Transcriptional regulatory circuits controlling mitochondrial

REVIEW

Transcriptional regulatory circuits

controlling mitochondrial biogenesis

and function

Daniel P. Kelly

1,3

and Richard C. Scarpulla

Center for Cardiovascular Research, Departments of Medicine, Molecular Biology & Pharmacology, and Pediatrics,

Washington University School of Medicine, St. Louis, Missouri 63119, USA;

Department of Cell and Molecular Biology,

Northwestern Medical School, Chicago, Illinois 60611, USA

We are witnessing a period of renewed interest in the

biology of the mitochondrion. The mitochondrion serves

a critical function in the maintenance of cellular energy

stores, thermogenesis, and apoptosis. Moreover, alter-

ations in mitochondrial function contribute to several

inherited and acquired human diseases and the aging

process. This review summarizes our understanding of

the transcriptional regulatory mechanisms involved in

the biogenesis and energy metabolic function of mito-

chondria in higher organisms.

The mitochondrial genome

A defining feature of eukaryotic cells is that they contain

nuclear and mitochondrial genomes sequestered into

distinct subcellular compartments. The mitochondrial

genetic system is comprised of a circular DNA genome

(mtDNA, ∼16.5 kb in vertebrates; Fig. 1), the enzymes

required for its transcription and replication, and the pro-

tein synthetic machinery necessary for the translation of

13 mitochondrial mRNAs (for review, see Garesse and

Vallejo 2001). These mRNAs, which account for the en-

tire protein-coding capacity of mtDNA, encode essential

subunits of respiratory complexes I, III, IV, and V. The

extrusion of protons through complexes I, III, and IV is

coupled to the sequential transfer of electrons to a series

of carriers of increasing redox potential resulting in an

electrochemical proton gradient across the inner mem-

brane. Complex V, comprised of an ATPase coupled to

an inner membrane proton channel, can dissipate the

proton gradient in the synthesis of ATP or can couple

proton pumping to ATP hydrolysis to maintain the gra-

dient. mtDNA also encodes for two ribosomal and 22

transfer RNAs, required for translation by mitoribo-

somes within the matrix.

The limited coding capacity of mtDNA necessitates

that nuclear genes make a major contribution to mito-

chondrial metabolic systems and molecular architecture

(Garesse and Vallejo 2001). One major class of nuclear

genes contributes catalytic and auxiliary proteins to the

mitochondrial enzyme systems. For example, the major-

ity of the 100 or so subunits of the respiratory apparatus

are nucleus-encoded. In addition, nucleus-encoded meta-

bolic enzymes necessary for the oxidation of pyruvate,

fatty acids (␤-oxidation cycle), and acetyl-CoA (tricar-

boxylic acid cycle), the biosynthesis of certain amino

acids, and the manufacture of heme, among others,

are localized to the mitochondrion. A second class of

nuclear genes encodes protein import and assembly fac-

tors. A third class contributes key proteins that are re-

quired for the replication and expression of the mito-

chondrial genome including nucleic acid polymerases,

RNA processing enzymes, transcription and replication

factors as well as tRNA-synthetases, translation factors,

and ribosomal subunits. Thus, the program regulating

mitochondrial biogenesis involves the coordinate ac-

tions of nuclear and mitochondrial genes.

Regulatory proteins involved in mitochondrial

gene transcription

In yeast, mtDNA transcription is initiated at ∼20 tran-

scriptional units throughout the genome (for review, see

Poyton and McEwen 1996). In vertebrates, transcription

is initiated bidirectionally at two promoters, P

and P

for heavy (H) and light strands (L), respectively, within

the D-loop regulatory region (Shadel and Clayton 1997;

Clayton 2000). The D-loop is the longest noncoding re-

gion in vertebrate mtDNA and contains, in addition to

and P

, the H-strand replication origin (O

; Fig. 1). In

the “strand asymmetric model” of mtDNA replication,

the RNA transcript initiated at P

is cleaved in the vi-

cinity of three evolutionarily conserved sequence blocks

(CSB I, II, and III), and H-strand replication is initiated at

the sites of these cleavages (Bogenhagen and Clayton

2003). Thus, transcription is coupled to DNA replication

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and the sites of RNA cleavage are transition sites be-

tween RNA and DNA synthesis. A decision must be

made to continue transcription through the CSBs or to

truncate the nascent RNA to initiate DNA replication.

After DNA synthesis begins, the nascent strand is often

terminated downstream from a conserved element re-

ferred to as a termination-associated sequence (TAS).

This event accounts for the triple-stranded D-loop struc-

ture and may be important in controlling mtDNA levels

(Brown and Clayton 2002).

The mitochondrial H- and L-strand transcriptional

units differ from most nuclear genes in that they are

polygenic. In addition to the RNA primer for H-strand

replication, P

also directs the synthesis of a transcript

that is processed to one mRNA and eight of the 22 tRNAs.

The polygenic transcript directed by P

is processed to

14 tRNAs, 12 mRNAs, and the two rRNAs (for review,

see Garesse and Vallejo 2001). Both promoters share a

critical upstream enhancer that serves as the recognition

site for mitochondrial transcription factor A or Tfam

(previously mtTF-1 and mtTFA), an HMG-box protein

that stimulates transcription through specific binding to

upstream enhancers. Like other HMG proteins, Tfam

can bend and unwind DNA, properties potentially linked

to its ability to stimulate transcription upon binding

DNA (Fisher et al. 1992). In addition to specific promoter

recognition, Tfam binds nonspecific DNA with high af-

finity. This property along with its abundance in mito-

chondria suggests that it plays a role in the stabilization

and maintenance of the mitochondrial chromosome

through its phased binding to nonpromoter sites (Parisi

et al. 1993).

Several lines of evidence indicate that Tfam is required

for mtDNA replication and maintenance. Tfam knock-

out mice display embryonic lethality and depletion of

mtDNA (Larsson et al. 1998). In addition, Tfam levels

correlate well with increased mtDNA in ragged-red

muscle fibers and decreased mtDNA levels in mtDNA-

depleted cells (Larsson et al. 1994; Poulton et al. 1994).

ABF2, a related HMG-box yeast factor, is required for

mtDNA maintenance and respiratory competence (Diff-

ley and Stillman 1991). Expression of Tfam can comple-

ment an ABF2 deficiency in yeast, suggesting that the

two proteins are functionally homologous (Parisi et al.

1993). Interestingly, despite this functional complemen-

tation, ABF2 lacks an activation domain present in Tfam

and does not stimulate transcription (Dairaghi et al.

1995).

Significant progress has been made in the character-

ization of the mtDNA transcription initiation machin-

ery. A vertebrate mitochondrial RNA polymerase and a

specificity factor that are required for mitochondrial-spe-

cific initiation were initially identified and characterized

in Xenopus laevis (Antoshechkin and Bogenhagen 1995;

Bogenhagen 1996). Although purification of the human

polymerase has been elusive, a human cDNA that en-

codes a protein with sequence similarity to yeast mito-

chondrial and phage polymerases has been identified in

database screenings (Tiranti et al. 1997). The encoded

protein localizes to mitochondria, suggesting that it is a

bona fide mitochondrial polymerase. A human mito-

chondrial transcription factor B (h-mtTFB) cDNA has

also been isolated, and the encoded protein has proper-

ties consistent with it being a functional homolog of the

Figure 1. Human mitochondrial DNA (mtDNA). The

genomic organization and structural features of human

mtDNA are depicted in a circular genomic map. The

D-loop regulatory region is expanded and shown above.

Protein coding and rRNA genes are interspersed with

22 tRNA genes (denoted by the single-letter amino acid

code). The D-loop regulatory region contains the L- and

H-strand promoters (P

and P

, respectively) along with

the origin of H-strand replication (O

). mtDNA trans-

cription complexes containing mitochondrial RNA poly-

merase, Tfam, and TFB are depicted in the expanded

D-loop along with the conserved sequence blocks (CSB

I, II, and III). The origin of L-strand replication (O

)is

displaced by approximately two-thirds of the genome

within a cluster of five tRNA genes. Protein-coding

genes include cytochrome oxidase (COX) subunits 1, 2,

and 3; NADH dehydrogenase (ND) subunits 1, 2, 3, 4,

4L, 5, and 6; ATP synthase (ATPS) subunits 6 and 8; and

cytochrome b (Cytb). ND6 and the eight tRNA genes

encoded on the L-strand are in bold type and under-

lined; all other genes are encoded on the H-strand.

Kelly and Scarpulla

358 GENES & DEVELOPMENT

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yeast specificity factor, sc-mtTFB (McCulloch et al.

2002). The protein is localized to mitochondria and can

bind DNA and stimulate transcription from an L-strand

promoter in vitro. Subsequently, two isoforms of h-mtTFB,

termed TFB1 and 2, were identified (Falkenberg et al.

2002). TFB1 is identical to the initial h-mtTFB isolate.

Like the yeast factor, both TFBs share sequence similari-

ties with rRNA dimethyltransferases, although the simi-

larity between TFB2 and this class of enzymes is weaker

than that of TFB1. Both TFB isoforms can support spe-

cific initiation from mitochondrial promoters in an in

vitro system containing purified recombinant proteins.

In this system, the TFB-dependent activation of tran-

scription depends on mitochondrial RNA polymerase

and Tfam (Fig. 1). Both TFBs interact with mitochondrial

RNA polymerase, but TFB1 has about one-tenth the

transcriptional activity of TFB2. In addition to binding

mitochondrial RNA polymerase, TFB1 also contacts the

C-terminal domain of Tfam (McCulloch and Shadel

2003). The region of contact between TFB1 and Tfam is

essential for transcriptional activation and corresponds

to a 29-amino-acid domain that was previously identi-

fied as a Tfam activation domain (Dairaghi et al. 1995).

This reinforces the distinction between Tfam and the

yeast HMG-box protein ABF2, which, like Tfam, is re-

quired for mtDNA maintenance but does not function as

a transcription factor.

Transcriptional regulators of nuclear encoded

mitochondrial proteins: the critical role of nuclear

respiratory factors 1 and 2

The cytochrome c and cytochrome oxidase genes have

served as the prototypes for identifying regulatory factors

that act on nuclear respiratory genes from both yeast and

mammalian cells. Early work in yeast demonstrated that

transcriptional regulation of the major cytochrome c iso-

form, CYC1, was mediated by oxygen and carbon sources

through the upstream activation sites, UAS1 and UAS2.

This work has been the subject of excellent reviews to

which the reader is referred for original citations (Zi-

tomer and Lowry 1992; Poyton and McEwen 1996).

The identification of nucleus-encoded transcription

factors required for the expression of the respiratory ap-

paratus in mammalian cells also began with the charac-

terization of the cytochrome c gene (for reviews, see

Scarpulla 1997, 1999). Interestingly, the mammalian cy-

tochrome c promoter has multiple recognition sites for

transcription factors that bear no obvious relationship to

those identified in yeast (Evans and Scarpulla 1988). A

potent cis-acting element, localized to the first intron,

consists of tandem Sp1 recognition sites that function

synergistically to maximize promoter activity. A second

cis-element binds transcription factors of the ATF/CREB

family (Evans and Scarpulla 1989). The cytochrome c

promoter also contains a recognition site for a transcrip-

tion factor designated nuclear respiratory factor 1, or

NRF-1 (Evans and Scarpulla 1989). NRF-1 is a 68-kD

polypeptide with the presence of a C-terminal transcrip-

tional activation domain comprised of glutamine-con-

taining clusters of hydrophobic amino acid residues

(Chau et al. 1992; Gugneja et al. 1996). Both endogenous

and recombinant proteins bind as a homodimer to pal-

indromic NRF-1 sites through guanine nucleotide con-

tacts over a single turn of the DNA helix (Virbasius et al.

1993a). Serine phosphorylation of the N-terminal do-

main of NRF-1 enhances both its DNA-binding (Gugneja

and Scarpulla 1997) and trans-activation functions (Her-

zig et al. 2000).

NRF-1 has been linked to the transcriptional control of

many genes involved in mitochondrial function and bio-

genesis (Table 1). NRF-1 target genes have been identi-

fied by characterization of functional NRF-1-binding

sites within their promoters. Many NRF-1 target genes

encode subunits of the five respiratory complexes (Vir-

basius et al. 1993a). However, the regulatory network

controlled by NRF-1 extends beyond the respiratory sub-

units to other classes of genes. These include genes in-

volved in assembly of the respiratory apparatus, con-

stituents of the mtDNA transcription and replication

machinery, mitochondrial and cytosolic enzymes of the

heme biosynthetic pathway, and components of mito-

chondrial protein import. Notably, Tfam is an NRF-1

target gene consistent with the postulate that NRF-1

plays an integrative role in nucleo–mitochondrial inter-

actions. This hypothesis has been reinforced by the re-

sults of several recent studies associating increases in

NRF-1 mRNA levels or DNA-binding activity with mi-

tochondrial biogenesis. NRF-1 and its coactivator PGC-1

(see below) are induced as part of the adaptation of skel-

etal muscle to exercise training (Murakami et al. 1998;

Baar et al. 2002). Similar results were obtained in cul-

tured myotubes in response to elevated calcium, which

mimics exercise-induced mitochondrial biogenesis (Ojuka

et al. 2003). Likewise, treatment of rats with a creatine

analog that induces muscle adaptations analogous to

those observed during exercise leads to the activation of

AMP-activated protein kinase and increased NRF-1-DNA

binding activity, cytochrome c content, and mitochon-

drial density (Bergeron et al. 2001). Both NRF-1 and Tfam

mRNAs are elevated in cells depleted of mtDNA, pre-

sumably as a response to increased oxidative stress (Mi-

randa et al. 1999). Lastly, NRF-1 and Tfam are up-regu-

lated in response to lipopolysaccharide-induced oxida-

tive damage to mitochondria, presumably to enhance

mtDNA levels and OXPHOS activity (Suliman et al.

2003).

Perhaps the strongest in vivo link between NRF-1 and

the control of mitochondrial function comes from the

results of targeted disruption of the NRF-1 gene in mice

(Huo and Scarpulla 2001). Homozygosity of the null al-

lele results in lethality between embryonic days 3.5 and

6.5 (E3.5 and E6.5). The null blastocysts fail to grow in

culture despite having a normal morphology. Homozy-

gous null blastocysts are defective in maintaining a mi-

tochondrial membrane potential and have severely re-

duced mtDNA levels. This is not accompanied by in-

creased apoptosis, making it unlikely that the reduction

in mtDNA is associated with a generalized increase in

DNA fragmentation. Moreover, the mature oocytes of

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heterozygous mothers have a normal complement of

mtDNA, supporting the argument against a defect in

mtDNA amplification during oogenesis. Therefore, the

mtDNA depletion occurs between fertilization and the

blastocyst stage and most likely results from the loss of

a NRF-1-dependent pathway of mtDNA maintenance.

Interestingly, Tfam-null embryos also exhibit severely

depleted levels of mtDNA but survive to E8.5–E10.5

(Larsson et al. 1998). Thus, it is likely that the early

mortality of NRF-1-null embryos results from the com-

bined effects of reduced levels of mtDNA and disruption

of other NRF-1-dependent functions.

Characterization of cytochrome oxidase genes led to

the identification of a second regulatory factor desig-

nated as NRF-2 (for review, see Scarpulla 1997, 1999). A

series of directly repeated NRF-2 sites within the mouse

COXIV promoter overlaps multiple transcription initia-

tion sites and contains additional binding sites for the

ETS-domain family of transcription factors (Virbasius

and Scarpulla 1991; Carter et al. 1992). The complex

binding the NRF-2 sites was purified to homogeneity

from HeLa cell nuclear extracts and is comprised of five

subunits. These include a DNA-binding ␣ subunit and

four others (␤

, ␤

, ␥

, and ␥

) that complex with ␣ but

alone do not bind DNA. The NRF-2 complexes activate

transcription through four directly repeated ETS-do-

main-binding sites in the COXVb promoter, suggesting

that NRF-2 may also act on multiple respiratory promot-

ers (Virbasius et al. 1993b).

Purification and molecular cloning of all five NRF-2

subunits established that NRF-2 is the human homolog

of mouse GABP (LaMarco and McKnight 1989) and that

the two additional human subunits, ␤

and ␥

, were mi-

nor splice variants of GABP subunits ␤

and ␤

(Gugneja

et al. 1995). The function of the non-DNA-binding sub-

units is twofold. First, the GABP␤

subunit, correspond-

ing to NRF-2␤

and NRF-2␤

(Gugneja et al. 1995), has

a dimerization domain that facilitates cooperative bind-

ing of a heterotetrameric complex to tandem binding

sites (Thompson et al. 1991). In solution, GABP exists as

Table 1. Nuclear and mitochondrial genes with NRF-1 and

NRF-2 recognition sites

NRF-1

NRF-2

Oxidative phosphorylation

Rat cytochrome c +

Human cytochrome c +

Complex I:

Human NADH dehydrogenase subunit

8 (TYKY) +

Complex II:

Human succinate dehydrogenase

subunit B + +

Human succinate dehydrogenase

subunit C + +

Human succinate dehydrogenase

subunit D + +

Complex III:

Human ubiquinone-binding protein +

Human core protein I +

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 + +

Human cytochrome oxidase subunit

VIIaL +

Bovine cytochrome oxidase subunit

VIIc +

Complex V:

Bovine ATP synthase ␥ subunit +

Human ATP synthase c subunit +

Human ATP synthase ␤ subunit +

mtDNA transcription and replication

Human Tfam + +

Mouse Tfam +

Rat Tfam +

Mouse MRP RNA +

Human MRP RNA +

Human TFB1 + +

Mouse TFB1 +

Human TFB2 + +

Mouse TFB2 +

HEME biosynthesis

Rat 5-aminolivulinate synthase +

Mouse uroporphyrinogen III synthase + +

Protein import and assembly

Human Tom 20 + +

Human Tom 70 +

Mouse chaperonin 10 +

Human SURF-1 +

Mouse COX17 + +

(continued)

Table 1. (continued)

NRF-1

NRF-2

Ion channels

Human VDAC3 +

Mouse VDAC3 +

Human VDAC1 +

Shuttles

Human glycerol phosphate

dehydrogenase +

Translation

Human mitochondrial ribosomal S12 + +

Original references for the majority of the indicated NRF-1

and/or NRF-2 target genes that are related to mitochondrial

function have been cited elsewhere (Scarpulla 1997, 2002). New

additions include human Tomm 70 (Blesa et al. 2003) and

mouse COX 17 (Takahashi et al. 2002).

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an ␣␤ heterodimer but is induced to form the heterote-

tramer ␣

␤

by DNA containing two or more binding

sites (Chinenov et al. 2000). The crystal structure of the

heterotetramer bound to DNA has been determined

(Batchelor et al. 1998). The second function of the non-

DNA-binding subunits is to contribute a transcriptional

activation domain. This domain resembles that found in

NRF-1 and has been localized to a region upstream from

the homodimerization domain (Gugneja et al. 1996).

Functional NRF-2 sites have now been identified in

several COX promoters as well as in many other genes

related to respiratory chain expression (for review, see

Scarpulla 2002). As with NRF-1, the list of respiratory

genes containing NRF-2 sites has expanded in recent

years (Table 1). These include genes for Tfam (Larsson

et al. 1998; Rantanen et al. 2001) and the newly discov-

ered TFB factors (Falkenberg et al. 2002; McCulloch et al.

2002) involved in mitochondrial transcription and DNA

replication (Rantanen et al. 2001). Genes encoding three

of the four human succinate dehydrogenase (complex II)

subunits also have both NRF-1 and NRF-2 sites in their

promoters (Au and Scheffler 1998; Elbehti-Green et al.

1998; Hirawake et al. 1999). In many cases, NRF-1 sites

are also present in NRF-2-dependent promoters, but this

is not a general rule. For example, several COX promot-

ers and the rodent Tfam (Choi et al. 2002) and TFB (Ran-

tanen et al. 2003) promoters do not have obvious NRF-1

consensus sites (Table 1). This contrasts with the human

Tfam (Virbasius and Scarpulla 1994) and TFB (R.C.

Scarpulla, unpubl.) promoters, which rely on functional

NRF-1 and NRF-2 recognition sites for their activities.

A subset of respiratory genes does not appear to be

regulated by NRF-1 or NRF-2. Other well-characterized

regulatory factors have been implicated in the expression

of these genes. The transcription factor Sp1 is associated

with the activation and/or repression of cytochrome c

(Li et al. 1996b) and adenine nucleotide translocase 2

genes (Li et al. 1996a), both of which lack NRF sites (Zaid

et al. 1999). Sp1 sites are also common to many GC-rich

promoters including those that are NRF-dependent. The

muscle-specific COX subunits, COXVIaH and COXVIII,

are also lacking NRF sites but depend on MEF-2 and/or

E-box consensus elements for their expression (Wan and

Moreadith 1995). Thus, the same or similar factors re-

quired for the expression of other muscle-specific genes

are linked to the regulation of these tissue-specific COX

subunits. In contrast, the promoter of the ubiquitously

expressed liver isoform, COXVIaL, depends on NRF-1

and NRF-2 as well as Sp1 for full activity (Seelan et al.

1996). This is consistent with the observation that in

gene pairs encoding ubiquitous and tissue-specific iso-

forms of a given protein, the NRF-1 site, when present, is

associated with the ubiquitously expressed gene (Virba-

sius et al. 1993a). Finally, the initiator element transcrip-

tion factor YY1 participates 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 helps confer a negative regulatory effect on COXVb

promoter activity (Basu et al. 1997). In the COXVIIc

promoter, two YY1 sites in conjunction with an NRF-2

site act as positive regulators of promoter activity (See-

lan and Grossman 1997). It is also important to note

that regulation of most nuclear genes encoding mito-

chondrial enzymes upstream of the respiratory chain is

NRF-1/NRF-2-independent. For example, genes encod-

ing mitochondrial fatty acid oxidation enzymes are regu-

lated by the peroxisome proliferator-activated receptor

alpha (PPAR␣) and other NRF-1-independent regulatory

pathways (Gulick et al. 1994). Thus, any unifying tran-

scriptional model of mitochondrial biogenesis needs to

account for the expression of genes that are NRF-inde-

pendent.

There are several reports suggesting that nuclear and

mitochondrial genes are controlled by common cis-act-

ing elements that are the targets of the same or simi-

lar transcription factors. Sequence similarities to the

OXBOX/REBOX (Haraguchi et al. 1994) and Mt (Suzuki

et al. 1995) elements have been localized to the mito-

chondrial D-loop. The ability of these elements and their

nuclear gene counterparts to bind proteins from crude

extracts with the same specificity has been taken as evi-

dence for shared regulatory factors between the two ge-

netic systems (Haraguchi et al. 1994). Similarly, other

nuclear factors, such as thyroid hormone receptors, have

been implicated in mitochondrial gene expression (for

review, see Wrutniak-Cabello et al. 2001). However,

there is no evidence that these proteins can use the mi-

tochondrial transcriptional machinery to direct mito-

chondrial gene expression.

The critical role of transcriptional coactivators

in the mitochondrial biogenic regulatory cascade:

The PPAR␥ coactivator-1 (PGC-1) family

As described above, the mitochondrial biogenic program

involves the integration of multiple transcriptional regu-

latory pathways controlling the expression of both nuclear

and mitochondrial genes. This highlights a mechanistic

enigma fundamental to the control of mitochondrial bio-

genesis. How is the activity of multiple transcription fac-

tors (e.g., NRF-1, NRF-2, PPAR␣, mtTFA) coordinately

regulated during the mitochondrial biogenic process?

Moreover, in the context of such complex integration,

how is cell- and tissue-specific function achieved? For

example, mitochondria within the brown adipocyte are

poised for uncoupled mitochondrial respiration, whereas

in other tissues such as heart, mitochondrial respiration

is largely coupled for high-level ATP production. To add

to the complexity, skeletal muscle is capable of support-

ing both coupled and uncoupled respiration. New insight

into this problem was provided by the discovery of the

transcriptional coactivator PPAR␥ coactivator 1␣ (PGC-

1␣) by Spiegelman and colleagues (Puigserver et al. 1998).

PGC-1

␣

was cloned in a yeast two-hybrid screen for

brown adipose-specific factors that interacted with the

adipogenic nuclear receptor PPAR␥ (Puigserver et al.

1998). PGC-1␣ serves as a direct transcriptional coacti-

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vator of PPAR␥ and is a member of a growing list of

proteins that coactivate transcription factors through di-

rect protein–protein interactions (for review, see Knutti

and Kralli 2001; Puigserver and Spiegelman 2003).

Transcriptional coactivators serve multiple functions

including modification of chromatin through posttrans-

lational histone acetylation, direct interaction with the

RNA polymerase II complex, mRNA processing, and re-

cruitment of other transcriptional coactivators (for re-

view, see Robyr et al. 2000; Belandia and Parker 2003).

Present evidence indicates that PGC-1␣ coactivates its

targets via recruitment of additional coactivators with

histone acetylase activity, such as SRC-1 (Puigserver

et al. 1999). In addition, the PGC-1␣ molecule contains

domains capable of interacting with and processing pre-

mRNA (Monsalve et al. 2000). PGC-1␣ also interacts di-

rectly with the TRAP/Mediator complex (Wallberg et al.

2003). Unlike most known transcriptional coactivators,

PGC-1␣ is unique in that it exhibits a tissue-enriched

expression pattern and is highly inducible (Puigserver

et al. 1998; Knutti and Kralli 2001; Puigserver and Spie-

gelman 2003). PGC-1

␣

is enriched in brown adipose,

heart, slow-twitch skeletal muscle, and kidney—tissues

with high-capacity mitochondrial systems. The expres-

sion of the PGC-1␣ gene is rapidly induced by cold ex-

posure, short-term exercise, and fasting; physiologic con-

ditions known to increase the demand on mitochondria

to produce heat or ATP (Puigserver et al. 1998; Wu et al.

1999; Goto et al. 2000; Lehman et al. 2000; Baar et al.

2002; Terada et al. 2002; Irrcher et al. 2003; Pilegaard

et al. 2003; Terada and Tabata 2003). These latter obser-

vations suggested that PGC-1␣ is involved in the physi-

ologic control of mitochondrial function.

Several lines of evidence indicate that the transcrip-

tional coactivator PGC-1␣ serves as a key regulator of

mitochondrial biogenesis in mammals. First, studies fo-

cused on the biologic function of PGC-1␣ revealed that it

activates the transcription of mitochondrial uncoupling

protein-1 (UCP-1) through interactions with the nuclear

hormone receptors PPAR␥ and thyroid hormone receptor

(Puigserver et al. 1998). These findings further supported

a role for PGC-1␣ in the process of mitochondrial un-

coupled respiration and thermogenesis in brown adipose

tissue. Second, forced expression studies in adipogenic

and myogenic mammalian cell lines demonstrated that

PGC-1␣ markedly induces the expression of NRF-1,

NRF-2, and Tfam (Wu et al. 1999). PGC-1␣ can also in-

teract directly with and coactivate NRF-1 on the Tfam

gene promoter. Third, studies in primary cardiac myo-

cytes in culture and in the hearts of transgenic mice have

demonstrated that overexpression of PGC-1

␣

up-regu-

lates the expression of genes involved in mitochondrial

fatty acid oxidation, most of which are PPAR␣ targets, in

addition to NRF-1 targets (Lehman et al. 2000). Cardiac-

specific overexpression of PGC-1

␣

in transgenic mice

leads to massive mitochondrial proliferation, ultimately

resulting in cardiomyopathy and death (Lehman et al.

2000). Interestingly, in neonatal cardiac myocytes in cul-

ture, PGC-1␣ induces mitochondria that support largely

coupled respiration consistent with the known ATP-gen-

erating function of this organelle in heart (Lehman et al.

2000). Lastly, forced expression of PGC-1

␣

in skeletal

muscle of transgenic mice triggers mitochondrial prolif-

eration and the formation of mitochondrial-rich type I,

oxidative (“slow-twitch”) muscle fibers (Lin et al.

2002b). Collectively, these results indicate that PGC-1␣

is capable of promoting mitochondrial biogenesis through

its coactivating effects on key factors such as NRF-1.

The gain-of-function studies described above provide

compelling evidence that PGC-1␣ serves as a transcrip-

tional coactivator to promote mitochondrial biogenesis

in postnatal mammalian tissues. Although NRF-1 is a

key target of PGC-1␣, it is clear that this transcription

factor does not control all of the components of the mi-

tochondrial biogenic response. Multiple PGC-1␣ targets

have now been identified, indicating that this coactiva-

tor serves as a pleiotropic regulator of multiple pathways

involved in cellular energy metabolism within and out-

side of the mitochondrion (Knutti and Kralli 2001; Puig-

server and Spiegelman 2003). Following the identifica-

tion of PPAR␥ as the initial PGC-1␣ transcription factor

target, a variety of additional members of the nuclear

receptor superfamily have been shown to interact with

PGC-1␣. This list includes PPAR␣ (Vega et al. 2000),

thyroid hormone receptor (Puigserver et al. 1998), reti-

noid receptors (Puigserver et al. 1998), glucocorticoid re-

ceptor (Knutti et al. 2000), estrogen receptor (Puigserver

et al. 1998; Knutti et al. 2000; Tcherepanova et al. 2000),

HNF-4 (Rhee et al. 2003), and estrogen-related receptors

(ERRs; Huss et al. 2002; Schreiber et al. 2003). In addi-

tion, several non-nuclear-receptor PGC-1␣ partners have

been identified, in addition to NRF-1, including myo-

cyte-enhancing factor-2 (MEF-2; Michael et al. 2001) and

FOX-01 (Puigserver et al. 2003). Although several of the

PGC-1␣ partners serve functions outside of the mito-

chondrion such as HNF-4 and FOX-01 (gluconeogenesis;

Rhee et al. 2003; Puigserver et al. 2003) and MEF-2 (glu-

cose transport; Michael et al. 2001), others are linked to

the mitochondrial biogenic transcriptional regulatory

program. For example, PGC-1␣ coactivates the nuclear

receptor PPAR␣, a key regulator of nuclear genes encod-

ing mitochondrial fatty acid oxidation enzymes (Vega

et al. 2000). More recently, PGC-1␣ was found to coac-

tivate the orphan nuclear receptors ERR␣ and ERR␥

(Huss et al. 2002; Schreiber et al. 2003). Although the

exact biologic function of ERRs has not been delineated,

ERR␣ and ERR␥ are enriched in tissues with high mito-

chondrial oxidative capacity including brown adipose

tissue and heart. In addition, medium-chain acyl-CoA

dehydrogenase (MCAD), a known PPAR␣ target that

catalyzes the initial step in mitochondrial fatty acid

␤-oxidation, is also regulated by ERR␣ (Sladek et al.

1997; Vega and Kelly 1997; Huss et al. 2002). These re-

sults suggest that ERR␣ and PPAR␣ may drive distinct

but overlapping mitochondrial pathways downstream of

PGC-1␣.

PGC-1␣ is now known to be a member of a family of

transcriptional coactivators. The first PGC-1␣ relative,

PGC-1-related coactivator (PRC), was identified through

a database search (Andersson and Scarpulla 2001). PRC

Kelly and Scarpulla

362 GENES & DEVELOPMENT

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contains several domains that are homologous to PGC-

1␣ including an acidic N-terminal region, an LXXLL mo-

tif for interacting with nuclear receptors, a proline-rich

region, and regions known to interact with RNA (Fig. 2).

Although overall homology between PGC-1␣ and PRC is

relatively low, the similarity of domains suggests related

function. In contrast to PGC-1␣,PRCis largely ubiqui-

tously expressed, is only slightly induced in response to

cold exposure, and is cell-cycle-regulated (Andersson and

Scarpulla 2001). However, functional studies indicate

that PRC may be capable of regulating mitochondrial

function in a manner similar to PGC-1␣. PRC interacts

directly with and coactivates NRF-1 via natural NRF-1

recognition sites in the 5-ALAS gene promoter (Anders-

son and Scarpulla 2001). Additional experiments have

revealed that PRC activates the transcription of another

known NRF-1 target, cytochrome c, but requires the co-

operation of other factors including CREB (Andersson

and Scarpulla 2001). A third member of the family, PGC-

␤

(also termed PGC-1-related estrogen receptor coacti-

vator or PERC), was also identified through database

searching (Kressler et al. 2002; Lin et al. 2002a). PGC-1␤

exhibits a greater degree of homology to PGC-1␣ than

PRC (Fig. 2). The expression pattern of PGC-1␤ exhibits

similarities with that of PGC-1␣ such as enrichment in

heart and brown adipose tissue. Furthermore, PGC-1

␤

induced by fasting but not in response to cold exposure

(Lin et al. 2002a). PGC-1␤ interacts with HNF-4␣, NRF-

1, and ERR␣ (Kressler et al. 2002; Lin et al. 2002a; Kamei

et al. 2003). PGC-1␤ also interacts with Host Cell Factor

(HCF), a cellular protein implicated in cell cycle regula-

tion (Lin et al. 2002a). The relevance of this latter in-

teraction to the regulation of mitochondrial function is

unknown.

The differences in regulation and tissue-expression

patterns of PGC-1 family members suggest that each

confers distinct biologic responses. In support of this

idea, recent work by the Spiegelman laboratory has pro-

vided evidence that PGC-1␣ and PGC-1␤ isoforms exert

coactivator-specific bioenergetic effects (St-Pierre et al.

2003). Specifically, overexpression studies in C

myotubes demonstrated that although both PGC-1␣ and

PGC-1␤ increase mitochondrial proton leak rates, cells

expressing PGC-1

␣

have a higher proportion of mito-

chondrial respiration linked to proton leak. PGC-1␤ was

shown to preferentially induce the expression of genes

involved in the removal of reactive oxygen species that

themselves could serve as activators of uncoupling

(St-Pierre et al. 2003).

Upstream signaling events involved in the control

of mitochondrial biogenesis: PGC-1

as an integrative coactivator

The observation that PGC-1

␣

gene expression is rapidly

induced by cold exposure in the brown adipose tissue of

mice spawned a series of observational studies aimed at

determining whether additional physiologic stimuli are

capable of modulating the expression of this coactivator.

It was subsequently shown that PGC-1

␣

gene expression

is induced by exercise in rodent and human skeletal

muscle (Goto et al. 2000; Baar et al. 2002; Terada et al.

2002; Terada and Tabata 2003; Pilegaard et al. 2003) and

by short-term starvation in the heart and liver of mice

(Lehman et al. 2000; Rhee et al. 2003). The transcrip-

tional regulatory mechanisms involved in the regulation

of PGC-1

␣

gene expression in response to physiologic

stimuli are only beginning to be understood. In one ex-

ample, the transcription factor CREB can promote he-

patic gluconeogenesis, in part through its induction of

PGC-1

␣

via direct binding to a functional CRE in the

PGC-1

␣

promoter (Herzig et al. 2001). More recently,

PGC-1

␣

transcription was shown to be regulated by

members of the MEF2 transcription factor family and

repressed by class II histone deacetylases (HDACs; Czu-

bryt et al. 2003).

Signal transduction pathways play a major role in the

physiologic regulation of mitochondrial function and

biogenesis; therefore, it is not surprising that PGC-1

␣

activity and expression are regulated by similar signaling

pathways. In tissues poised for mitochondrial thermo-

genesis, such as brown adipose, the ␤-adrenergic/cAMP

pathway is upstream of the PGC-1␣-mediated regulation

of targets such as UCP-1 (Puigserver et al. 1998). A sig-

Figure 2. The PGC-1 family of coactiva-

tors. Schematic representation of the pri-

mary structures of PGC-1␣, PRC, and PGC-

1␤. The leucine-rich (LXXLL) domains criti-

cal for interaction with nuclear receptors

are also shown. Additional specific shared

domains are indicated as denoted by the key

at the bottom.

Regulation of mitochondrial biogenesis

GENES & DEVELOPMENT 363

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nificant body of evidence focused largely on skeletal

muscle indicates that in response to contractile activity,

calcium-dependent signaling pathways trigger a cascade

of regulatory events leading to increased formation of

oxidative fiber types and a marked increase in mitochon-

drial number and function (Holloszy and Coyle 1984;

Chin et al. 1998). Several important gain-of-function

studies have now provided evidence for regulatory links

between calcineurin A, calcium/calmodulin-dependent

protein kinase (CaMK), PGC-1␣, and skeletal muscle mi-

tochondrial biogenesis. First, overexpression of CaMK in

the skeletal muscle of transgenic mice triggers a robust

mitochondrial biogenesis associated with an induction

of PGC-1

␣

expression (Wu et al. 2002). Second, overex-

pression of PGC-1

␣

in the skeletal muscle of transgenic

mice leads to the formation of slow-twitch skeletal

muscle fibers and an induction of genes involved in mi-

tochondrial oxidative metabolism (Lin et al. 2002b).

Third, studies performed in myogenic cell lines indicate

that both calcineurin A and CaMK are capable of acti-

vating PGC-1

␣

gene transcription (Handschin et al.

2003). The calcineurin A-mediated activation of PGC-1

␣

transcription is dependent on MEF2 response elements,

whereas CaMK-mediated regulation requires CREB-bind-

ing sites.

Several other signal transduction pathways have been

implicated in the control of PGC-1 expression and activ-

ity. p38 MAPK activates PGC-1

␣

by releasing repression

of an unidentified factor and by increasing PGC-1␣ pro-

tein stability (Knutti et al. 2001; Puigserver et al. 2001).

p38 MAPK can also activate the PGC-1␣ partner,

PPAR␣, suggesting that activation of this signaling path-

way influences mitochondrial fatty acid oxidation

(Barger et al. 2001). However, the role of the p38 MAPK

pathway in regulating mitochondrial biogenesis is not

known. More recently, evidence has emerged that nitric

oxide (NO) activates mitochondrial biogenesis in a vari-

ety of cell types including adipocytes, and HeLa cells

(Nisoli et al. 2003). The mitochondrial thermogenic re-

sponse is significantly altered in mice lacking eNOS.

This NO effect is dependent on cGMP and linked to

PGC-1

␣

activation. These results raise the intriguing

possibility that mitochondrial biogenesis is one of the

important effects of NO activation. Given the known

role of NO as a vasodilator, it is tempting to speculate

that this key upstream regulatory pathway coordinately

regulates downstream events including an increase in

the capacity to use oxygen in mitochondria.

Summary

Over the past decade, significant new insight has been

gained into the circuitry of molecular regulatory cas-

cades controlling mitochondrial biogenesis and function

(Fig. 3). The interdependence of nuclear and mitochon-

drial genomes has evolved with the emergence of the

mitochondrion as a eukaryotic organelle. It is likely that

the complexity of the mammalian organism mandates a

complex regulatory network that provides for the dy-

namic coordinate control of nuclear and mitochondrial

genes during development and in the adult. This regula-

tory circuitry not only triggers mitochondrial biogenesis

in response to developmental and physiologic cues, but

also confers cell- and tissue-specific features. New in-

sight into the dynamic control of mitochondrial function

and biogenesis has been provided by the identification of

relevant transcription factors, transcriptional coactiva-

tors, and upstream signaling events. However, the

mechanisms involved in the control of cell-specific mi-

tochondrial phenotypes and the full cast of transcrip-

tional regulatory factors comprise an exciting investiga-

tive frontier. New experimental approaches such as the

Figure 3. PGC-1 serves a central integra-

tive role in the transcriptional regulatory

cascade upstream of the mitochondrial bio-

genic response. A schematic representation

of the mitochondrial biogenic regulatory

cascade, including known PGC-1 partners

and putative upstream signaling pathways.

Kelly and Scarpulla

364 GENES & DEVELOPMENT

Cold Spring Harbor Laboratory Press on September 1, 2024 - Published by genesdev.cshlp.orgDownloaded from

delineation of tissue-specific mitochondrial proteomes

(Mootha et al. 2003) should provide an excellent frame-

work for future studies aimed at understanding the mo-

lecular events involved in defining the mitochondrial

phenotype.

Acknowledgments

Special thanks to Mary Wingate for assistance with manuscript

preparation and Janice Huss for critical review of the manu-

script. Work in the authors’ laboratories is supported by United

States Public Health Service Grants DK45416, HL58493,

HL57278, HL61006 to D.P.K. and GM32525 to R.C.S. from the

National Institutes of Health.

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Transcriptional regulatory circuits controlling mitochondrial

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