Drosophila Mitochondrial Transcription Factor B2 Regulates Mitochondrial DNA Copy Number and Transcription in Schneider Cells

Matsushima, Yohkazu; Garesse, Rafael; Kaguni, Laurie S.

doi:10.1074/jbc.m401643200

Cited by 77 publications

(77 citation statements)

References 55 publications

(47 reference statements)

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“…These results indicate that mtDNA is in excess of that needed for normal mitochondrial transcription in Schneider cells. Similar results were obtained upon either suppression or overexpression of the mitochondrial transcription factor A (25,30,31). These data contrast with our finding that mitochondrial transcription factor B2 regulates directly both mtDNA copy number and transcription (25), whereas mitochondrial transcription factor B1 affects neither mtDNA nor transcript levels (26).…”

Section: Discussioncontrasting

confidence: 57%

Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Matsushima

Kaguni

2007

Journal of Biological Chemistry

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We report the cloning and molecular analysis of Drosophila mitochondrial DNA helicase (d-mtDNA helicase) homologous to human TWINKLE, which encodes one of the genes responsible for autosomal dominant progressive external ophthalmoplegia. An RNA interference construct was designed that reduces expression of d-mtDNA helicase to an undetectable level in Schneider cells. RNA interference knockdown of d-mtDNA helicase decreases the copy number of mitochondrial DNA (mtDNA) ϳ5-fold. In a corollary manner, overexpression of d-mtDNA helicase increases mtDNA levels 1.4-fold. Overexpression of helicase active site mutants K388A and D483A results in a severe depletion of mtDNA and a dominant negative lethal phenotype. Overexpression of mutants analogous to human autosomal dominant progressive external ophthalmoplegia mutations shows differential effects. Overexpression of I334T and A442P mutants yields a dominant negative effect as for the active site mutants. In contrast, overexpression of A326T, R341Q, and W441C mutants results in increased mtDNA copy number, as observed with wild-type overexpression. Our dominant negative analysis of d-mtDNA helicase in cultured cells provides a tractable model for understanding human autosomal dominant progressive external ophthalmoplegia mutations.One of the important functions of mitochondria is the production of ATP by the oxidative phosphorylation pathway. Animal mitochondrial DNA (mtDNA) 2 encodes 13 polypeptides involved in oxidative phosphorylation, whereas all of the factors essential for mtDNA replication are encoded in the nuclear genome (1). In general, mutations in these factors result in both loss of mtDNA integrity via base substitution mutations, duplications, and deletions and mtDNA depletion (2).Autosomal dominant progressive external ophthalmoplegia (adPEO) is a human mitochondrial disorder associated with the presence of multiple deletions of mtDNA (2-5). The disease is manifest in adulthood, typically at 20 -40 years of age; symptoms include muscle weakness, wasting, exercise intolerance, ataxia, hearing loss, cardiomyopathy, and peripheral neuropathy (2). Most of the adPEO families carry heterozygous mutations in one of three genes: ANT1 (the adenine nucleotide translocator 1), POLG (mitochondrial DNA polymerase), and TWINKLE (mtDNA helicase) (2, 6 -8).TWINKLE protein shares high homology with the bacteriophage T7 gene 4 protein (T7 gp4), which contains both helicase and primase catalytic activities located in its carboxyl-and amino-terminal halves, respectively (7). Like other DNA helicases of the DnaB family, T7 gp4 functions as a hexameric ring (9 -11). The amino acid sequence of the helicase domain of T7 gp4 is well conserved in TWINKLE, whereas that of the primase domain is not. TWINKLE exhibits 5Ј-3Ј DNA helicase activity on double-stranded DNA substrates and functions in reconstituted mtDNA replication forks in vitro (12, 13). It co-localizes with mtDNA in structures designated as mitochondrial nucleoids in vivo (7). Recently, TWINKLE was demonstrated to modu...

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Section: Discussioncontrasting

confidence: 57%

Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Matsushima

Kaguni

2007

Journal of Biological Chemistry

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“…The TFB1M isoform is transiently down-regulated relative to that of the TFB2M isoform in serum-stimulated quiescent fibroblasts, suggesting that the latter is favored in the transition to proliferative growth (19). RNA-mediated interference knockdown of the Drosophila B2 isoform results in reduced mtDNA transcription and copy number (43). This contrasts with RNA-mediated interference knockdown of the B1 isoform, which has no effect on mtDNA transcription or replication but does result in reduced mitochondrial translation (44).…”

Section: Discussioncontrasting

confidence: 44%

PGC-1-related Coactivator Complexes with HCF-1 and NRF-2β in Mediating NRF-2(GABP)-dependent Respiratory Gene Expression

Vercauteren

Gleyzer

Scarpulla

2008

Journal of Biological Chemistry

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The PGC-1 family of regulated coactivators (PGC-1␣, PGC-1␤, and PRC) plays an important role in directing respiratory gene expression in response to environmental signals. Here, we show that PRC and PGC-1␣ differ in their interactions with nuclear hormone receptors but are highly similar in their direct binding to several nuclear transcription factors implicated in the expression of the respiratory chain. Surprisingly, neither coactivator binds NRF-2(GABP), a multisubunit transcriptional activator associated with the expression of many respiratory genes. However, the NRF-2 subunits and PRC are co-immunoprecipitated from cell extracts indicating that the two proteins exist in a complex in vivo. Several lines of evidence indicate that HCF-1 (host cell factor 1), a major chromatin component, mediates the association between PRC and NRF-2. Both PRC and NRF-2␤ bind HCF-1 in vitro, and the molecular determinants required for the interactions of each with HCF-1 are also required for PRC trans-activation through promoter-bound NRF-2. These determinants include a consensus HCF-1 binding site on PRC and the NRF-2 activation domain. In addition, PRC and NRF-2␤ can complex with HCF-1 in vivo, and all three associate with NRF-2-dependent nuclear genes that direct the expression of the mitochondrial transcription factors, TFB1M and TFB2M. Finally, short hairpin RNA-mediated knock down of PRC protein levels leads to reduced expression of TFB2M mRNA and mitochondrial transcripts for cytochrome oxidase II (COXII) and cytochrome b. These changes in gene expression coincide with a marked reduction in cytochrome oxidase activity. The results are consistent with a pathway whereby PRC regulates NRF-2-dependent genes through a multiprotein complex involving HCF-1.

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“…However, unlike T7 RNA polymerase, which does not require any transcription factors, efficient promoter-specific initiation by human POLRMT in vitro requires the high mobility group box transcription factor h-mtTFA/TFAM (referred to as h-mtTFA from this point forward) and one of two rRNA methyltransferase-related transcription factors, h-mtTFB1 and h-mtTFB2 (11)(12)(13)(14). Based on work in Drosophila (15,16), cultured human cells (17,18), and mice (19), it is becoming clear that, whereas both h-mtTFB1 and h-mtTFB2 can bind POLRMT and activate transcription in vitro, h-mtTFB2 is probably the primary transcription factor in vivo, whereas h-mtTFB1 is the primary rRNA methyltransferase critical for mitochondrial ribosome biogenesis and translation. However, both proteins have retained both activities (20,21) and act in concert to promote normal mitochondrial biogenesis, gene expression, and activity (17).…”

mentioning

confidence: 99%

Core human mitochondrial transcription apparatus is a regulated two-component system in vitro

Shutt

Lodeiro

Cotney

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

114

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The core human mitochondrial transcription apparatus is currently regarded as an obligate three-component system comprising the bacteriophage T7-related mitochondrial RNA polymerase, the rRNA methyltransferase-related transcription factor, h-mtTFB2, and the high mobility group box transcription/DNA-packaging factor, h-mtTFA/TFAM. Using a faithful recombinant human mitochondrial transcription system from Escherichia coli, we demonstrate that specific initiation from the mtDNA promoters, LSP and HSP1, only requires mitochondrial RNA polymerase and h-mtTFB2 in vitro. When h-mtTFA is added to these basal components, LSP exhibits a much lower threshold for activation and a larger amplitude response than HSP1. In addition, when LSP and HSP1 are together on the same transcription template, h-mtTFA-independent transcription from HSP1 and h-mtTFA-dependent transcription from both promoters is enhanced and a higher concentration of h-mtTFA is required to stimulate HSP1. Promoter competition experiments revealed that, in addition to LSP competing transcription components away from HSP1, additional cis-acting signals are involved in these aspects of promoter regulation. Based on these results, we speculate that the human mitochondrial transcription system may have evolved to differentially regulate transcription initiation and transcription-primed mtDNA replication in response to the amount of h-mtTFA associated with nucleoids, which could begin to explain the heterogeneity of nucleoid structure and activity in vivo. Furthermore, this study sheds new light on the evolution of mitochondrial transcription components by showing that the human system is a regulated two-component system in vitro, and thus more akin to that of budding yeast than thought previously.h-mtTFA/TFAM | mtDNA | nucleoid | POLRMT | h-mtTFB2/TFB2M

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Drosophila Mitochondrial Transcription Factor B2 Regulates Mitochondrial DNA Copy Number and Transcription in Schneider Cells

Cited by 77 publications

References 55 publications

Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

PGC-1-related Coactivator Complexes with HCF-1 and NRF-2β in Mediating NRF-2(GABP)-dependent Respiratory Gene Expression

Core human mitochondrial transcription apparatus is a regulated two-component system in vitro

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