution.

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Bone Marrow Mast Cell Antibody-Targetable Cell Surface Protein Expression Profiles in Systemic Mastocytosis

International Journal of Molecular Sciences ◽

10.3390/ijms20030552 ◽

2019 ◽

Vol 20 (3) ◽

pp. 552 ◽

Cited By ~ 2

Author(s):

Noelia Dasilva-Freire ◽

Andrea Mayado ◽

Cristina Teodosio ◽

María Jara-Acevedo ◽

Iván Álvarez-Twose ◽

...

Keyword(s):

Bone Marrow ◽

Cell Surface ◽

Systemic Mastocytosis ◽

Expression Profiles ◽

Surface Membrane ◽

Surface Protein ◽

Surface Proteins ◽

Significant Fraction ◽

Surface Protein Expression ◽

Protein Expression Profiles

Despite recent therapeutic advances, systemic mastocytosis (SM) remains an incurable disease due to limited complete remission (CR) rates even after novel therapies. To date, no study has evaluated the expression on SM bone marrow mast cells (BMMC) of large panel of cell surface suitable for antibody-targeted therapy. In this study, we analyzed the expression profile of six cell-surface proteins for which antibody-based therapies are available, on BMMC from 166 SM patients vs. 40 controls. Overall, variable patterns of expression for the markers evaluated were observed among SM BMMC. Thus, CD22, CD30, and CD123, while expressed on BMMC from patients within every subtype of SM, showed highly variable patterns with a significant fraction of negative cases among advanced SM (aggressive SM (ASM), ASM with an associated clonal non-MC lineage disease (ASM-AHN) and MC leukemia (MCL)), 36%, 46%, and 39%, respectively. In turn, CD25 and FcεRI were found to be expressed in most cases (89% and 92%) in virtually all BMMC (median: 92% and 95%) from both indolent and advanced SM, but with lower/absent levels in a significant fraction of MC leukemia (MCL) and both in MCL and well-differentiated SM (WDSM) patients, respectively. In contrast, CD33 was the only marker expressed on all BMMC from every SM patient. Thus, CD33 emerges as the best potentially targetable cell-surface membrane marker in SM, particularly in advanced SM.

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Sex-Dependent Molecular Mechanisms of Lipotoxic Injury in Brain Microvasculature: Implications for Dementia

International Journal of Molecular Sciences ◽

10.3390/ijms21218146 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8146

Author(s):

Saivageethi Nuthikattu ◽

Dragan Milenkovic ◽

John C. Rutledge ◽

Amparo C. Villablanca

Keyword(s):

Transcription Factors ◽

Cognitive Function ◽

Gene Networks ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Density Lipoprotein ◽

Bioinformatic Analysis ◽

Specific Pattern ◽

Specific Gene ◽

Protein Coding

Cardiovascular risk factors and biologic sex play a role in vascular dementia which is characterized by progressive reduction in cognitive function and memory. Yet, we lack understanding about the role sex plays in the molecular mechanisms whereby lipid stress contributes to cognitive decline. Five-week-old low-density lipoprotein deficient (LDL-R −/−) male and female mice and C57BL/6J wild types (WT) were fed a control or Western Diet for 8 weeks. Differential expression of protein coding and non-protein coding genes (DEG) were determined in laser captured hippocampal microvessels using genome-wide microarray, followed by bioinformatic analysis of gene networks, pathways, transcription factors and sex/gender-based analysis (SGBA). Cognitive function was assessed by Y-maze. Bioinformatic analysis revealed more DEGs in females (2412) compared to males (1972). Hierarchical clusters revealed distinctly different sex-specific gene expression profiles irrespective of diet and genotype. There were also fewer and different biologic responses in males compared to females, as well as different cellular pathways and gene networks (favoring greater neuroprotection in females), together with sex-specific transcription factors and non-protein coding RNAs. Hyperlipidemic stress also resulted in less severe cognitive dysfunction in females. This sex-specific pattern of differential hippocampal microvascular RNA expression might provide therapeutic targets for dementia in males and females.

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EgJUB1 and EgERF113 transcription factors as potential master regulators of defense response in Elaeis guineensis against the hemibiotrophic Ganoderma boninense

BMC Plant Biology ◽

10.1186/s12870-020-02812-7 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Nurshafika Mohd Sakeh ◽

Siti Nor Akmar Abdullah ◽

Mohammad Nazri Abdul Bahari ◽

Azzreena Mohamad Azzeme ◽

Noor Azmi Shaharuddin ◽

...

Keyword(s):

Transcription Factors ◽

Oil Palm ◽

Defense Response ◽

Fungal Pathogen ◽

Defense Mechanism ◽

Elaeis Guineensis ◽

Expression Profiles ◽

Present Report ◽

Ganoderma Boninense ◽

Cell Wall Modification

Abstract Background Hemibiotrophic pathogen such as the fungal pathogen Ganoderma boninense that is destructive to oil palm, manipulates host defense mechanism by strategically switching from biotrophic to necrotrophic phase. Our previous study revealed two distinguishable expression profiles of oil palm genes that formed the basis in deducing biotrophic phase at early interaction which switched to necrotrophic phase at a later stage of infection. Results The present report is a continuing study from our previous published transcriptomic profiling of oil palm seedlings against G. boninense. We focused on identifying differentially expressed genes (DEGs) encoding transcription factors (TFs) from the same RNA-seq data; resulting in 106 upregulated and 108 downregulated TFs being identified. The DEGs are involved in four established defense-related pathways responsible for cell wall modification, reactive oxygen species (ROS)-mediated signaling, programmed cell death (PCD) and plant innate immunity. We discovered upregulation of JUNGBRUNNEN 1 (EgJUB1) during the fungal biotrophic phase while Ethylene Responsive Factor 113 (EgERF113) demonstrated prominent upregulation when the palm switches to defense against necrotrophic phase. EgJUB1 was shown to have a binding activity to a 19 bp palindromic SNBE1 element, WNNYBTNNNNNNNAMGNHW found in the promoter region of co-expressing EgHSFC-2b. Further in silico analysis of promoter regions revealed co-expression of EgJUB1 with TFs containing SNBE1 element with single nucleotide change at either the 5th or 18th position. Meanwhile, EgERF113 binds to both GCC and DRE/CRT elements promoting plasticity in upregulating the downstream defense-related genes. Both TFs were proven to be nuclear-localized based on subcellular localization experiment using onion epidermal cells. Conclusion Our findings demonstrated unprecedented transcriptional reprogramming of specific TFs potentially to enable regulation of a specific set of genes during different infection phases of this hemibiotrophic fungal pathogen. The results propose the intricacy of oil palm defense response in orchestrating EgJUB1 during biotrophic and EgERF113 during the subsequent transition to the necrotrophic phase. Binding of EgJUB1 to SNBE motif instead of NACBS while EgERF113 to GCC-box and DRE/CRT motifs is unconventional and not normally associated with pathogen infection. Identification of these phase-specific oil palm TFs is important in designing strategies to tackle or attenuate the progress of infection.

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Gene Expression Profiles Associated with Radio-Responsiveness in Locally Advanced Rectal Cancer

Biology ◽

10.3390/biology10060500 ◽

2021 ◽

Vol 10 (6) ◽

pp. 500

Author(s):

Jeeyong Lee ◽

Junhye Kwon ◽

DaYeon Kim ◽

Misun Park ◽

KwangSeok Kim ◽

...

Keyword(s):

Candidate Genes ◽

Expression Profiles ◽

Cell Cycle Control ◽

Methylation Status ◽

Locally Advanced ◽

Gene Expression Profiles ◽

Advanced Rectal Cancer ◽

Bioinformatic Analysis ◽

Locally Advanced Rectal Cancer ◽

Genes Encoding

LARC patients were sorted according to their radio-responsiveness and patient-derived organoids were established from the respective cancer tissues. Expression profiles for each group were obtained using RNA-seq. Biological and bioinformatic analysis approaches were used in deciphering genes and pathways that participate in the radio-resistance of LARC. Thirty candidate genes encoding proteins involved in radio-responsiveness–related pathways, including the immune system, DNA repair and cell-cycle control, were identified. Interestingly, one of the candidate genes, cathepsin E (CTSE), exhibited differential methylation at the promoter region that was inversely correlated with the radio-resistance of patient-derived organoids, suggesting that methylation status could contribute to radio-responsiveness. On the basis of these results, we plan to pursue development of a gene chip for diagnosing the radio-responsiveness of LARC patients, with the hope that our efforts will ultimately improve the prognosis of LARC patients.

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The PKD-Dependent Biogenesis of TGN-to-Plasma Membrane Transport Carriers

Cells ◽

10.3390/cells10071618 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1618

Author(s):

Yuichi Wakana ◽

Felix Campelo

Keyword(s):

Cell Surface ◽

Membrane Trafficking ◽

Surface Protein ◽

Membrane Contact Sites ◽

Tissue Organization ◽

Constitutive Secretion ◽

Membrane Contact ◽

Golgi Network ◽

Organelle Membrane ◽

And Control

Membrane trafficking is essential for processing and transport of proteins and lipids and to establish cell compartmentation and tissue organization. Cells respond to their needs and control the quantity and quality of protein secretion accordingly. In this review, we focus on a particular membrane trafficking route from the trans-Golgi network (TGN) to the cell surface: protein kinase D (PKD)-dependent pathway for constitutive secretion mediated by carriers of the TGN to the cell surface (CARTS). Recent findings highlight the importance of lipid signaling by organelle membrane contact sites (MCSs) in this pathway. Finally, we discuss our current understanding of multiple signaling pathways for membrane trafficking regulation mediated by PKD, G protein-coupled receptors (GPCRs), growth factors, metabolites, and mechanosensors.

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Screening Driving Transcription Factors in the Processing of Gastric Cancer

Gastroenterology Research and Practice ◽

10.1155/2016/8431480 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Guangzhong Xu ◽

Kai Li ◽

Nengwei Zhang ◽

Bin Zhu ◽

Guosheng Feng

Keyword(s):

Gastric Cancer ◽

Transcription Factors ◽

Regulatory Network ◽

Target Genes ◽

Transcriptional Regulatory Network ◽

Expression Profiles ◽

Functional Enrichment ◽

Regulatory Mechanisms ◽

Integrated Analysis ◽

Transcriptional Regulatory

Background. Construction of the transcriptional regulatory network can provide additional clues on the regulatory mechanisms and therapeutic applications in gastric cancer.Methods. Gene expression profiles of gastric cancer were downloaded from GEO database for integrated analysis. All of DEGs were analyzed by GO enrichment and KEGG pathway enrichment. Transcription factors were further identified and then a global transcriptional regulatory network was constructed.Results. By integrated analysis of the six eligible datasets (340 cases and 43 controls), a bunch of 2327 DEGs were identified, including 2100 upregulated and 227 downregulated DEGs. Functional enrichment analysis of DEGs showed that digestion was a significantly enriched GO term for biological process. Moreover, there were two important enriched KEGG pathways: cell cycle and homologous recombination. Furthermore, a total of 70 differentially expressed TFs were identified and the transcriptional regulatory network was constructed, which consisted of 566 TF-target interactions. The top ten TFs regulating most downstream target genes were BRCA1, ARID3A, EHF, SOX10, ZNF263, FOXL1, FEV, GATA3, FOXC1, and FOXD1. Most of them were involved in the carcinogenesis of gastric cancer.Conclusion. The transcriptional regulatory network can help researchers to further clarify the underlying regulatory mechanisms of gastric cancer tumorigenesis.

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AP2/ERF and R2R3-MYB family transcription factors: potential associations between temperature stress and lipid metabolism in Auxenochlorella protothecoides

Biotechnology for Biofuels ◽

10.1186/s13068-021-01881-6 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Guanlan Xing ◽

Jinyu Li ◽

Wenli Li ◽

Sin Man Lam ◽

Hongli Yuan ◽

...

Keyword(s):

Transcription Factors ◽

Lipid Metabolism ◽

Temperature Stress ◽

Expression Profiles ◽

Critical Factor ◽

Biofuel Production ◽

Systematic Analysis ◽

Triacylglycerol Accumulation ◽

Response To Temperature ◽

R2r3 Myb

Abstract Background Both APETALA2/Ethylene Responsive Factor (AP2/ERF) superfamily and R2R3-MYB family were from one of the largest diverse families of transcription factors (TFs) in plants, and played important roles in plant development and responses to various stresses. However, no systematic analysis of these TFs had been conducted in the green algae A. protothecoides heretofore. Temperature was a critical factor affecting growth and lipid metabolism of A. protothecoides. It also remained largely unknown whether these TFs would respond to temperature stress and be involved in controlling lipid metabolism process. Results Hereby, a total of six AP2 TFs, six ERF TFs and six R2R3-MYB TFs were identified and their expression profiles were also analyzed under low-temperature (LT) and high-temperature (HT) stresses. Meanwhile, differential adjustments of lipid pathways were triggered, with enhanced triacylglycerol accumulation. A co-expression network was built between these 18 TFs and 32 lipid-metabolism-related genes, suggesting intrinsic associations between TFs and the regulatory mechanism of lipid metabolism. Conclusions This study represented an important first step towards identifying functions and roles of AP2 superfamily and R2R3-MYB family in lipid adjustments and response to temperature stress. These findings would facilitate the biotechnological development in microalgae-based biofuel production and the better understanding of photosynthetic organisms’ adaptive mechanism to temperature stress.

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Purinergic receptor stimulation increases membrane trafficking in brown adipocytes.

The Journal of General Physiology ◽

10.1085/jgp.108.5.393 ◽

1996 ◽

Vol 108 (5) ◽

pp. 393-404 ◽

Cited By ~ 27

Author(s):

P A Pappone ◽

S C Lee

Keyword(s):

Membrane Trafficking ◽

Energy Homeostasis ◽

Purinergic Receptor ◽

Sympathetic Innervation ◽

Surface Membrane ◽

Cytosolic Calcium ◽

Membrane Capacitance ◽

Adrenergic Stimulation ◽

Membrane Expression ◽

Brown Adipocytes

Stimulation of brown adipocytes by their sympathetic innervation plays a major role in body energy homeostasis by regulating the energy-wasting activity of the tissue. The norepinephrine released by sympathetic activity acts on adrenergic receptors to activate a variety of metabolic and membrane responses. Since sympathetic stimulation may also release vesicular ATP, we tested brown fat cells for ATP responses. We find that micromolar concentrations of extracellular ATP initiates profound changes in the membrane trafficking of brown adipocytes. ATP elicited substantial increases in total cell membrane capacitance, averaging approximately 30% over basal levels and occurring on a time scale of seconds to minutes. The membrane capacitance increase showed an agonist sensitivity of 2-methylthio-ATP > or = ATP > ADP > > adenosine, consistent with mediation by a P2r type purinergic receptor. Membrane capacitance increases were not seen when cytosolic calcium was increased by adrenergic stimulation, and capacitance responses to ATP were similar in the presence and absence of extracellular calcium. These results indicate that increases in cytosolic calcium alone do not mediate the membrane response to ATP. Photometric assessment of surface-accessible membrane using the dye FM1-43 showed that ATP caused an approximate doubling of the amount of membrane actively trafficking with the cell surface. The discrepancy in the magnitudes of the capacitance and fluorescence changes suggests that ATP both activates exocytosis and alters other aspects of membrane handling. These findings suggest that secretion, mobilization of membrane transporters, and/or surface membrane expression of receptors may be regulated in brown adipocytes by P2r purinergic receptor activity.

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Uncontrolled Diabetes Mellitus Has No Major Influence on the Platelet Transcriptome

BioMed Research International ◽

10.1155/2018/8989252 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Thomas G. Nührenberg ◽

Marco Cederqvist ◽

Federico Marini ◽

Christian Stratz ◽

Björn A. Grüning ◽

...

Keyword(s):

Diabetes Mellitus ◽

Next Generation Sequencing ◽

Platelet Reactivity ◽

Platelet Rich Plasma ◽

Expression Profiles ◽

Bioinformatic Analysis ◽

Differentially Expressed ◽

Major Influence ◽

Next Generation ◽

Generation Sequencing

Background. Diabetes mellitus (DM) has been associated with increased platelet reactivity as well as increased levels of platelet RNAs in plasma. Here, we sought to evaluate whether the platelet transcriptome is altered in the presence of uncontrolled DM. Methods. Next-generation sequencing (NGS) was performed on platelet RNA for 5 patients with uncontrolled DM (HbA1c 9.0%) and 5 control patients (HbA1c 5.5%) with otherwise similar clinical characteristics. RNA was isolated from leucocyte-depleted platelet-rich plasma. Libraries of platelet RNAs were created separately for long RNAs after ribosomal depletion and for small RNAs from total RNA, followed by next-generation sequencing. Results. Platelets in both groups demonstrated RNA expression profiles characterized by absence of leukocyte-specific transcripts, high expression of well-known platelet transcripts, and in total 6,343 consistently detectable transcripts. Extensive statistical bioinformatic analysis yielded 12 genes with consistently differential expression at a lenient FDR < 0.1, thereof 8 protein-coding genes and 2 genes with known expression in platelets (MACF1 and ITGB3BP). Three of the four differentially expressed noncoding genes were YRNAs (RNY1, RNY3, and RNY4) which were all downregulated in DM. 23 miRNAs were differentially expressed between the two groups. Of the 13 miRNAs with decreased expression in the diabetic group, 8 belonged to the DLK1–DIO3 gene region on chromosome 14q32.2. Conclusions. In this study, uncontrolled DM had a remote impact on different components of the platelet transcriptome. Increased expression of MACF1, together with supporting predicted mRNA-miRNA interactions as well as reduced expression of RNYs in platelets, may reflect subclinical platelet activation in uncontrolled DM.

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Genomic Architecture of Cells in Tissues (GeACT): Study of Human Mid-gestation Fetus

10.1101/2020.04.12.038000 ◽

2020 ◽

Author(s):

Feng Tian ◽

Fan Zhou ◽

Xiang Li ◽

Wenping Ma ◽

Honggui Wu ◽

...

Keyword(s):

Transcription Factors ◽

Single Cell ◽

Human Cell ◽

Expression Profiles ◽

Single Cells ◽

Cell Types ◽

List Type ◽

Cell Type ◽

Genomic Architecture ◽

Gene Modules

SummaryBy circumventing cellular heterogeneity, single cell omics have now been widely utilized for cell typing in human tissues, culminating with the undertaking of human cell atlas aimed at characterizing all human cell types. However, more important are the probing of gene regulatory networks, underlying chromatin architecture and critical transcription factors for each cell type. Here we report the Genomic Architecture of Cells in Tissues (GeACT), a comprehensive genomic data base that collectively address the above needs with the goal of understanding the functional genome in action. GeACT was made possible by our novel single-cell RNA-seq (MALBAC-DT) and ATAC-seq (METATAC) methods of high detectability and precision. We exemplified GeACT by first studying representative organs in human mid-gestation fetus. In particular, correlated gene modules (CGMs) are observed and found to be cell-type-dependent. We linked gene expression profiles to the underlying chromatin states, and found the key transcription factors for representative CGMs.HighlightsGenomic Architecture of Cells in Tissues (GeACT) data for human mid-gestation fetusDetermining correlated gene modules (CGMs) in different cell types by MALBAC-DTMeasuring chromatin open regions in single cells with high detectability by METATACIntegrating transcriptomics and chromatin accessibility to reveal key TFs for a CGM

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