An N-ethyl-N-Nitrosourea Mutagenesis Screen in Mice Reveals a Mutation in Nuclear Respiratory Factor 1 (Nrf1) Altering the DNA Methylation State and Correct Embryonic Development

We have established a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify novel genes playing a role in epigenetic regulation in mammals. We hypothesize that the ENU mutagenesis screen will lead to the discovery of unknown genes responsible of the maintenance of the epigenetic state as the genes found are modifiers of variegation of the transgene green fluorescent protein (GFP) expression in erythrocytes, which are named MommeD. Here we report the generation of a novel mutant mouse line, MommeD46, that carries a new missense mutation producing an amino acid transversion (L71P) in the dimerization domain of Nuclear Respiratory Factor 1 (Nrf1). The molecular characterization of the mutation reveals a decrease in the Nrf1 mRNA levels and a novel role of Nrf1 in the maintenance of the DNA hypomethylation in vivo. The heritability of the mutation is consistent with paternal imprinting and haploinsufficiency. Homozygous mutants display embryonic lethality at 14.5 days post-coitum and developmental delay. This work adds a new epi-regulatory role to Nrf1 and uncovers unknown phenotypical defects of the Nrf1 hypomorph. The generated mouse line represents a valuable resource for studying NRF1-related diseases.

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Identification of the Sex of Pre-implantation Mouse Embryos Using a Marked Y Chromosome and CRISPR/Cas9

Scientific Reports ◽

10.1038/s41598-019-50731-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Xiuling Zhao ◽

Wei Wei ◽

Hong Pan ◽

Junyu Nie ◽

Dongrong Chen ◽

...

Keyword(s):

Y Chromosome ◽

Fluorescent Protein ◽

Farm Animals ◽

Mouse Line ◽

Protein Markers ◽

Mammalian Embryo ◽

Non Invasive ◽

A Genome ◽

Wild Type Female ◽

Green Fluorescent

Abstract Although numerous attempts have been made to alter the sex ratio of the progeny of mammals, the limitations of current technologies have prevented their widespread use in farm animals. The presence or absence of a Y chromosome determines whether a mammalian embryo develops as a male or female, and non-invasive genetic reporters such as fluorescence protein markers have been intensively applied in a variety of fields of research. To develop a non-invasive and instantaneous method for advance determination of the sex of embryos, we developed a Y chromosome-linked eGFP mouse line that stably expresses green fluorescent protein under the control of the CAG promoter. The development of the CRISPR/Cas9 system has made it easy to deliver an exogenous gene to a specific locus of a genome, and linking a tracer to the Y chromosome has simplified the process of predicting the sex of embryos collected by mating a Y-Chr-eGFP transgenic male with a wild-type female. XY embryos appeared green, under a fluorescence microscope, and XX embryos did not. Y chromosome-linked genes were amplified by nested PCR to further confirm the accuracy of this method, and the simultaneous transplantation of green and non-green embryos into foster mothers indicated that 100% accuracy was achieved by this method. Thus, the Y-Chr-eGFP mouse line provides an expeditious and accurate approach for sexing pre-implantation embryos and can be efficiently used for the pre-selection of sex.

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A new live-cell reporter strategy to simultaneously monitor mitochondrial biogenesis and morphology

Scientific Reports ◽

10.1038/srep17217 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 15

Author(s):

Linn Iren Hodneland Nilsson ◽

Ina Katrine Nitschke Pettersen ◽

Julie Nikolaisen ◽

David Micklem ◽

Hege Avsnes Dale ◽

...

Keyword(s):

Mitochondrial Biogenesis ◽

Fluorescent Protein ◽

Live Cell ◽

Mitochondrial Morphology ◽

Reporter Construct ◽

Nuclear Respiratory Factor ◽

Nuclear Respiratory Factor 1 ◽

Vital Functions ◽

Dependent Protein ◽

Green Fluorescent

Abstract Changes in mitochondrial amount and shape are intimately linked to maintenance of cell homeostasis via adaptation of vital functions. Here, we developed a new live-cell reporter strategy to simultaneously monitor mitochondrial biogenesis and morphology. This was achieved by making a genetic reporter construct where a master regulator of mitochondrial biogenesis, nuclear respiratory factor 1 (NRF-1), controls expression of mitochondria targeted green fluorescent protein (mitoGFP). HeLa cells with the reporter construct demonstrated inducible expression of mitoGFP upon activation of AMP-dependent protein kinase (AMPK) with AICAR. We established stable reporter cells where the mitoGFP reporter activity corresponded with mitochondrial biogenesis both in magnitude and kinetics, as confirmed by biochemical markers and confocal microscopy. Quantitative 3D image analysis confirmed accordant increase in mitochondrial biomass, in addition to filament/network promoting and protecting effects on mitochondrial morphology, after treatment with AICAR. The level of mitoGFP reversed upon removal of AICAR, in parallel with decrease in mtDNA. In summary, we here present a new GFP-based genetic reporter strategy to study mitochondrial regulation and dynamics in living cells. This combinatorial reporter concept can readily be transferred to other cell models and contexts to address specific physiological mechanisms.

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Origins of interspecies variation in mammalian muscle metabolic enzymes

Physiological Genomics ◽

10.1152/physiolgenomics.00025.2011 ◽

2011 ◽

Vol 43 (14) ◽

pp. 873-883 ◽

Cited By ~ 3

Author(s):

K. M. Kocha ◽

C. E. Genge ◽

C. D. Moyes

Keyword(s):

Tibialis Anterior ◽

Regulatory Element ◽

Element Distribution ◽

Peroxisome Proliferator ◽

Mrna Levels ◽

Peroxisome Proliferator Activated Receptor ◽

Nuclear Respiratory Factor ◽

Interspecies Differences ◽

Interspecies Variation ◽

Nuclear Respiratory Factor 1

Do the transcriptional mechanisms that control an individual's mitochondrial content, PGC1α (peroxisome proliferator-activated receptor γ coactivator-1α) and NRF1 (nuclear respiratory factor-1), also cause differences between species? We explored the determinants of cytochrome c oxidase (COX) activities in muscles from 12 rodents differing 1,000-fold in mass. Hindlimb muscles differed in scaling patterns from isometric (soleus, gastrocnemius) to allometric (tibialis anterior, scaling coefficient = −0.16). Consideration of myonuclear domain reduced the differences within species, but interspecies differences remained. For tibialis anterior, there was no significant scaling relationship in mRNA/g for COX4-1, PGC1α, or NRF1, yet COX4-1 mRNA/g was a good predictor of COX activity (r2 = 0.55), PGC1α and NRF1 mRNA correlated with each other (r2 = 0.42), and both could predict COX4-1 mRNA (r2 = 0.48 and 0.52) and COX activity (r2 = 0.55 and 0.49). This paradox was resolved by multivariate analysis, which explained 90% of interspecies variation, about equally partitioned between mass effects and PGC1α (or NRF1) mRNA levels, independent of mass. To explore the determinants of PGC1α mRNA, we analyzed 52 mammalian PGC1α proximal promoters and found no size dependence in regulatory element distribution. Likewise, the activity of PGC1α promoter reporter genes from 30 mammals showed no significant relationship with body mass. Collectively, these studies suggest that not all muscles scale equivalently, but for those that show allometric scaling, transcriptional regulation of the master regulators, PGC1α and NRF1, does not account for scaling patterns, though it does contribute to interspecies differences in COX activities independent of mass.

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A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals

Function ◽

10.1093/function/zqab012 ◽

2021 ◽

Vol 2 (3) ◽

Cited By ~ 1

Author(s):

Nelly Redolfi ◽

Elisa Greotti ◽

Giulia Zanetti ◽

Tino Hochepied ◽

Cristina Fasolato ◽

...

Keyword(s):

Transgenic Mouse ◽

Fluorescent Protein ◽

Ex Vivo ◽

Brain Slices ◽

Resonance Energy ◽

Mouse Line ◽

Isolated Cells ◽

Genomic Locus ◽

Transgenic Mouse Line

AbstractMitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

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PGC-1-Related Coactivator, a Novel, Serum-Inducible Coactivator of Nuclear Respiratory Factor 1-Dependent Transcription in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.11.3738-3749.2001 ◽

2001 ◽

Vol 21 (11) ◽

pp. 3738-3749 ◽

Cited By ~ 238

Author(s):

Ulf Andersson ◽

Richard C. Scarpulla

Keyword(s):

Transcriptional Activation ◽

Mammalian Cells ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Peroxisome Proliferator ◽

Nuclear Respiratory Factor ◽

Nuclear Respiratory Factor 1 ◽

Cell Extracts

ABSTRACT The thermogenic peroxisome proliferator-activated receptor γ (PPAR-γ) coactivator 1 (PGC-1) has previously been shown to activate mitochondrial biogenesis in part through a direct interaction with nuclear respiratory factor 1 (NRF-1). In order to identify related coactivators that act through NRF-1, we searched the databases for sequences with similarities to PGC-1. Here, we describe the first characterization of a 177-kDa transcriptional coactivator, designated PGC-1-related coactivator (PRC). PRC is ubiquitously expressed in murine and human tissues and cell lines; but unlike PGC-1, PRC was not dramatically up-regulated during thermogenesis in brown fat. However, its expression was down-regulated in quiescent BALB/3T3 cells and was rapidly induced by reintroduction of serum, conditions where PGC-1 was not detected. PRC activated NRF-1-dependent promoters in a manner similar to that observed for PGC-1. Moreover, NRF-1 was immunoprecipitated from cell extracts by antibodies directed against PRC, and both proteins were colocalized to the nucleoplasm by confocal laser scanning microscopy. PRC interacts in vitro with the NRF-1 DNA binding domain through two distinct recognition motifs that are separated by an unstructured proline-rich region. PRC also contains a potent transcriptional activation domain in its amino terminus adjacent to an LXXLL motif. The spatial arrangement of these functional domains coincides with those found in PGC-1, supporting the conclusion that PRC and PGC-1 are structurally and functionally related. We conclude that PRC is a functional relative of PGC-1 that operates through NRF-1 and possibly other activators in response to proliferative signals.

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Transgenic Mouse Line with Green-fluorescent Protein-labeled Centrin 2 allows Visualization of the Centrosome in Living Cells

Transgenic Research ◽

10.1023/b:trag.0000026071.41735.8e ◽

2004 ◽

Vol 13 (2) ◽

pp. 155-164 ◽

Cited By ~ 60

Author(s):

Holden Higginbotham ◽

Stephanie Bielas ◽

Teruyuki Tanaka ◽

Joseph G. Gleeson

Keyword(s):

Green Fluorescent Protein ◽

Transgenic Mouse ◽

Fluorescent Protein ◽

Living Cells ◽

Mouse Line ◽

Transgenic Mouse Line ◽

Green Fluorescent

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The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

Journal of Virology ◽

10.1128/jvi.00384-16 ◽

2016 ◽

Vol 90 (13) ◽

pp. 5860-5875 ◽

Cited By ~ 21

Author(s):

Eva Maria Borst ◽

Rudolf Bauerfeind ◽

Anne Binz ◽

Thomas Min Stephan ◽

Sebastian Neuber ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Viral Genome ◽

Fluorescent Protein ◽

Unit Length ◽

Start Codon ◽

Nuclear Targeting ◽

Reading Frame ◽

A Genome ◽

Genome Encapsidation ◽

Insight Into

ABSTRACTSeveral essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging.IMPORTANCEThe essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.

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