scholarly journals bHLH11 negatively regulates Fe homeostasis by its EAR motifs recruiting corepressors in Arabidopsis

2020 ◽  
Author(s):  
Yang Li ◽  
Rihua Lei ◽  
Mengna Pu ◽  
Yuerong Cai ◽  
Chengkai Lu ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Signal Sequence ◽  
Negative Regulator ◽  
Fine Tuning ◽  
Nuclear Accumulation ◽  
Loss Of Function ◽  
Enhanced Sensitivity ◽  
Crucial Component ◽  
Regulation Function ◽  
Fe Homeostasis

ABSTRACTIron (Fe) homeostasis is essential for plant growth and development. Although tremendous progress has been made in understanding the maintenance of Fe homeostasis in plants, the underlying molecular mechanisms remain elusive. Recently, bHLH11 was reported to function as a negative regulator. However, the molecular mechanism by which bHLH11 regulates Fe homeostasis is unclear. Here, we generated two bhlh11 loss-of-function mutants which displayed the enhanced sensitivity to excessive Fe. bHLH11 is located in the cytoplasm and nucleus due to lack of a nuclear location signal sequence, and its interaction partners, bHLH IVc transcription factors (TFs) (bHLH34, bHLH104, bHLH105 and bHLH115) facilitate its nuclear accumulation. bHLH11 exerts its negative regulation function by recruiting the corepressors TOPLESS/TOPLESS-RELATED. Moreover, bHLH11 antagonizes the transactivity of bHLH IVc TFs towards bHLH Ib genes (bHLH38, bHLH39, bHLH100 and bHLH101). This work indicates that bHLH11 is a crucial component of Fe homeostasis signaling network, playing a pivotal role in the fine-tuning of Fe homeostasis.

scholarly journals Deficiency of Lipin2 Results in Enhanced NF-κB Signaling and Osteoclast Formation in RAW-D Murine Macrophages

2021 ◽  
Vol 22 (6) ◽  
pp. 2893
Author(s):  
Asami Watahiki ◽  
Seira Hoshikawa ◽  
Mitsuki Chiba ◽  
Hiroshi Egusa ◽  
Satoshi Fukumoto ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Inflammatory Responses ◽  
Osteoclast Formation ◽  
Fine Tuning ◽  
Nuclear Accumulation ◽  
Innate Immune Responses ◽  
Osteoclastic Resorption ◽  
Bone Disorder ◽  
Proinflammatory Responses ◽  
Potential Therapeutic Intervention

Lipin2 is a phosphatidate phosphatase that plays critical roles in fat homeostasis. Alterations in Lpin2, which encodes lipin2, cause the autoinflammatory bone disorder Majeed syndrome. Lipin2 limits lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. However, little is known about the precise molecular mechanisms underlying its anti-inflammatory function. In this study, we attempted to elucidate the molecular link between the loss of lipin2 function and autoinflammatory bone disorder. Using a Lpin2 knockout murine macrophage cell line, we showed that lipin2 deficiency enhances innate immune responses to LPS stimulation through excessive activation of the NF-κB signaling pathway, partly because of TAK1 signaling upregulation. Lipin2 depletion also enhanced RANKL-mediated osteoclastogenesis and osteoclastic resorption activity accompanied by NFATc1 dephosphorylation and increased nuclear accumulation. These results suggest that lipin2 suppresses the development of autoinflammatory bone disorder by fine-tuning proinflammatory responses and osteoclastogenesis in macrophages. Therefore, this study provides insights into the molecular pathogenesis of monogenic autoinflammatory bone disorders and presents a potential therapeutic intervention.

scholarly journals An auxin signaling network translates low-sugar-state input into compensated cell enlargement in the fugu5 cotyledon

PLoS Genetics ◽  
2021 ◽  
Vol 17 (8) ◽  
pp. e1009674
Author(s):  
Hiromitsu Tabeta ◽  
Shunsuke Watanabe ◽  
Keita Fukuda ◽  
Shizuka Gunji ◽  
Mariko Asaoka ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Molecular Mechanisms ◽  
Genetic Interaction ◽  
Mitotic Cell ◽  
Fine Tuning ◽  
Auxin Signaling ◽  
Cell Enlargement ◽  
Loss Of Function ◽  
Nutrient Reserves

In plants, the effective mobilization of seed nutrient reserves is crucial during germination and for seedling establishment. The Arabidopsis H+-PPase-loss-of-function fugu5 mutants exhibit a reduced number of cells in the cotyledons. This leads to enhanced post-mitotic cell expansion, also known as compensated cell enlargement (CCE). While decreased cell numbers have been ascribed to reduced gluconeogenesis from triacylglycerol, the molecular mechanisms underlying CCE remain ill-known. Given the role of indole 3-butyric acid (IBA) in cotyledon development, and because CCE in fugu5 is specifically and completely cancelled by ech2, which shows defective IBA-to-indoleacetic acid (IAA) conversion, IBA has emerged as a potential regulator of CCE. Here, to further illuminate the regulatory role of IBA in CCE, we used a series of high-order mutants that harbored a specific defect in IBA-to-IAA conversion, IBA efflux, IAA signaling, or vacuolar type H+-ATPase (V-ATPase) activity and analyzed the genetic interaction with fugu5–1. We found that while CCE in fugu5 was promoted by IBA, defects in IBA-to-IAA conversion, IAA response, or the V-ATPase activity alone cancelled CCE. Consistently, endogenous IAA in fugu5 reached a level 2.2-fold higher than the WT in 1-week-old seedlings. Finally, the above findings were validated in icl–2, mls–2, pck1–2 and ibr10 mutants, in which CCE was triggered by low sugar contents. This provides a scenario in which following seed germination, the low-sugar-state triggers IAA synthesis, leading to CCE through the activation of the V-ATPase. These findings illustrate how fine-tuning cell and organ size regulation depend on interplays between metabolism and IAA levels in plants.

scholarly journals MADS-box factor AGL16 negatively regulates drought resistance via stomatal density and stomatal movement

2019 ◽  
Author(s):  
Ping-Xia Zhao ◽  
Zi-Qing Miao ◽  
Jing Zhang ◽  
Qian-Qian Liu ◽  
Cheng-Bin Xiang
Keyword(s):  
Drought Stress ◽  
Drought Resistance ◽  
Molecular Mechanisms ◽  
Stomatal Density ◽  
Stomatal Closure ◽  
Negative Regulator ◽  
Stomatal Development ◽  
Mads Box ◽  
Loss Of Function ◽  
Research Attention

ABSTRACTDrought is one of the most severe environmental factors limiting plant growth and productivity. Plants respond to drought by closing stomata to reduce water loss. The molecular mechanisms underlying plant drought resistance are very complex and yet to be fully understood. While much research attention has been focused on the positive regulation of stomatal closure, less is known about its negative regulation, equally important in this reversible process. Here we show that the MADS-box transcriptional factor AGL16 acts as a negative regulator in drought resistance by regulating both stomatal density and movement. Loss-of-function mutantagl16was more resistant to drought stress with higher relative water content, which was attributed to a reduced leaf stomatal density and more sensitive stomatal closure due to a higher leaf ABA level compared with wild type, whileAGL16overexpression lines displayed the opposite phenotypes.AGL16is preferentially expressed in guard cells and down regulated in response to drought stress. The expression ofCYP707A3andAAO3in ABA metabolism andSDD1in stomatal development was altered by AGL16 as shown inagl16and overexpression lines. Chromatin immunoprecipitation, transient transactivation, and yeast-one-hybrid assays demonstrated that AGL16 bound the CArG motif in the promoter of theCYP707A3,AAO3, andSDD1to regulate their transcription, and therefore alter leaf stomatal density and ABA level. Taken together, AGL16 acts as a negative regulator of drought resistance by modulating leaf stomatal density and ABA accumulation.

CBIO-04. G-QUADRUPLEX STABILIZATION ENHANCES REPLICATION STRESS AND DNA DAMAGE IN ATRX-DEFICIENT HIGH-GRADE GLIOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi27-vi28
Author(s):  
Sharvari Dharmaiah ◽  
Vasudev Tadimeti ◽  
Prit Benny Malgulwar ◽  
Christian Alvarez ◽  
Ahsan Farooqi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Molecular Mechanisms ◽  
Standard Of Care ◽  
High Grade Glioma ◽  
Replication Stress ◽  
High Grade ◽  
Loss Of Function ◽  
Enhanced Sensitivity ◽  
Alternative Lengthening ◽  
Dna Secondary Structures

Abstract Loss of function mutations in α-thalassaemia/mental retardation X-linked (ATRX) are a critical molecular hallmark for invariably fatal high-grade glioma (HGG). Mutational inactivation of histone chaperone ATRX leads to accumulations of abnormal DNA secondary structures known as G-quadruplexes (G4s), thereby inducing replication stress and DNA damage. As G4s arise at GC-rich regions (i.e., pericentromeric and telomeric regions), ATRX-deficiency alters genome-wide accessibility of chromatin, leads to transcriptional dysregulation, and induces alternative lengthening of telomeres (ALT). Our goal is to target ATRX deficiency through G4 stabilizers, which represent a class of novel small molecule compounds that selectively bind to and stabilize G4 structures. However, the genomic consequences and efficacy of this therapy for ATRX-deficient HGG are poorly understood. We therefore sought to evaluate the molecular mechanisms that drive selective lethality in patient-derived ATRX-deficient glioma stem cells (GSCs), following G4 stabilization. We found that ATRX-deficient GSCs demonstrate dose-dependent enhanced sensitivity to G4 stabilization, compared to ATRX-intact controls. Cell viability assays confirmed the specificity of our G4 stabilizer in comparison to other commonly used G4 stabilizers. Interestingly, G4 stabilization activated p53-independent apoptosis in ATRX-deficient GSCs. Furthermore, ATRX-deficient GSCs exhibit upregulated expression of both ATR and ATM pathways upon G4 stabilization, indicating enhanced replication stress and DNA damage via double-stranded breaks, respectively. Our preliminary findings suggest that ATR and ATM activation leads to the inhibition of transcription factor NF-κB, which in turn drives apoptosis. Lastly, our data indicate that G4 stabilization perturbs the ALT phenotype in ATRX-deficient GSCs, likely contributing to telomeric dysfunction. Taken together, these findings suggest that G4 stabilizers could synergize with ionizing radiation, the standard of care, as they are both DNA-damaging therapies. Our work defines mechanisms of action and efficacy of a novel therapeutic strategy for ATRX-deficient HGG, with strong implications for other ATRX-deficient cancers.

scholarly journals OsIRO3 Plays an Essential Role in Iron Deficiency Responses and Regulates Iron Homeostasis in Rice

Plants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1095
Author(s):  
Wujian Wang ◽  
Jun Ye ◽  
Yanran Ma ◽  
Ting Wang ◽  
Huixia Shou ◽  
...  
Keyword(s):  
Iron Homeostasis ◽  
Molecular Mechanisms ◽  
Sufficient Conditions ◽  
Negative Regulator ◽  
Fe Deficiency ◽  
Plant Growth And Development ◽  
E Box ◽  
Young Leaves ◽  
Related Transcription Factor ◽  
Fe Homeostasis

Iron (Fe) homeostasis is essential for plant growth and development, and it is strictly regulated by a group of transcriptional factors. Iron-related transcription factor 3 (OsIRO3) was previously identified as a negative regulator for Fe deficiency response in rice. However, the molecular mechanisms by which OsIRO3 regulate Fe homeostasis is unclear. Here, we report that OsIRO3 is essential for responding to Fe deficiency and maintaining Fe homeostasis in rice. OsIRO3 is expressed in the roots, leaves, and base nodes, with a higher level in leaf blades at the vegetative growth stage. Knockout of OsIRO3 resulted in a hypersensitivity to Fe deficiency, with severe necrosis on young leaves and defective root development. The iro3 mutants accumulated higher levels of Fe in the shoot under Fe-deficient conditions, associated with upregulating the expression of OsNAS3, which lead to increased accumulation of nicotianamine (NA) in the roots. Further analysis indicated that OsIRO3 can directly bind to the E-box in the promoter of OsNAS3. Moreover, the expression of typical Fe-related genes was significantly up-regulated in iro3 mutants under Fe-sufficient conditions. Thus, we conclude that OsIRO3 plays a key role in responding to Fe deficiency and regulates NA levels by directly, negatively regulating the OsNAS3 expression.

Fli1 Promotes Vascular Morphogenesis by Regulating Endothelial Potential of Multipotent Myogenic Progenitors

2021 ◽  
Author(s):  
Anwarul Ferdous ◽  
Sarvjeet Singh ◽  
Yuxuan Luo ◽  
Md J Abedin ◽  
Nan Jiang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Vascular System ◽  
Ectopic Expression ◽  
Molecular Techniques ◽  
Fine Tuning ◽  
Mdx Mice ◽  
Loss Of Function ◽  
Differentiation Potential ◽  
Reciprocal Regulation ◽  
Vascular Morphogenesis

Rationale: Fetal growth and survival depend critically on proper development and integrity of the vascular system. Fli1 (Friend leukemia integration 1), a member of the Ets family of transcription factors, plays critical roles in vascular morphogenesis and homeostasis at mid-gestation, the developmental stage at which expression of its upstream regulator, Etv2, ceases. However, molecular mechanisms of Fli1 action in vascular morphogenesis remain incompletely understood. Objective: To dissect molecular mechanisms of vascular morphogenesis governed by Fli1. Methods and Results: Utilizing Fli1 promoter-driven lineage-specific LacZ expression, Fli1 loss-of-function strategies, and a series of molecular techniques, we demonstrate that Fli1 expression in multipotent myogenic progenitor cells (MPCs) occurs independent of Etv2, and loss of Fli1 expression results in a significant increase in LacZ+ cells in mesoderm within somites and limb buds, leading to reciprocal regulation of the expression of several key endothelial and myogenic genes and vascular abnormalities. Conversely, embryos with conditional Fli1 gain-of-function in MPCs manifested aberrant vasculogenesis with lack of myogenesis. Mechanistically, elevated Fli1 activity in myoblasts and in adult MPCs (also called satellite cells) of X-linked muscular dystrophic mdx mice markedly induced endothelial, but attenuated myogenic, gene expression and differentiation. Importantly, ectopic expression of Myf5 or MyoD, two key myogenic regulators, in Fli1-expressing myoblasts restored their differentiation potential, indicating that levels of Fli1 and myogenic regulators in MPCs inversely regulate their endothelial versus myogenic potential. Conclusions: Fli1 governs vascular morphogenesis by regulating endothelial potential by inversely regulating endothelial versus myogenic programs in MPCs. Our data uncover an important and previously unrecognized mechanism of vascular morphogenesis governed by Fli1 and highlight the physiological significance of the fine tuning of Fli1 activity in multipotent progenitors for proper vascular and muscle morphogenesis during development and disease.

scholarly journals TET2 deficiency leads to stem cell factor–dependent clonal expansion of dysfunctional erythroid progenitors

Blood ◽  
2018 ◽  
Vol 132 (22) ◽  
pp. 2406-2417 ◽  
Author(s):  
Xiaoli Qu ◽  
Shijie Zhang ◽  
Shihui Wang ◽  
Yaomei Wang ◽  
Wei Li ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Molecular Mechanisms ◽  
Erythroid Differentiation ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation

Abstract Myelodysplastic syndromes (MDSs) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Anemia is the defining cytopenia of MDS patients, yet the molecular mechanisms for dyserythropoiesis in MDSs remain to be fully defined. Recent studies have revealed that heterozygous loss-of-function mutation of DNA dioxygenase TET2 is 1 of the most common mutations in MDSs and that TET2 deficiency disturbs erythroid differentiation. However, mechanistic insights into the role of TET2 on disordered erythropoiesis are not fully defined. Here, we show that TET2 deficiency leads initially to stem cell factor (SCF)–dependent hyperproliferation and impaired differentiation of human colony-forming unit–erythroid (CFU-E) cells, which were reversed by a c-Kit inhibitor. We further show that this was due to increased phosphorylation of c-Kit accompanied by decreased expression of phosphatase SHP-1, a negative regulator of c-Kit. At later stages, TET2 deficiency led to an accumulation of a progenitor population, which expressed surface markers characteristic of normal CFU-E cells but were functionally different. In contrast to normal CFU-E cells that require only erythropoietin (EPO) for proliferation, these abnormal progenitors required SCF and EPO and exhibited impaired differentiation. We termed this population of progenitors “marker CFU-E” cells. We further show that AXL expression was increased in marker CFU-E cells and that the increased AXL expression led to increased activation of AKT and ERK. Moreover, the altered proliferation and differentiation of marker CFU-E cells were partially rescued by an AXL inhibitor. Our findings document an important role for TET2 in erythropoiesis and have uncovered previously unknown mechanisms by which deficiency of TET2 contributes to ineffective erythropoiesis.

scholarly journals MicroRNA-322 (miR-322) and Its Target Protein Tob2 Modulate Osterix (Osx) mRNA Stability

2013 ◽  
Vol 288 (20) ◽  
pp. 14264-14275 ◽  
Author(s):  
Beatriz Gámez ◽  
Edgardo Rodríguez-Carballo ◽  
Ramon Bartrons ◽  
José Luis Rosa ◽  
Francesc Ventura
Keyword(s):  
Osteoblast Differentiation ◽  
Primary Cultures ◽  
Transcriptional Regulators ◽  
Negative Regulator ◽  
Fine Tuning ◽  
Loss Of Function ◽  
Murine Bone Marrow ◽  
Regulatory Circuit ◽  
Reporter Assays ◽  
Mirna Expression Profiling

Osteogenesis depends on a coordinated network of signals and transcription factors such as Runx2 and Osterix. Recent evidence indicates that microRNAs (miRNAs) act as important post-transcriptional regulators in a large number of processes, including osteoblast differentiation. In this study, we performed miRNA expression profiling and identified miR-322, a BMP-2-down-regulated miRNA, as a regulator of osteoblast differentiation. We report miR-322 gain- and loss-of-function experiments in C2C12 and MC3T3-E1 cells and primary cultures of murine bone marrow-derived mesenchymal stem cells. We demonstrate that overexpression of miR-322 enhances BMP-2 response, increasing the expression of Osx and other osteogenic genes. Furthermore, we identify Tob2 as a target of miR-322, and we characterize the specific Tob2 3′-UTR sequence bound by miR-322 by reporter assays. We demonstrate that Tob2 is a negative regulator of osteogenesis that binds and mediates degradation of Osx mRNA. Our results demonstrate a new molecular mechanism controlling osteogenesis through the specific miR-322/Tob2 regulation of specific target mRNAs. This regulatory circuit provides a clear example of a complex miRNA-transcription factor network for fine-tuning the osteoblast differentiation program.

scholarly journals Circular RNA mmu_circ_0005019 inhibits fibrosis of cardiac fibroblasts and reverses electrical remodeling of cardiomyocytes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Na Wu ◽  
Chengying Li ◽  
Bin Xu ◽  
Ying Xiang ◽  
Xiaoyue Jia ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cardiac Fibroblasts ◽  
Circular Rna ◽  
Protective Role ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Candidate Target ◽  
Paired Control ◽  
Reporter Assays ◽  
And Migration

Abstract Background Circular RNA (circRNA) have been reported to play important roles in cardiovascular diseases including myocardial infarction and heart failure. However, the role of circRNA in atrial fibrillation (AF) has rarely been investigated. We recently found a circRNA hsa_circ_0099734 was significantly differentially expressed in the AF patients atrial tissues compared to paired control. We aim to investigate the functional role and molecular mechanisms of mmu_circ_0005019 which is the homologous circRNA in mice of hsa_circ_0099734 in AF. Methods In order to investigate the effect of mmu_circ_0005019 on the proliferation, migration, differentiation into myofibroblasts and expression of collagen of cardiac fibroblasts, and the effect of mmu_circ_0005019 on the apoptosis and expression of Ito, INA and SK3 of cardiomyocytes, gain- and loss-of-function of cell models were established in mice cardiac fibroblasts and HL-1 atrial myocytes. Dual-luciferase reporter assays and RIP were performed to verify the binding effects between mmu_circ_0005019 and its target microRNA (miRNA). Results In cardiac fibroblasts, mmu_circ_0005019 showed inhibitory effects on cell proliferation and migration. In cardiomyocytes, overexpression of mmu_circ_0005019 promoted Kcnd1, Scn5a and Kcnn3 expression. Knockdown of mmu_circ_0005019 inhibited the expression of Kcnd1, Kcnd3, Scn5a and Kcnn3. Mechanistically, mmu_circ_0005019 exerted biological functions by acting as a miR-499-5p sponge to regulate the expression of its target gene Kcnn3. Conclusions Our findings highlight mmu_circ_0005019 played a protective role in AF development and might serve as an attractive candidate target for AF treatment.

scholarly journals Production, characterization, and cross-reactivity of a polyclonal antibody against Arabidopsis TARGET OF RAPAMYCIN

2019 ◽  
Vol 62 (1) ◽  
Author(s):  
Gyeong-Im Shin ◽  
Sun Young Moon ◽  
Song Yi Jeong ◽  
Myung Geun Ji ◽  
Joon-Yung Cha ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cross Reactivity ◽  
Target Of Rapamycin ◽  
Loss Of Function ◽  
Anticancer Activities ◽  
Purification Method ◽  
E Coli ◽  
Related Family ◽  
Large Gene ◽  
Truncated Variants

AbstractTARGET OF RAPAMYCIN (TOR), a member of the phosphatidylinositol 3-kinase-related family of protein kinases, is encoded by a single, large gene and is evolutionarily conserved in all eukaryotes. TOR plays a role as a master regulator that integrates nutrient, energy, and stress signaling to orchestrate development. TOR was first identified in yeast mutant screens, as its mutants conferred resistance to rapamycin, an antibiotic with immunosuppressive and anticancer activities. In Arabidopsis thaliana, the loss-of-function tor mutant displays embryo lethality, but the precise mechanisms of TOR function are still unknown. Moreover, a lack of reliable molecular and biochemical assay tools limits our ability to explore TOR functions in plants. Here, we produced a polyclonal α-TOR antibody using two truncated variants of TOR (1–200 and 1113–1304 amino acids) as antigens because recombinant full-length TOR is challenging to express in Escherichia coli. Recombinant His-TOR1−200 and His-TOR1113−1304 proteins were individually expressed in E. coli, and a mixture of proteins (at a 1:1 ratio) was used for immunizing rabbits. Antiserum was purified by an antigen-specific purification method, and the purified polyclonal α-TOR antibody successfully detected endogenous TOR proteins in wild-type Arabidopsis and TOR orthologous in major crop plants, including tomato, maize, and alfalfa. Moreover, our α-TOR antibody is useful for coimmunoprecipitation assays. In summary, we generated a polyclonal α-TOR antibody that detects endogenous TOR in various plant species. Our antibody could be used in future studies to determine the precise molecular mechanisms of TOR, which has largely unknown multifunctional roles in plants.

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