In vertebrates, mitochondrial DNA (mtDNA) transcription is initiated bidirectionally from closely spaced promoters, HSP and LSP, within the D-loop regulatory region. Early studies demonstrated that mtDNA transcription requires mitochondrial RNA polymerase and Tfam, a DNA binding stimulatory factor that is required for mtDNA maintenance. Recently, mitochondrial transcription specificity factors (TFB1M and TFB2M), which markedly enhance mtDNA transcription in the presence of Tfam and mitochondrial RNA polymerase, have been identified in mammalian cells. Here, we establish that the expression of human TFB1M and TFB2M promoters is governed by nuclear respiratory factors (NRF-1 and NRF-2), key transcription factors implicated in mitochondrial biogenesis. In addition, we show that NRF recognition sites within both TFB promoters are required for maximal trans activation by the PGC-1 family coactivators, PGC-1␣ and PRC. The physiological induction of these coactivators has been associated with the integration of NRFs and other transcription factors in a program of mitochondrial biogenesis. Finally, we demonstrate that the TFB genes are up-regulated along with Tfam and either PGC-1␣ or PRC in cellular systems where mitochondrial biogenesis is induced. Moreover, ectopic expression of PGC-1␣ is sufficient to induce the coordinate expression of all three nucleus-encoded mitochondrial transcription factors along with nuclear and mitochondrial respiratory subunits. These results support the conclusion that the coordinate regulation of nucleus-encoded mitochondrial transcription factors by NRFs and PGC-1 family coactivators is essential to the control of mitochondrial biogenesis.The biogenesis of mitochondria requires the expression of a large number of genes encoded by both nuclear and mitochondrial genetic systems (10). However, because the protein coding capacity of mitochondrial DNA (mtDNA) is limited to 13 respiratory subunits, nuclear genes must provide the vast majority of products required for mitochondrial oxidative functions and biosynthetic capacity. In addition, nuclear genes must play a predominant role in controlling mitochondrial transcription, translation, and DNA replication.Understanding the transcription and replication of mtDNA has been a major focus (6, 29). The majority of evidence points to a mechanism of bidirectional replication where the replication origins for the two strands, termed heavy (H) and light (L) based on their buoyant densities, are displaced by about twothirds of the genome. The D-loop regulatory region contains bidirectional promoters, HSP and LSP, for transcribing H and L strands as well as the H-strand replication origin (O H ). The activities of both HSP and LSP require a 15-nucleotide conserved sequence motif that defines the core promoter. In addition, the two promoters share an upstream enhancer that serves as the recognition site for Tfam (previously called mtTF-1 and mtTFA), a high-mobility-group (HMG) box protein that stimulates transcription through specific binding to the ups...

PGC-1-related coactivator (PRC) was initially characterized as a transcriptional coactivator that shares structural and functional features with PGC-1␣. Both coactivators interact with nuclear respiratory factor 1 (NRF-1) and activate NRF-1 target genes required for respiratory chain expression. Here, we establish that PRC belongs to the class of immediate early genes that are rapidly induced in the transition from quiescence to proliferative growth. As observed for other members of this class, the rapid serum induction of PRC mRNA does not require de novo protein synthesis and inhibition of protein synthesis stabilizes PRC mRNA, leading to its superinduction. Previous work indicated that PRC activation of cytochrome c expression occurs through cis-acting elements that bind both NRF-1 and CREB. Here, we demonstrate that, like NRF-1, CREB binds PRC in vitro and exists in a complex with PRC in cell extracts. Both CREB and NRF-1 bind the same sites on PRC, and the interaction with CREB requires the CREB b-Zip DNA binding domain. Moreover, a CREB/NRF-1 interaction domain on PRC is required for its trans activation of the cytochrome c promoter and a PRC subfragment containing this domain inhibits respiratory growth on galactose when expressed in trans from a lentivirus vector. Finally, PRC associates with the cytochrome c promoter in vivo and its occupancy of the promoter is markedly elevated in response to serum induction of quiescent fibroblasts. The results establish that PRC is an immediate early gene product that can target key transcription factors as an early event in the program of cellular proliferation.Mitochondrial biogenesis relies upon the integrated expression of both the nucleo-cytosolic and the mitochondrial genetic system (7). Although mitochondria have their own DNA (mtDNA), which, in vertebrates, is a covalently closed circular molecule of approximately 16.5 kilobases, the protein coding capacity of this genome is limited to 13 polypeptide subunits of respiratory chain complexes I, III, IV, and V. The only other products of mtDNA expression are the tRNAs and rRNAs of the mitochondrial translation system. This arrangement necessitates that nuclear genes encode the majority of respiratory chain subunits as well as all of the gene products required for the transcription and replication of mtDNA.In recent years, there have been significant inroads into understanding the transcriptional mechanisms that contribute to nucleo-mitochondrial interactions in mammalian systems (15). Key components of the mitochondrial transcriptional machinery have been characterized (8). These include a single mitochondrial RNA polymerase (POLRMT); a stimulatory factor (Tfam), which binds DNA and is required for maintenance of the mitochondrial genome; specificity factors (TFB1M and -2M) that interact with Tfam and the polymerase; and a termination factor (mTERF), which may help regulate the rRNA/mRNA ratio. In addition, several nuclear transcription factors that act on nuclear genes required for mitochondrial function have b...

PRC, a member of the PGC-1 coactivator family, is responsive to serum growth factors and up-regulated in proliferating cells. Here, we investigated its in vivo role by stably silencing PRC expression with two different short hairpin RNAs (shRNA1 and shRNA4) that were lentivirally introduced into U2OS cells. shRNA1 transductants exhibited nearly complete knockdown of PRC protein, whereas shRNA4 transductants expressed PRC protein at ϳ15% of the control level. Complete PRC silencing by shRNA1 resulted in a severe inhibition of respiratory growth; reduced expression of respiratory protein subunits from complexes I, II, III, and IV; markedly lower complex I and IV respiratory enzyme levels; and diminished mitochondrial ATP production. Surprisingly, shRNA1 transductants exhibited a striking proliferation of abnormal mitochondria that were devoid of organized cristae and displayed severe membrane abnormalities. Although shRNA4 transductants had normal respiratory subunit expression and a moderately diminished respiratory growth rate, both transductants showed markedly reduced growth on glucose accompanied by inhibition of G 1 /S cell cycle progression. Microarray analysis revealed striking overlaps in the genes affected by PRC silencing in the two transductants, and the functional identities of these overlapping genes were consistent with the observed mitochondrial and cell growth phenotypes. The consistency between phenotype and PRC expression levels in the two independent transductant lines argues that the defects result from PRC silencing and not from off target effects. These results support a role for PRC in the integration of pathways directing mitochondrial respiratory function and cell growth.Mitochondria are semiautonomous organelles that function as important sites of biological oxidation and ATP production. Central to this function is the electron transport chain and oxidative phosphorylation system, which is composed predominantly of five multisubunit protein complexes embedded in the mitochondrial inner membrane (1, 2). Mitochondria contain their own genetic system directed by a covalently closed circular DNA genome whose entire protein coding capacity is devoted to the production of 13 protein subunits of respiratory complexes I, III, IV, and V. Although essential, these subunits account for only a fraction of the protein composition with the majority of the respiratory subunits of nuclear origin (3, 4). Its bigenomic expression makes the respiratory apparatus unique among mitochondrial oxidative functions and poses an important biological problem in understanding the coordination of nuclear and mitochondrial gene expression.A number of nucleus-encoded regulatory factors have been associated with the transcriptional control of both nuclear and mitochondrial genes that specify the respiratory apparatus in mammalian systems. Initial work identified nuclear respiratory factors, NRF-1 and NRF-2(GABP), as activators of nuclear cytochrome c and cytochrome oxidase genes. These factors have subsequently been ass...

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.

Background: PRC is a transcriptional coactivator involved in respiratory chain expression and cell growth. Results: PRC protein levels are induced in response to various forms of metabolic stress leading to the activation of a program of inflammatory gene expression. Conclusion: PRC can function as a sensor of metabolic stress. Significance: Elucidating the molecular basis of chronic inflammatory responses may contribute to our understanding of cancer and degenerative diseases.