Differential expression of mRNA in human thyroid cells depleted of mitochondrial DNA by ethidium bromide treatment

1999 ◽  
Vol 97 (2) ◽  
pp. 207-213 ◽  
Author(s):  
A. W. THOMAS ◽  
A. MAJID ◽  
E. J. SHERRATT ◽  
J. W. GAGG ◽  
J. C. ALCOLADO
Keyword(s):  
Gene Expression ◽  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Nuclear Gene ◽  
Simian Virus 40 ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Mtdna Mutations ◽  
Nuclear Gene Expression

A wide variety of human diseases have been associated with defects in mitochondrial DNA (mtDNA). The exact mechanism by which specific mtDNA mutations cause disease is unknown and, although the disparate phenotypes might be explained on the basis of impaired mitochondrial gene function alone, the role of altered nuclear gene expression must also be considered. In recent years, the experimental technique of depleting cells of mtDNA by culturing them with ethidium bromide has become a popular method of studying mitochondrial disorders. However, apart from depleting mtDNA, ethidium bromide may have many other intracellular and nuclear effects. The aim of the present study was to investigate the effects of ethidium bromide treatment on nuclear gene expression. A simian-virus-40-transformed human thyroid cell line was depleted of mtDNA by culture in ethidium bromide, and differential display reverse transcriptase–PCR (DDRT-PCR) was then employed to compare mRNA expression between wild-type, mtDNA-replete (ρ+) and ethidium bromide-treated, mtDNA-depleted (ρ0) cells. Expression of the majority of nuclear-encoded genes, including those for subunits involved in oxidative phosphorylation, remained unaffected by the treatment. Seven clones were found to be underexpressed; three of the clones showed significant similarity with sequences of the human genes encoding RNase L inhibitor, human tissue factor and ARCN1 (archain vesicle transport protein 1), a highly conserved species which is related to vesicle structure and trafficking proteins. We conclude that the effects of ethidium bromide treatment on nuclear gene expression are not simply limited to changes in pathways directly associated with known mitochondrial function. Further studies will be required to elucidate which of these changes are due to mtDNA depletion, ATP deficiency or other disparate effects of ethidium bromide exposure. Given that most genes appear unaffected, the results suggest that depleting cells of mtDNA by ethidium bromide treatment is a valuable approach for the study of mitochondrial mutations by cybrid techniques.

scholarly journals HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression

2016 ◽  
Vol 2 (1) ◽  
Author(s):  
Manti Guha ◽  
Satish Srinivasan ◽  
Kip Guja ◽  
Edison Mejia ◽  
Miguel Garcia-Diaz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mitochondrial Dna ◽  
Transcriptional Activation ◽  
Nuclear Gene ◽  
C2c12 Cells ◽  
Retrograde Signaling ◽  
Epigenetic Mechanism ◽  
Mitochondrial Stress ◽  
Nuclear Gene Expression ◽  
Mitochondrial Dna Copy Number

Abstract Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies.

scholarly journals Balanced mitochondrial and cytosolic translatomes underlie the biogenesis of human respiratory complexes

2021 ◽  
Author(s):  
Iliana C Soto ◽  
Mary Couvillion ◽  
Erik McShane ◽  
Katja G Hansen ◽  
J. Conor Moran ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Mitochondrial Dna ◽  
Nuclear Gene ◽  
Cell Types ◽  
Ribosome Profiling ◽  
Nuclear Gene Expression ◽  
Leigh’S Syndrome ◽  
Cellular Compartments ◽  
Translation Systems

Oxidative phosphorylation (OXPHOS) complexes consist of nuclear and mitochondrial DNA- encoded subunits. Their biogenesis requires cross-compartment gene regulation to mitigate the accumulation of disproportionate subunits. To determine how human cells coordinate mitochondrial and nuclear gene expression processes, we established an optimized ribosome profiling approach tailored for the unique features of the human mitoribosome. Analysis of ribosome footprints in five cell types revealed that average mitochondrial synthesis rates corresponded precisely to cytosolic rates across OXPHOS complexes. Balanced mitochondrial and cytosolic synthesis did not rely on rapid feedback between the two translation systems. Rather, LRPPRC, a gene associated with Leigh's syndrome, is required for the reciprocal translatomes and maintains cellular proteostasis. Based on our findings, we propose that human mitonuclear balance is enabled by matching OXPHOS subunit synthesis rates across cellular compartments, which may represent a vulnerability for cellular proteostasis.

scholarly journals Characterization of a thyroid-specific enhancer located 5.5 kilobase pairs upstream of the human thyroid peroxidase gene.

1990 ◽  
Vol 10 (12) ◽  
pp. 6216-6224 ◽  
Author(s):  
F Kikkawa ◽  
F J Gonzalez ◽  
S Kimura
Keyword(s):  
Hepg2 Cells ◽  
Simian Virus 40 ◽  
Human Thyroid ◽  
Thyroid Peroxidase ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Enhancer Element ◽  
Peroxidase Gene ◽  
Nuclear Extracts ◽  
Human Thyroid Peroxidase

A 6.3-kbp segment of DNA, upstream of the human thyroid peroxidase gene, and various deletions thereof were linked to a promoterless bacterial chloramphenicol acetyltransferase reporter gene. These constructs were analyzed by transfection and expression in rat FRTL-5 thyroid cells and in human hepatoma HepG2 cells to localize sequences that are important for thyroid cell-specific expression of the thyroid peroxidase gene. A thyroid-specific enhancer element, capable of activating enhancerless simian virus 40 promoter expression in FRTL-5 cells, was localized to a 230-bp region approximately 5.5 kbp upstream of the human thyroid peroxidase gene transcription start site. DNase I footprinting, using nuclear extracts prepared from FRTL-5 cells, revealed three regions within the 230-bp fragment; none of these regions were protected by nuclear extracts from HepG2 cells. Gel mobility shift assays, using double-stranded oligonucleotides corresponding to the three protected regions, further confirmed the existence of factors in FRTL-5 cells, but not HepG2 cells, able to specifically bind to the enhancer sequences. These results suggest the presence of three cis-acting DNA elements in the human thyroid peroxidase gene enhancer that interact with thyroid-specific trans-acting factors.

Characterization of a thyroid-specific enhancer located 5.5 kilobase pairs upstream of the human thyroid peroxidase gene

1990 ◽  
Vol 10 (12) ◽  
pp. 6216-6224
Author(s):  
F Kikkawa ◽  
F J Gonzalez ◽  
S Kimura
Keyword(s):  
Hepg2 Cells ◽  
Simian Virus 40 ◽  
Human Thyroid ◽  
Thyroid Peroxidase ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Enhancer Element ◽  
Peroxidase Gene ◽  
Nuclear Extracts ◽  
Human Thyroid Peroxidase

A 6.3-kbp segment of DNA, upstream of the human thyroid peroxidase gene, and various deletions thereof were linked to a promoterless bacterial chloramphenicol acetyltransferase reporter gene. These constructs were analyzed by transfection and expression in rat FRTL-5 thyroid cells and in human hepatoma HepG2 cells to localize sequences that are important for thyroid cell-specific expression of the thyroid peroxidase gene. A thyroid-specific enhancer element, capable of activating enhancerless simian virus 40 promoter expression in FRTL-5 cells, was localized to a 230-bp region approximately 5.5 kbp upstream of the human thyroid peroxidase gene transcription start site. DNase I footprinting, using nuclear extracts prepared from FRTL-5 cells, revealed three regions within the 230-bp fragment; none of these regions were protected by nuclear extracts from HepG2 cells. Gel mobility shift assays, using double-stranded oligonucleotides corresponding to the three protected regions, further confirmed the existence of factors in FRTL-5 cells, but not HepG2 cells, able to specifically bind to the enhancer sequences. These results suggest the presence of three cis-acting DNA elements in the human thyroid peroxidase gene enhancer that interact with thyroid-specific trans-acting factors.

Differential expression of mRNA in human thyroid cells depleted of mitochondrial DNA by ethidium bromide treatment

1999 ◽  
Vol 97 (2) ◽  
pp. 207 ◽  
Author(s):  
A.W. THOMAS ◽  
A. MAJID ◽  
E.J. SHERRATT ◽  
J.W. GAGG ◽  
J.C. ALCOLADO
Keyword(s):  
Mitochondrial Dna ◽  
Differential Expression ◽  
Ethidium Bromide ◽  
Human Thyroid ◽  
Thyroid Cells

scholarly journals Cytokine Release and Focal Adhesion Proteins in Normal Thyroid Cells Cultured on the Random Positioning Machine

2017 ◽  
Vol 43 (1) ◽  
pp. 257-270 ◽  
Author(s):  
Elisabeth Warnke ◽  
Jessica Pietsch ◽  
Sascha Kopp ◽  
Johann Bauer ◽  
Jayashree Sahana ◽  
...  
Keyword(s):  
Gene Expression ◽  
Focal Adhesion ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Multicellular Spheroids ◽  
Thyroid Cells ◽  
Random Positioning Machine ◽  
Adhesion Proteins ◽  
Focal Adhesion Proteins

Background/Aims: Spaceflight impacts on the function of the thyroid gland in vivo. In vitro normal and malignant thyrocytes assemble in part to multicellular spheroids (MCS) after exposure to the random positioning machine (RPM), while a number of cells remain adherent (AD). We aim to elucidate possible differences between AD and MCS cells compared to 1g-controls of normal human thyroid cells. Methods: Cells of the human follicular epithelial thyroid cell line Nthy-ori 3-1 were incubated for up to 72 h on the RPM. Afterwards, they were investigated by phase-contrast microscopy, quantitative real-time PCR and by determination of cytokines released in their supernatants. Results: A significant up-regulation of IL6, IL8 and CCL2 gene expression was found after a 4h RPM-exposure, when the whole population was still growing adherently. MCS and AD cells were detected after 24 h on the RPM. At this time, a significantly reduced gene expression in MCS compared to 1g-controls was visible for IL6, IL8, FN1, ITGB1, LAMA1, CCL2, and TLN1. After a 72 h RPM-exposure, IL-6, IL-8, and TIMP-1 secretion rates were increased significantly. Conclusion: Normal thyrocytes form MCS within 24 h. Cytokines seem to be involved in the initiation of MCS formation via focal adhesion proteins.

scholarly journals Modulation of ACE-2 mRNA by inflammatory cytokines in human thyroid cells: a pilot study

Endocrine ◽  
2021 ◽  
Author(s):  
Francesca Coperchini ◽  
Gianluca Ricci ◽  
Laura Croce ◽  
Marco Denegri ◽  
Rubina Ruggiero ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Combination Treatment ◽  
Primary Cultures ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Mrna Levels ◽  
Thyroid Cells ◽  
Tnf Α ◽  
Ifn Γ ◽  
Pro Inflammatory Cytokines

Abstract Introduction Angiotensin-converting-enzyme-2 (ACE-2) was demonstrated to be the receptor for cellular entry of SARS-CoV-2. ACE-2 mRNA was identified in several human tissues and recently also in thyroid cells in vitro. Purpose Aim of the present study was to investigate the effect of pro-inflammatory cytokines on the ACE-2 mRNA levels in human thyroid cells in primary cultures. Methods Primary thyroid cell cultures were treated with IFN-γ and TNF-α alone or in combination for 24 h. ACE-2 mRNA levels were measured by RT-PCR. As a control, the levels of IFN-γ inducible chemokine (CXCL10) were measured in the respective cell culture supernatants. Results The mean levels of ACE-2 mRNA increased after treatment with IFN-γ and TNF-α in all the thyroid cell preparations, while the combination treatment did not consistently synergically increase ACE-2-mRNA. At difference, CXCL10 was consistently increased by IFN-γ and synergically further increased by the combination treatment with IFN-γ + TNF-α, with respect to IFN-γ alone. Conclusions The results of the present study show that IFN-γ and, to a lesser extent TNF-α consistently increase ACE-2 mRNA levels in NHT primary cultures. More interestingly, the combined stimulation (proven to be effective according to the synergic effect registered for CXCL10) produces different responses in terms of ACE-2 mRNA modulation. These results would suggest that elevated levels of pro-inflammatory cytokines could facilitate the entering of the virus in cells by further increasing ACE-2 expression and/or account for the different degree of severity of SARS-COV-2 infection. This hypothesis deserves to be confirmed by further specific studies.

scholarly journals Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1518
Author(s):  
Maria Qatato ◽  
Vaishnavi Venugopalan ◽  
Alaa Al-Hashimi ◽  
Maren Rehders ◽  
Aaron D. Valentine ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Human Thyroid ◽  
Apical Plasma Membrane ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Trace Amine ◽  
Rat Thyroid

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

Chloroplast Redox Control of Nuclear Gene Expression—A New Class of Plastid Signals in Interorganellar Communication

2003 ◽  
Vol 5 (1) ◽  
pp. 95-101 ◽  
Author(s):  
Thomas Pfannschmidt ◽  
Katia Schütze ◽  
Vidal Fey ◽  
Irena Sherameti ◽  
Ralf Oelmüller
Keyword(s):  
Gene Expression ◽  
Nuclear Gene ◽  
Redox Control ◽  
New Class ◽  
Nuclear Gene Expression

Cytoplasmic modification of nuclear gene expression

1983 ◽  
Vol 57 (1) ◽  
Author(s):  
JerryW. Shay
Keyword(s):  
Gene Expression ◽  
Nuclear Gene ◽  
Nuclear Gene Expression
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