Transcriptional activators and coactivators in the nuclear control of mitochondrial function in mammalian cells

Gene 286 (2002) 81–89 www.elsevier.com/locate/gene

Transcriptional activators and coactivators in the nuclear control of mitochondrial function in mammalian cells Richard C. Scarpulla* Department of Cell and Molecular Biology, Northwestern Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA Received 3 July 2001; accepted 7 November 2001 Received by M.N. Gadaleta

Abstract The biogenesis and function of mitochondria rely upon the regulated expression of nuclear genes. Recent evidence points to both transcriptional activators and coactivators as important mediators of mitochondrial maintenance and proliferation. Several sequence-specific activators including NRF-1, NRF-2, Sp1, YY1, CREB and MEF-2/E-box factors, among others, have been implicated in respiratory chain expression. Notably, recognition sites for NRF-1, NRF-2 and Sp1 are common to most nuclear genes encoding respiratory subunits, mitochondrial transcription and replication factors, as well as certain heme biosynthetic enzymes and components of the protein import machinery. Moreover, genetic evidence supports a role for NRF-1 in the maintenance of mtDNA during embryonic development. Despite these advances, the means by which multiple transcription factors are integrated into a program of mitochondrial biogenesis remains an open question. New insight into this problem came with the discovery of the transcriptional coactivator, PGC-1. This cofactor is cold inducible in brown fat and interacts with multiple transcription factors to orchestrate a program of adaptive thermogenesis. As part of this program, PGC1 can up-regulate nuclear genes that are required for mitochondrial biogenesis in part through a direct interaction with NRF-1. Ectopic expression of PGC-1 induces the expression of respiratory subunit mRNAs and leads to mitochondrial proliferation in both cultured cells and transgenic mice. More recently, PRC was characterized as a novel coactivator that shares certain structural similarities with PGC-1 including an activation domain, an RS domain and an RNA recognition motif. However, unlike PGC-1, PRC is not induced significantly during thermogenesis but rather is cell-cycle regulated in cultured cells under conditions where PGC-1 is not expressed. PRC has a transcriptional specificity that is very similar to PGC-1, especially in its interaction with NRF-1 and in the activation of NRF-1 target genes. These regulated coactivators may provide a means for integrating sequence-specific activators in the biogenesis and function of mitochondria under diverse physiological conditions. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Transcription factors; Coactivators; NRF-1; PRC; PGC-1; Mitochondria

1. Introduction Although mitochondria participate in a large number of essential cellular functions, they are best known as the major sites of ATP production through electron transfer and oxidative phosphorylation (Hatefi, 1985). Mitochondria have their own genetic system but in vertebrates the protein coding capacity of mtDNA is limited to only 13 subunits of the respiratory apparatus. Thus, the organelle is semiauAbbreviations: 5-ALAS, 5-aminolevulinate synthase; COX, cytochrome c oxidase; CREB, cAMP response element binding protein; MRP, mitochondrial RNA processing; mtDNA, mitochondrial DNA; NRF, nuclear respiratory factor; PGC-1, PPARg coactivator-1; PPAR, peroxisome proliferator activated receptor; PRC, PGC-1-related coactivator; Tfam, mitochondrial transcription factor A; UCP, uncoupling protein; YY1, yingyang factor 1 * Tel.: 11-312-503-2946; fax: 11-312-503-0798. E-mail address: [email protected] (R.C. Scarpulla).

tonomous, relying upon nuclear genes to provide the majority of the constituents necessary for its metabolic systems and molecular architecture (Attardi and Schatz, 1988). One major class of nuclear genes contributes catalytic and auxiliary proteins to the mitochondrial enzyme systems. In the case of the oxidative phosphorylation system, the majority of the 100 or so respiratory subunits are nucleus encoded. A second class of nuclear genes encodes protein import and assembly factors that are necessary for the proper localization of membrane bound and soluble proteins (Neupert, 1997). A third class contributes key enzymes and factors that are necessary for the replication and expression of the mitochondrial genome (reviewed in Shadel and Clayton, 1993, 1997). A subclass of this group includes the mtDNA and RNA polymerases, a transcription termination factor, the MRP endonuclease and the mitochondrial transcription factor, Tfam. MRP endonuclease cleaves nascent

0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00809-5

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transcripts to generate primers for heavy strand replication while Tfam stimulates transcription from the bidirectional promoters within the D-loop regulatory region. As might be expected, the abundance of mitochondria and the expression of the respiratory apparatus are subject to regulation. Tissue- and cell-specific requirements for oxidative energy production are reflected in the number of organelles and respiratory chains (Stotz, 1939). Muscle mitochondria proliferate in response to exercise training (Holloszy, 1967) and electrical stimulation (Williams et al., 1987). Thyroid hormones are known to increase metabolic rate and mitochondrial enzyme levels (Tata et al., 1963). Both pre- and postnatal changes in mitochondrial biogenesis have been documented. Mitochondria and mtDNA are amplified many fold during oogenesis (Michaels et al., 1982) and embryonic replication is not reinitiated until after the blastocyst stage of development (Piko and Taylor, 1987; Piko and Matsumoto, 1976). At birth the production of mitochondria and respiratory enzymes is induced as the neonate adapts to extrauterine conditions by processes that involve both transcriptional and post-transcriptional mechanisms (Izquierdo et al., 1995; Luis et al., 1993). In certain mitochondrial diseases, defective mitochondria proliferate in diseased muscle fibers, the so-called ragged red fiber (Moraes et al., 1992). This proliferation is thought to be a nuclear compensatory response to genetic lesions in mtDNA leading to defective oxidative phosphorylation. Finally, mitochondrial biogenesis also occurs in the brown fat of rodents during adaptation to cold temperatures in a process termed adaptive thermogenesis (reviewed in Lowell and Spiegelman, 2000). As these examples illustrate, mitochondria are dynamic organelles that respond to environmental and developmental signals in meeting cellular energy demands.

2. Nuclear transcription factors in respiratory chain expression 2.1. Nuclear respiratory factors The respiratory apparatus is of particular interest in understanding nucleo-mitochondrial interactions because it is the only mitochondrial enzyme system that is comprised of protein subunits encoded by both nuclear and mitochondrial genes (Attardi and Schatz, 1988). One approach to identifying potential transcriptional mechanisms governing the expression of the respiratory chain is to determine whether nuclear genes expressing respiratory subunits rely on common cis-acting elements and trans-acting factors. Such an analysis of cytochrome c and cytochrome oxidase promoters led to the discovery of the nuclear respiratory factors, NRF-1 (Evans and Scarpulla, 1989, 1990) and NRF-2 (Virbasius et al., 1993a; Virbasius and Scarpulla, 1991). NRF-1 was initially characterized as an activator of cytochrome c expression (Evans and Scarpulla, 1989) and

subsequently found to act on many genes encoding subunits from all five respiratory complexes (reviewed in Scarpulla, 1997) (Table 1). Similarly, NRF-2 was identified as an activator of COXIV transcription (Virbasius and Scarpulla, Table 1 NRF-1 and NRF-2 recognition sites in nuclear genes required for respiratory chain expression Target gene and category

NRF-1 a

Oxidative phosphorylation Rat cytochrome c Human cytochrome c

1 1

Complex I Human NADH dehydrogenase subunit 8 (TYKY)

1

Complex II Human succinate dehydrogenase subunit B Human succinate dehydrogenase subunit C Human succinate dehydrogenase subunit D

1 1 1

Complex III Human ubiquinone binding protein Human core protein I

1 1

Complex IV Rat cytochrome oxidase subunit IV Mouse cytochrome oxidase subunit IV Mouse cytochrome oxidase subunit Vb Rat cytochrome oxidase subunit Vb Human/primate cytochrome oxidase subunit Vb Rat cytochrome oxidase subunit VIc Human cytochrome oxidase subunit VIaL Bovine cytochrome oxidase subunit VIIaL Bovine cytochrome oxidase subunit VIIc

NRF-2 a

1 1 1 1 1 1

Complex V Bovine ATP synthase g subunit Human ATP synthase c subunit

1 1

mtDNA transcription and replication Human Tfam Mouse MRP RNA Human MRP RNA

1 1 1

HEME biosynthesis Rat 5-aminolevulinate synthase Mouse uroporphyrinogen III synthase

1 1

Protein import Human Tom 20 Mouse chaperonin 10

1 1

1 1

1 1 1 1 1 1 1 1

1

1

1

a Original references for most of the indicated genes containing NRF-1 and/or NRF-2 sites are cited in Scarpulla (1997). New additions to the list include: human NADH dehydrogenase subunit 8 (de Sury et al., 1998), human succinate dehydrogenase subunits B (Au and Scheffler, 1998), C (Elbehti-Green et al., 1998), and D (Hirawake et al., 1999), human cytochrome oxidase subunit VIaL (Hu¨ ttemann et al., 2000; Wong-Riley et al., 2000), bovine cytochrome oxidase subunit VIIc (Seelan and Grossman, 1997), human ATP synthase c subunit (Dyer and Walker, 1993), mouse uroporphyrinogen III synthase (Aizencang et al., 2000), human Tom 20 (Hernandez et al., 1999), and mouse chaperonin 10 (Fletcher et al., 2001).

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1991) and is now known to act on a subset of nuclear respiratory genes, many of which also contain NRF-1 sites (Table 1). In addition, one or both of these factors have been associated with the expression of mitochondrial transcription and replication factors (Virbasius and Scarpulla, 1994; Evans and Scarpulla, 1990), heme biosynthetic enzymes (Aizencang et al., 2000; Braidotti et al., 1993) and certain protein import factors (Hernandez et al., 1999) (Table 1). Most notable is the presence of functional NRF-1 sites in MRP RNA (Evans and Scarpulla, 1990), Tfam (Virbasius and Scarpulla, 1994) and 5 0 -aminolevulinate synthase genes (Braidotti et al., 1993). MRP RNA and Tfam are required for the coupled transcription and replication of mtDNA while 5 0 -ALAS is the rate-limiting heme biosynthetic enzyme of the mitochondrial matrix. Thus, NRF-1 control over these functions has the potential to link the expression and function of respiratory proteins encoded by both genomes. NRF-1 and NRF-2 have been purified to homogeneity (Virbasius et al., 1993a; Chau et al., 1992) and cDNAs have been isolated for NRF-1 (Virbasius et al., 1993b) and multiple NRF-2 subunits (Gugneja et al., 1995). NRF2 is the human homologue of mouse GABP, a protein that was originally characterized as an activator of Herpes virus gene expression (Thompson et al., 1991; LaMarco et al., 1991). The crystal structure of the GABPa/b heterodimer bound to DNA has been resolved, thus providing important insights into the DNA binding specificity of ETS domain family members (Batchelor et al., 1998). The basic properties of these factors have been discussed in recent reviews (Scarpulla, 1996, 1997, 1999; Graves, 1998). A key question is whether nuclear respiratory factors function in a physiological context to regulate respiratory gene expression. Recently, changes in respiratory chain constituents and their activities were monitored upon serum-induced entry of quiescent fibroblasts to the cell cycle. The induction of cytochrome c was a relatively early event in the response to serum stimulation and this induction coincided with an elevation in the rate of respiration using glutamate plus malate or ascorbate plus TMPD as substrates (Herzig et al., 2000). No changes in citrate synthase, COX activity or COXIV mRNA and protein levels were observed within 12 h of the restoration of serum. The results indicate that cytochrome c levels control the respiratory rate in preparation for cell division before the synthesis of new respiratory chains. Cytochrome c promoter activity was also induced in this system and the maximal induction required both NRF-1 and CREB recognition sites (Herzig et al., 2000). This was accompanied by a rapid and transient phosphorylation of CREB and a more delayed but stable phosphorylation of NRF-1. The activation of CREB by phosphorylation at ser133 is known to occur as an early response to serum growth factors (Shaywitz and Greenberg, 1999). It is of interest that cytochrome c is the only known promoter

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among respiratory genes to have functional CREB sites. Thus, the existence of these sites most likely accounts for the rapid differential response of the cytochrome c gene to serum. It is notable in this context that cytochrome c expression is also cAMP inducible (Gopalakrishnan and Scarpulla, 1994) and that a rapid increase in respiratory rate follows the cAMP-dependent phosphorylation of the 18 kDa subunit of complex I (Scacco et al., 2000). NRF-1 is phosphorylated on multiple serine residues within a concise amino-terminal domain and the phosphorylation increases the in vitro DNA binding activity of the protein (Gugneja and Scarpulla, 1997). Phosphorylation also enhances the ability of NRF-1 to trans-activate NRF-1-dependent promoters in a heterologous insect cell system (Herzig et al., 2000). These results suggest that the phosphorylation of NRF-1 may serve to regulate its activity in response to physiological signals. Thus, the rapid and stable induction of cytochrome c upon entry to the cell cycle seems to depend upon the sequential activation of CREB and NRF-1. The phosphorylated form of NRF-1 may participate in the synthesis of additional respiratory chain components at later stages in the cycle. Genetic evidence linking NRF-1 to the maintenance of mitochondria in vivo came from the construction of a targeted disruption of the NRF-1 locus in mice. A portion of the NRF-1 gene that encodes the nuclear localization signal and the DNA binding and dimerization domains was replaced by a b-galactosidase-neomycin cassette (Huo and Scarpulla, 2001). Heterozygous animals displayed a mild phenotype consisting of smaller size, higher mortality and a defect in succinate oxidation in kidney (Huo and Scarpulla, unpublished data). In contrast, homozygous null mice died between embryonic days 3.5 and 6.5 and their blastocysts were unable to grow in vitro despite having a normal morphology. The NRF-1 gene was expressed during oogenesis and in 2.5- and 3.5-day embryos and the embryonic expression did not result from maternal carryover. These results demonstrate that NRF-1 provides a vital function during early embryonic development. Consistent with the proposed role for NRF-1, homozygous null blastocysts were defective in maintaining a mitochondrial membrane potential and had reduced mtDNA levels relative to nuclear DNA (Huo and Scarpulla, 2001). No difference in apoptosis was detected in mutant blastocysts compared to wild-type making it unlikely that the reduction in mtDNA resulted from increased apoptosis in the NRF-1 null embryos. In addition, the amplification of mtDNA during oogenesis was unaffected by the heterozygosity of the mothers of NRF-1 (2/2) offspring. The homozygous nulls arose from mature oocytes that had a normal complement of mtDNA. Therefore, the depletion of mtDNA occurred between fertilization and the blastocyst stage and most likely resulted from the loss of a NRF-1dependent pathway of mtDNA maintenance. Interestingly, embryos from mice with a targeted disruption of the Tfam gene also exhibit severely depleted levels of mtDNA but survive to embryonic days 8.5–10.5 (Larsson

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et al., 1998). Thus, it seems unlikely that the lethality of NRF-1 null embryos between days 3.5 and 6.5 results solely from the reduced levels of mtDNA. It is likely that the disruption of other NRF-1-dependent functions accounts for the early lethality of these embryos. Moreover, loss of NRF-1 is not expected to completely eliminate Tfam expression because other transcription factors are known to act on the Tfam promoter (Virbasius and Scarpulla, 1994). Other genes whose expression is more dependent on NRF-1 would be more likely candidates for explaining both the lethality and mtDNA-deficient phenotypes. For example, the 5-ALAS promoter is almost completely dependent on tandem NRF-1 sites in vitro (Braidotti et al., 1993). Ectopic expression of UCP-1 in HeLa cells led to the NRF1-dependent induction of 5-ALAS mRNA suggesting that NRF-1 can regulate 5-ALAS expression in vivo (Li et al., 1999). Alternatively, the observed phenotypes in the NRF1 (2/2) embryos may result from the cumulative effects of a modest reduction in expression of many genes that depend upon NRF-1 for full activity. 2.2. Other transcription factors Although NRF sites are present in the majority of known genes encoding respiratory subunits there are several genes in which these sites are absent (reviewed in Scarpulla, 1999). In some cases, recognition sites for other well-characterized regulatory factors have been identified. The transcription factor Sp1 has been implicated in the activation and/or repression of cytochrome c1 (Li et al., 1996a) and adenine nucleotide translocase 2 genes (Li et al., 1996b), both of which lack NRF sites (Zaid et al., 1999). Sp1 sites are also common to nearly all GC-rich promoters including those that are NRF-dependent (Seelan et al., 1996; Virbasius and Scarpulla, 1991, 1994; Evans and Scarpulla, 1989). The muscle-specific COX subunits, COXVIaH and COXVIII, are also devoid of NRF sites but are dependent on MEF-2 and/or E-box consensus elements for their expression (Lenka et al., 1996; Wan and Moreadith, 1995). It is reasonable that these subunits would be governed by the same or similar factors required for the expression of other musclespecific genes. In contrast, the promoter of the ubiquitously expressed liver isoform, COXVIaL, depends upon NRF-1 and NRF-2 as well as Sp1 for full activity (Seelan et al., 1996). This is in keeping with the observation that in gene pairs encoding ubiquitous and tissue-specific isoforms of a given protein, the NRF-1 site, when present, is associated with the ubiquitously expressed gene (Virbasius et al., 1993b). Finally, there is also evidence for the participation of the initiator element transcription factor YY1 in the expression of certain COX genes. Functional YY1 binding sites have been detected in the promoters of genes encoding COXVb (Basu et al., 1997) and COXVIIc (Seelan and Grossman, 1997). Multiple YY1 sites in the COXVb promoter bind YY1 and possibly other factors and at least one of these sites help confer a negative regulatory effect on

COXVb promoter activity (Basu et al., 1997). In the COXVIIc promoter, two YY1 sites in conjunction with a NRF-2 site act as positive regulators of promoter activity (Seelan and Grossman, 1997). The significance of these findings to COX expression awaits further analysis. 3. Transcriptional coactivators in the integrated expression of respiratory genes It is important to understand the mechanisms by which diverse regulatory factors that are involved in the expression of the respiratory chain as well as other mitochondrial constituents can be integrated into a program of mitochondrial biogenesis. Such a program would be useful in the implementation of physiological and developmental processes that depend upon changes in energy metabolism. One such process is adaptive thermogenesis. In the brown adipose tissue of rodents, cold exposure triggers a series of events leading to the induction and activation of UCP-1 and the biogenesis of mitochondria, both of which are essential characteristics of the thermogenic response (reviewed in Lowell and Spiegelman, 2000). The result is heat production through the dissipation of the electrochemical proton gradient in uncoupled mitochondria. A cold inducible, brown fatspecific enhancer in the UCP-1 gene is the putative target of multiple regulatory factors that are ubiquitously expressed (reviewed in Silva and Rabelo, 1997). These include thyroid hormone and retinoic acid receptors, an undefined cAMPresponsive factor and the peroxisome proliferation activator receptor (PPAR) g. The latter is thought to have a regulatory role in brown fat development as well as in UCP-1 expression. Interestingly, none of these factors have been linked directly to the expression of the genes of the mitochondrial respiratory apparatus. Thus, these observations prompt two fundamental questions. First, how can ubiquitously expressed transcriptional regulators, that are involved in diverse cellular processes, account for the brown fat-specific expression of UCP-1? Second, how is mitochondrial biogenesis integrated with the expression of UCP-1 in the thermogenic program? 3.1. PGC-1 and adaptive thermogenesis A potential inroad to these questions came with the discovery of the transcriptional coactivator, PGC-1 (Puigserver et al., 1998). The PGC-1 cDNA was cloned from a library derived from a differentiated brown fat cell line by yeast two-hybrid screening using PPARg as bait. Although the protein was not closely related to others in the databases, it did have an RNA recognition motif and an RS domain (depicted in Fig. 1) that are characteristic of RNA splicing factors (Tacke and Manley, 1999; Shamoo et al., 1995). PGC-1 interacted with PPARg and other members of the nuclear hormone receptor superfamily including thyroid hormone receptor b, retinoic acid receptor a and estrogen receptor a suggesting that it may play a role in transcrip-

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Fig. 1. Comparison of structural similarities between PRC and PGC-1. The alignment of compositional and sequence similarities between PRC and PGC-1 is depicted.

tional activation. PGC-1 mRNA was induced in the brown fat of mice upon exposure to cold and treatment with badrenergic agonists which mimic the cold-induced sympathetic enervation of brown fat (Puigserver et al., 1998). In support of a gene regulatory function, PGC-1 enhanced the PPARg and thyroid hormone receptor b-dependent transactivation of the UCP-1 promoter (summarized in Fig. 2). Most notably, ectopic expression of PGC-1 in pre-adipocytes and myoblasts induced both nuclear and mitochondrial gene expression and led to an increase in mtDNA (Wu et al., 1999; Puigserver et al., 1998). In differentiated myotubules, PGC-1 expression was accompanied by increases in both total respiratory capacity and mitochondrial uncoupling (Wu et al., 1999). These results were consistent with the notion that PGC-1 is a potent transcriptional coactivator of adaptive thermogenesis. If PGC-1 could integrate mitochondrial uncoupling and biogenesis at the level of gene expression one might expect that it act as a coactivator of NRF-1 and/or NRF-2-dependent transcription. In co-transfection experiments, PGC-1 was able to induce the NRF-1-dependent expression of a NRF-1 reporter that consisted of tandem NRF-1 recognition sites linked to a luciferase cassette (Wu et al., 1999). The NRF-1 site in the human Tfam promoter was also required for its activation by PGC-1. Activation of the 5-ALAS promoter by PGC-1 also occurred through its NRF-1 sites

(Andersson and Scarpulla, unpublished data). The effects of PGC-1 were much less pronounced with NRF-2-dependent transcription but PGC-1 could induce both NRF-1 and NRF2 mRNAs in myoblasts. The transcriptional effects of PGC1 are most likely mediated through a direct interaction with NRF-1. NRF-1 was co-immunoprecipitated with PGC-1 and PGC-1 interacted directly with NRF-1 in an in vitro binding assay. Both NRF-1 and PPARg bound to an overlapping region of PGC-1 that is downstream from its amino-terminal activation domain but upstream of the RS domain (Fig. 1). Perhaps most compelling, expression of PGC-1 was able to induce mitochondrial biogenesis and this induction could be inhibited by a dominant negative allele of NRF-1 that lacked its transcriptional activation domain. As depicted in Fig. 2, these results link NRF-1 directly to PGC-1-mediated mitochondrial biogenesis. Since these initial findings, additional evidence has reinforced the conclusion that PGC-1 is a transcriptional coactivator involved in the biogenesis of mitochondria. PGC-1 was found to have a potent activation domain that is recruited to the promoter through docking with a cognate transcription factor (Knutti et al., 2000; Puigserver et al., 1999). This interaction is thought to result in a conformational change that facilitates the binding of additional coactivators, SRC-1 and p300 (Puigserver et al., 1999). These augment transcriptional activity presumably through their histone modifying func-

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tions. In addition, although the RS domain and RNA recognition motifs are not required for coactivator function, they are involved in the coupling of transcription to mRNA splicing (Monsalve et al., 2000). The mRNA processing activity requires that PGC-1 be associated with a promoter through its interaction with transcriptional activators. As summarized in Fig. 2, PGC-1 has also been implicated in the induction of fatty acid oxidation enzymes through its ability to interact with and trans-activate through PPARa (Vega et al., 2000). Many of the PPARa target genes that have been identified are involved in the oxidation of fatty acids (Lemberger et al., 1996; Gulick et al., 1994). The demonstration that PGC-1 can utilize PPARa in gene activation is a significant finding because fatty acid oxidation is a major mitochondrial function that is important for both thermogenesis and the generation of myocardial energy. The mitochondrial biogenic potential of PGC-1 has been further reinforced by studying the effects of its forced expression in both cultured cardiac myocytes and in the hearts of transgenic mice. Expression of PGC-1 from an adenovirus vector in neonatal cardiac myocytes resulted in the induction of nuclear genes representing several mitochondrial pathways including the citric acid cycle, electron transport and oxidative phosphorylation and fatty acid oxidation (Lehman et al., 2000). It also increased mitochondrial number and oxygen consumption through coupled respiration. Moreover, mitochondrial proliferation was induced in the postnatal hearts of transgenic mice by the

cardiac-specific expression of PGC-1 directed by a cardiac a-MHC promoter. The enlargement and proliferation of mitochondria was accompanied by a phenotype characteristic of dilated cardiomyopathy. These observations may be reflective of a physiological pathway of cardiac development because the same authors observed an induction of PGC-1 in the early postnatal period where the mitochondrial enzymatic machinery is also up-regulated. The results are consistent with a broad role for PGC-1 as a master regulator of mitochondrial biogenesis. 3.2. PGC-1-related coactivator (PRC) The tissue restricted expression of PGC-1 mRNA as well as its low expression in pre-adipocyte and myoblast cell lines (Wu et al., 1999; Puigserver et al., 1998) prompted the question of whether other PGC-1-like coactivators exist. Such related molecules might provide a similar function to that of PGC-1 but may be controlled by different regulatory pathways. A database search failed to reveal molecules with extended regions of significant sequence similarity with PGC-1. However, one large but partial cDNA sequence, deposited as KIAA0595 in the HUGE database (Kazusa DNA Research Institute) (Nagase et al., 1998), contained a region of significant sequence similarity with PGC-1 located in the carboxy-terminal domain comprising the RNA recognition motif. Cloning of the full-length cDNA revealed additional similarities with

Fig. 2. Summary of activator–coactivator interactions in the regulation of mitochondrial function. PGC-1 is induced by thermogenic signals involving sympathetic enervation and activation of the b3-adrenergic receptor. PRC is induced by unidentified mediators of cell proliferation. NRF-1 in conjunction with NRF-2 and other factors regulate the expression of the mitochondrial respiratory chain and both coactivators can interact directly with NRF-1 in activating NRF-1 target genes. In addition, PGC-1 can induce the enzymes of fatty acid oxidation through its interaction with PPARa and UCP-1 expression through PPARg and TRb. PRC can bind CREB and activate CRE-dependent promoters.

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PGC-1 including an acidic amino-terminal region, an LXXLL signature for nuclear receptor coactivators, a proline-rich region and an RS-rich region in addition to the RNA recognition motif (Andersson and Scarpulla, 2001). Although not highly conserved in sequence, the spatial conservation of these features was suggestive of related function (Fig. 1). Despite its structural relatedness to PGC-1, the expression pattern of PRC was significantly different (Andersson and Scarpulla, 2001). PRC was localized to the nucleoplasm as might be expected of a transcriptional coactivator but its mRNA level did not vary much from tissue to tissue. A possible exception is human skeletal muscle where PRC mRNA levels were somewhat higher. In contrast to PGC1, PRC mRNA was not enriched in brown fat relative to white fat and was only slightly induced in brown fat upon cold exposure. However, PRC was cell-cycle regulated in BALB/3T3 cells under conditions where PGC-1 was not detected. PRC was markedly induced upon serum treatment of quiescent fibroblasts and down-regulated when cells became contact inhibited for growth. Thus, the expression of PRC differed from PGC-1 in that it was responsive to proliferative signals rather than to the thermogenic signals that control PGC-1 expression (Fig. 2). Although PRC differed from PGC-1 in its tissue specificity and response to physiological signals it was identical in its ability to utilize NRF-1 for the trans-activation of NRF-1 target genes (Andersson and Scarpulla, 2001). NRF-1 and PRC were co-immunoprecipitated from cell extracts and interacted specifically in an in vitro binding assay. As with PGC-1, PRC required the NRF-1 DNA binding domain for its interaction with NRF-1. The activation of NRF-1-dependent promoters by PRC required a potent trans-activation domain localized to its amino-terminal region and functional NRF-1 sites in the target promoter. This was particularly evident for an artificial promoter comprised of four tandem NRF-1 sites and for the natural 5-ALAS promoter, which is highly dependent on two tandem NRF-1 sites for expression. The results with the cytochrome c promoter were more complex in that mutation of the NRF-1 site alone only partially reduced the trans-activation by PRC. Additional mutations in the CREB sites were required to reduce PRC activation to near baseline suggesting that CREB or a CREB-related factor is also a target for PRC. In contrast, mutation of the Sp1 sites reduced promoter activity but had no effect on PRC activation. This result is consistent with selectivity on the part of PRC for specific trans-activators. More recent experiments show that PRC and PGC-1 can interact with CREB but not with Sp1 in vitro and that PRC can trans-activate a CREB-dependent promoter (Andersson and Scarpulla, unpublished data). It is of interest in this context that CREB and NRF-1 but not Sp1 participate in the response of the cytochrome c promoter to serum activation (Herzig et al., 2000). Thus, it appears that PRC is a growth-regulated coactivator that may coordinate the activities of multiple transcription factors required for cell growth.

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Several important questions remain to be answered. First, is PRC able to induce mitochondrial biogenesis in living systems? Initial attempts to overexpress PRC have not been successful presumably because of its instability (Andersson and Scarpulla, 2001). Although its ability to trans-activate through NRF-1 is identical to that of PGC-1, it is important to know whether this is reflected in the in vivo function of PRC. Secondly, do PRC and PGC-1 differ in their transcription factor specificities? One might predict that the two coactivators operate on overlapping rather than identical sets of transcriptional activators and perhaps help recruit different coactivators to the transcription complex. This is because the proliferative and thermogenic programs may require the expression of many common genes (e.g. those involved in mitochondrial biogenesis) but also those specific for each pathway (e.g. UCP-1). Third, is the RNA splicing activity of PGC-1 conserved in PRC? This might be the case because of the conservation of the RS and RNA recognition motifs. Fourth, what are the specific effectors of PRC regulation? For example, if thyroid hormones induce the expression of PRC it might explain how these hormones affect the expression of nuclear respiratory genes without actually binding to their promoters via thyroid hormone receptors. Finally, what governs activator–coactivator interactions? Regulated posttranscriptional modifications of activator and/or coactivator may facilitate or inhibit interactions.

4. Summary The available evidence indicates that the transcriptional regulation of mitochondrial biogenesis in mammalian cells involves the interplay of DNA binding transcription factors and regulated coactivators (Fig. 2). The nuclear respiratory factors along with Sp1 are the most prevalent activators of respiratory chain expression and considerable biochemical and genetic evidence points to NRF-1 as a key regulator both in vitro and in vivo. However, other factors such as muscle-specific activators, CREB and YY1, are also utilized and other important mitochondrial functions rely upon additional factors, for example PPARa and the enzymes of fatty acid oxidation. The coactivators PGC-1 and PRC are of special interest because their expression varies in response to key physiological signals governing thermogenesis and cell growth, respectively. Both of these processes involve the proliferation of mitochondria and both coactivators are potent regulators of NRF-1-dependent gene expression. PGC-1 in particular utilizes a number of transcription factors that have been implicated in various aspects of adaptive thermogenesis including UCP-1 induction, mitochondrial proliferation and fatty acid oxidation (Fig. 2). Most importantly, ectopic expression of PGC-1 in both cultured cells and transgenic mice leads to the proliferation of mitochondria. It is clear that the transcriptional regulation of respiratory gene expression can be modulated at several levels. The

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regulated expression of a particular coactivator by a specific physiological signal can confer specificity to the response. The interaction of such cofactors with certain DNA binding transcription factors and their recruitment of other histone modifying coactivators to the transcription complex represent an additional level of amplification and specificity. Finally, the occupancy of target promoters by a given set of DNA binding regulatory proteins may determine the type of coactivator(s) that becomes engaged in the expression of a given set of genes. Understanding these interactions should yield insights into the control of complex biological processes such as mitochondrial biogenesis. Acknowledgements I thank Dr Lei Huo for critical reading of the manuscript. Work in the author’s laboratory was supported by United States Public Health Service Grant GM32525-19 from the National Institutes of Health. References Aizencang, G.I., Bishop, D.F., Forrest, D., Astrin, K.H., Desnick, R.J., 2000. Uroporphyrinogen III synthase. An alternative promoter controls erythroid-specific expression in the murine gene. J. Biol. Chem. 275, 2295–2304. Andersson, U., Scarpulla, R.C., 2001. PGC-l-related coactivator, a novel, serum-inducible coactivator of nuclear respiratory factor 1-dependent transcription in mammalian cells. Mol. Cell. Biol. 21, 3738–3749. Attardi, G., Schatz, G., 1988. Biogenesis of mitochondria. Annu. Rev. Cell Biol. 4, 289–333. Au, H.C., Scheffler, I.E., 1998. Promoter analysis of the human succinate dehydrogenase iron-protein gene. Both nuclear respiratory factors NRF-1 and NRF-2 are required. Eur. J. Biochem. 251, 164–174. Basu, A., Lenka, N., Mullick, J., Avadhani, N.G., 1997. Regulation of murine cytochrome oxidase Vb gene expression in different tissues and during myogenesis – role of a YY-1 factor-binding negative enhancer. J. Biol. Chem. 272, 5899–5908. Batchelor, A.H., Piper, D.E., De la Brousse, F.C., McKnight, S.L., Wolberger, C., 1998. The structure of GABPa/b: an ETS domain ankyrin repeat heterodimer bound to DNA. Science 279, 1037–1041. Braidotti, G., Borthwick, I.A., May, B.K., 1993. Identification of regulatory sequences in the gene for 5-aminolevulinate synthase from rat. J. Biol. Chem. 268, 1109–1117. Chau, C.A., Evans, M.J., Scarpulla, R.C., 1992. Nuclear respiratory factor 1 activation sites in genes encoding the gamma-subunit of ATP synthase, eukaryotic initiation factor 2a, and tyrosine aminotransferase. Specific interaction of purified NRF-1 with multiple target genes. J. Biol. Chem. 267, 6999–7006. de Sury, R., Martinez, P., Procaccio, V., Lunardi, J., Issartel, J.P., 1998. Genomic structure of the human NDUFS8 gene coding for the ironsulfur TYKY subunit of the mitochondrial NADH:ubiquinone oxidoreductase. Gene 215, 1–10. Dyer, M.R., Walker, J.E., 1993. Sequences of members of the human gene family for the c subunit of mitochondrial ATP synthase. Biochem. J. 293, 51–64. Elbehti-Green, A., Au, H.C., Mascarello, J.T., Ream-Robinson, D., Scheffler, I.E., 1998. Characterization of the human SDHC gene encoding one of the integral membrane proteins of succinate-quinone oxidoreductase in mitochondria. Gene 213, 133–140. Evans, M.J., Scarpulla, R.C., 1989. Interaction of nuclear factors with

multiple sites in the somatic cytochrome c promoter. Characterization of upstream NRF-1, ATF and intron Sp1 recognition sites. J. Biol. Chem. 264, 14361–14368. Evans, M.J., Scarpulla, R.C., 1990. NRF-1:a trans-activator of nuclearencoded respiratory genes in animal cells. Genes Dev. 4, 1023–1034. Fletcher, B.H., Cassidy, A.I., Summers, K.M., Cavanagh, A.C., 2001. The murine chaperonin 10 gene family contains an intronless, putative gene for early pregnancy factor, Cpn10-rs1. Mamm. Genome 12, 133–140. Gopalakrishnan, L., Scarpulla, R.C., 1994. Differential regulation of respiratory chain subunits by a CREB-dependent signal transduction pathway. Role of cyclic AMP in cytochrome c and COXIV gene expression. J. Biol. Chem. 269, 105–113. Graves, B.J., 1998. Inner workings of a transcription factor partnership. Science 279, 1000–1002. Gugneja, S., Scarpulla, R.C., 1997. Serine phosphorylation within a concise amino-terminal domain in nuclear respiratory factor 1 enhances DNA binding. J. Biol. Chem. 272, 18732–18739. Gugneja, S., Virbasius, J.V., Scarpulla, R.C., 1995. Four structurally distinct, non-DNA-binding subunits of human nuclear respiratory factor 2 share a conserved transcriptional activation domain. Mol. Cell. Biol. 15, 102–111. Gulick, T., Cresci, S., Caira, T., Moore, D.D., Kelly, D.P., 1994. The peroxisome proliferator-activated receptor regulates mitochondrial fatty acid oxidative enzyme gene expression. Proc. Natl. Acad. Sci. USA 91, 11012–11016. Hatefi, Y., 1985. The mitochondrial electron transport chain and oxidative phosphorylation system. Annu. Rev. Biochem. 54, 1015–1069. Hernandez, J.M., Giner, P., Hernandez-Yago, J., 1999. Gene structure of the human mitochondrial outer membrane receptor Tom20 and evolutionary study of its family of processed pseudogenes. Gene 239, 283–291. Herzig, R.P., Scacco, S., Scarpulla, R.C., 2000. Sequential serum-dependent activation of CREB and NRF-1 leads to enhanced mitochondrial respiration through the induction of cytochrome c. J. Biol. Chem. 275, 13134–13141. Hirawake, H., Taniwaki, M., Tamura, A., Amino, H., Tomitsuka, E., Kita, K., 1999. Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinateubiquinone oxidoreductase. Biochim. Biophys. Acta 1412, 295–300. Holloszy, J.O., 1967. Biochemical adaptations in muscle. Effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle. J. Biol. Chem. 242, 2278–2282. Huo, L., Scarpulla, R.C., 2001. Mitochondrial DNA instability and periimplantation lethality associated with targeted disruption of nuclear respiratory factor 1 in mice. Mol. Cell. Biol. 21, 644–654. Hu¨ ttemann, M., Mu¨ hlenbein, N., Schmidt, T.R., Grossman, L.I., Kadenbach, B., 2000. Isolation and sequence of the human cytochrome c oxidase subunit VIIaL gene. Biochim. Biophys. Acta 1492, 252–258. Izquierdo, J.M., Ricart, J., Ostronoff, L.K., Egea, G., Cuezva, J.M., 1995. Changing patterns of transcriptional and post-transcriptional control of b-F1-ATPase gene expression during mitochondrial biogenesis in liver. J. Biol. Chem. 270, 10342–10350. Knutti, D., Kaul, A., Kralli, A., 2000. A tissue-specific coactivator of steroid receptors, identified in a functional genetic screen. Mol. Cell. Biol. 20, 2411–2422. LaMarco, K., Thompson, C.C., Byers, B.P., Walton, E.M., McKnight, S.L., 1991. Identification of Ets- and notch-related subunits in GA binding protein. Science 253, 789–792. Larsson, N.G., Wang, J.M., Wilhelmsson, H., Oldfors, A., Rustin, P., Lewandoski, M., Barsh, G.S., Clayton, D.A., 1998. Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice. Nat. Genet. 18, 231–236. Lehman, J.J., Barger, P.M., Kovacs, A., Saffitz, J.E., Medeiros, D.M., Kelly, D.P., 2000. Peroxisome proliferator-activated receptor g coactivator-1 promotes cardiac mitochondrial biogenesis. J. Clin. Invest. 106, 847–856. Lemberger, T., Desvergne, B., Wahli, W., 1996. Peroxisome proliferator-

R.C. Scarpulla / Gene 286 (2002) 81–89 activated receptors: a nuclear receptor signalling pathway in lipid physiology. Annu. Rev. Cell Biol. 12, 335–363. Lenka, N., Basu, A., Mullick, J., Avadhani, N.G., 1996. The role of an E box binding basic helix loop helix protein in the cardiac muscle-specific expression of the rat cytochrome oxidase subunit VIII gene. J. Biol. Chem. 271, 30281–30289. Li, B., Holloszy, J.O., Semenkovich, C.F., 1999. Respiratory uncoupling induces d-aminolevulinate synthase expression through a nuclear respiratory factor 1-dependent mechanism in HeLa cells. J. Biol. Chem. 274, 17534–17540. Li, R., Luciakova, K., Nelson, B.D., 1996a. Expression of the human cytochrome c1 gene is controlled through multiple Sp1-binding sites and an initiator region. Eur. J. Biochem. 241, 649–656. Li, R., Hodny, Z., Luciakova, K., Barath, P., Nelson, B.D., 1996b. Sp1 activates and inhibits transcription from separate elements in the proximal promoter of the human adenine nucleotide translocase 2 (ANT2) gene. J. Biol. Chem. 271, 18925–18930. Lowell, B.B., Spiegelman, B.M., 2000. Towards a molecular understanding of adaptive thermogenesis. Nature 404, 652–660. Luis, A.M., Isquierdo, J.M., Ostronoff, L.K., Salinas, M., Santaren, J.F., Cuezva, J.M., 1993. Translational regulation of mitochondrial differentiation in neonatal rat liver. J. Biol. Chem. 268, 1868–1875. Michaels, G.S., Hauswirth, W.W., Laipis, P.J., 1982. Mitochondrial DNA copy number in bovine oocytes and somatic cells. Dev. Biol. 94, 246– 251. Monsalve, M., Wu, Z., Adelmant, G., Puigserver, P., Fan, M., Spiegelman, B.M., 2000. Direct coupling of transcription and mRNA processing through the thermogenic coactivator PGC-1. Mol. Cell 6, 307–316. Moraes, C.T., Ricci, E., Bonilla, E., DiMauro, S., Schon, E.A., 1992. The mitochondrial tRNA Leu(UUR) mutation in mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS): genetic, biochemical, and morphological correlations in skeletal muscle. Am. J. Hum. Genet. 50, 934–949. Nagase, T., Ishikawa, K., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., Ohara, O., 1998. Prediction of the coding region sequences of unidentified human genes. IX. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro. DNA Res. 5, 31–39. Neupert, W., 1997. Protein import into mitochondria. Annu. Rev. Biochem. 66, 863–917. Piko, L., Matsumoto, L., 1976. Number of mitochondria and some properties of mitochondrial DNA in the mouse egg. Dev. Biol. 49, 1–10. Piko, L., Taylor, K.D., 1987. Amounts of mitochondrial DNA and abundance of some mitochondrial gene transcripts in early mouse embryos. Dev. Biol. 123, 364–374. Puigserver, P., Wu, Z., Park, C.W., Graves, R., Wright, M., Spiegelman, B.M., 1998. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92, 829–839. Puigserver, P., Adelmant, C., Wu, Z.D., Fan, M., Xu, J.M., O’Malley, B., Spiegelman, B.M., 1999. Activation of PPARgamma coactivator-1 through transcription factor docking. Science 286, 1368–1371. Scacco, S., Vergari, R., Scarpulla, R.C., Technikova-Dobrova, Z., Sardanelli, A., Lambo, R., Lorusso, V., Papa, S., 2000. cAMP-dependent phosphorylation of the nuclear encoded 18-kDa (IP) subunit of respiratory complex I and activation of the complex in serum-starved mouse fibroblast cultures. J. Biol. Chem. 275, 17578–17582. Scarpulla, R.C., 1996. Nuclear respiratory factors and the pathways of nuclear-mitochondrial interaction. Trends Cardiovasc. Med. 6, 39–45. Scarpulla, R.C., 1997. Nuclear control of respiratory chain expression in mammalian cells. J. Bioenerg. Biomembr. 29, 109–119. Scarpulla, R.C., 1999. Nuclear transcription factors in cytochrome c and cytochrome oxidase expression. In: Papa, S., Guerrieri, F., Tager, J. (Eds.). Frontiers of Cellular Bioenergetics: Molecular Biology, Biochemistry, and Physiopathology, Plenum, London, pp. 553–591.

89

Seelan, R.S., Grossman, L.I., 1997. Structural organization and promoter analysis of the bovine cytochrome c oxidase subunit VIIc gene – a functional role for YY1. J. Biol. Chem. 272, 10175–10181. Seelan, R.S., Gopalakrishnan, L., Scarpulla, R.C., Grossman, L.I., 1996. Cytochrome c oxidase subunit VIIa liver isoform: characterization and identification of promoter elements in the bovine gene. J. Biol. Chem. 271, 2112–2120. Shadel, G.S., Clayton, D.A., 1993. Mitochondrial transcription initiation. Variation and conservation. J. Biol. Chem. 268, 16083–16086. Shadel, G.S., Clayton, D.A., 1997. Mitochondrial DNA maintenance in vertebrates. Annu. Rev. Biochem. 66, 409–435. Shamoo, Y., Abdul-Manan, N., Williams, K.R., 1995. Multiple RNA binding domains (RBDs) just don’t add up. Nucleic Acids Res. 23, 725–728. Shaywitz, A.J., Greenberg, M.E., 1999. CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu. Rev. Biochem. 68, 821–861. Silva, J.E., Rabelo, R., 1997. Regulation of uncoupling protein gene expression. Eur. J. Endocrinol. 136, 251–264. Stotz, E., 1939. The estimation and distribution of cytochrome oxidase and cytochrome c in rat tissues. J. Biol. Chem. 131, 555–565. Tacke, R., Manley, J.L., 1999. Determinants of SR protein specificity. Curr. Opin. Cell Biol. 11, 358–362. Tata, J.R., Ernster, L., Lindberg, O., Arrhenius, E., Pederson, S., Hedman, R., 1963. The action of thyroid hormones at the cell level. Biochem. J. 86, 408–428. Thompson, C.C., Brown, T.A., McKnight, S.L., 1991. Convergence of Etsand notch-related structural motifs in a heteromeric DNA binding complex. Science 253, 762–768. Vega, R.B., Huss, J.M., Kelly, D.P., 2000. The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor a in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes. Mol. Cell. Biol. 20, 1868–1876. Virbasius, J.V., Scarpulla, R.C., 1991. Transcriptional activation through ETS domain binding sites in the cytochrome c oxidase subunit IV gene. Mol. Cell. Biol. 11, 5631–5638. Virbasius, J.V., Scarpulla, R.C., 1994. Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis. Proc. Natl. Acad. Sci. USA 91, 1309–1313. Virbasius, J.V., Virbasius, C.A., Scarpulla, R.C., 1993a. Identity of GABP with NRF-2, a multisubunit activator of cytochrome oxidase expression, reveals a cellular role for an ETS domain activator of viral promoters. Genes Dev. 7, 380–392. Virbasius, C.A., Virbasius, J.V., Scarpulla, R.C., 1993b. NRF-1, an activator involved in nuclear-mitochondrial interactions, utilizes a new DNAbinding domain conserved in a family of developmental regulators. Genes Dev. 7, 2431–2445. Wan, B., Moreadith, R.W., 1995. Structural characterization and regulatory element analysis of the heart isoform of cytochrome c oxidase VIa. J. Biol. Chem. 270, 26433–26440. Williams, R.S., Garcia-Moll, M., Mellor, J., Salmons, S., Harlan, W., 1987. Adaptation of skeletal muscle to increased contractile activity. J. Biol. Chem. 262, 2764–2767. Wong-Riley, M., Guo, A., Bachman, N.J., Lomax, M.I., 2000. Human COX6A1 gene: promoter analysis, cDNA isolation and expression in the monkey brain. Gene 247, 63–75. Wu, Z., Puigserver, P., Andersson, U., Zhang, C., Adelmant, G., Mootha, V., Troy, A., Cinti, S., Lowell, B., Scarpulla, R.C., Spiegelman, B.M., 1999. Mechanisms controlling mitochondrial biogenesis and function through the thermogenic coactivator PGC-1. Cell 98, 115–124. Zaid, A., Li, R., Luciakova, K., Barath, P., Nery, S., Nelson, B.D., 1999. On the role of the general transcription factor Sp1 in the activation and repression of diverse mammalian oxidative phosphorylation genes. J. Bioenerg. Biomembr. 31, 129–135.