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Physiol Rev 88: 611– 638, 2008;
doi:10.1152/physrev.00025.2007.
Transcriptional Paradigms in Mammalian Mitochondrial
Biogenesis and Function
RICHARD C. SCARPULLA
Department of Cell and Molecular Biology, Northwestern Medical School, Chicago, Illinois
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I. Introduction
A. Overview
B. Pathophysiology
II. The Mitochondrial Genetic System: mtDNA Structure and Inheritance
III. Transcription of mtDNA
A. Mitochondrial transcriptional units
B. Tfam
C. mtTFB
D. Transcription termination and mitochondrial gene expression
IV. Nuclear Control of Mitochondrial Function
A. Nuclear transcription factors governing respiratory gene expression
B. Nuclear coactivators in mitochondrial biogenesis
V. Lessons From Mouse Gene Knockouts
A. Transcription factor knockouts
B. Coactivator knockouts
VI. Retrograde Pathways
A. The RTG pathway in yeast
B. Mammalian retrograde regulation
VII. Conclusion
Scarpulla RC. Transcriptional Paradigms in Mammalian Mitochondrial Biogenesis and Function. Physiol Rev 88:
611– 638, 2008; doi:10.1152/physrev.00025.2007.—Mitochondria contain their own genetic system and undergo a
unique mode of cytoplasmic inheritance. Each organelle has multiple copies of a covalently closed circular DNA
genome (mtDNA). The entire protein coding capacity of mtDNA is devoted to the synthesis of 13 essential subunits
of the inner membrane complexes of the respiratory apparatus. Thus the majority of respiratory proteins and all of
the other gene products necessary for the myriad mitochondrial functions are derived from nuclear genes.
Transcription of mtDNA requires a small number of nucleus-encoded proteins including a single RNA polymerase
(POLRMT), auxiliary factors necessary for promoter recognition (TFB1M, TFB2M) and activation (Tfam), and a
termination factor (mTERF). This relatively simple system can account for the bidirectional transcription of mtDNA
from divergent promoters and key termination events controlling the rRNA/mRNA ratio. Nucleomitochondrial
interactions depend on the interplay between transcription factors (NRF-1, NRF-2, PPAR␣, ERR␣, Sp1, and others)
and members of the PGC-1 family of regulated coactivators (PGC-1␣, PGC-1␤, and PRC). The transcription factors
target genes that specify the respiratory chain, the mitochondrial transcription, translation and replication machinery, and protein import and assembly apparatus among others. These factors are in turn activated directly or
indirectly by PGC-1 family coactivators whose differential expression is controlled by an array of environmental
signals including temperature, energy deprivation, and availability of nutrients and growth factors. These transcriptional paradigms provide a basic framework for understanding the integration of mitochondrial biogenesis and
function with signaling events that dictate cell- and tissue-specific energetic properties.
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0031-9333/08 $18.00 Copyright © 2008 the American Physiological Society
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I. INTRODUCTION
A. Overview
FIG. 1. Summary of protein subunits of the five
respiratory chain complexes encoded by nuclear and
mitochondrial genes. Depicted is a schematic of the
five respiratory complexes (I–V) embedded in the
lipid bilayer of the inner mitochondrial membrane.
Dissociable electron carriers cytochrome c (Cyt c)
and coenzyme Q (Q) are also shown. Arrows (green)
show the pathway of electrons from the various electron donors. Broken arrows (blue) show the sites of
proton pumping from the matrix side to the cytosolic
side by complexes I, III, and IV. The red arrow shows
the flow of protons through complex V from the cytosolic side to the matrix coupled to the synthesis of
ATP. Indicated above each complex are the number
of protein subunits encoded by nuclear (nDNA) and
mitochondrial (mtDNA) genomes.
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Mitochondria are ubiquitous membrane-bound organelles that are a defining feature of the eukaryotic cell.
The organelle is comprised of a soluble matrix surrounded by a double membrane, an ion impermeable
inner membrane, and a permeable outer membrane. Early
biochemists recognized the importance of mitochondria
as the sites of aerobic oxidation of metabolic fuels. It is
now well established that they contribute to many important functions including pyruvate and fatty acid oxidation,
nitrogen metabolism, and heme biosynthesis among others. Most notably, the mitochondrion is the site of the
electron transport chain and oxidative phosphorylation
system that provides the bulk of cellular energy in the
form of ATP (16, 88). Most of the chemical bond energy
from the oxidation of fats and carbohydrates is converted
to the reducing power of NADH and FADH2 within the
mitochondrial matrix. The respiratory apparatus consists
of a series of electrogenic proton pumps that convert this
reducing potential to an electrochemical proton gradient
across the inner membrane (Fig. 1). The electrochemical
potential of this gradient is converted via the ATP synthase to the high-energy phosphate bonds of ATP. In
addition, the gradient can be dissipated in specialized
brown adipocytes by uncoupling proteins to generate
heat.
Mitochondria and their chloroplast cousins are
unique among eukaryotic extranuclear organelles in that
they contain their own genetic system. In vertebrates, this
system is based on a mitochondrial genome consisting of
a circular double-stranded DNA (mtDNA) (Fig. 2). The
gene complement of mtDNA comprises but a small fraction of the number of genes necessary to account for the
molecular architecture and biological functions of the
organelle. Because of this limited coding capacity, mitochondria are genetically semiautonomous in that they rely
heavily on the expression of nuclear genes for all of their
biological functions. For example, the majority of protein
subunits that comprise the five inner membrane complexes of the electron transport chain and oxidative phosphorylation system are nucleus encoded (Fig. 1). The
largest of these, complex I, the NADH dehydrogenase, has
39 of its 46 subunits specified by nuclear genes, whereas
the smallest, the succinate dehydrogenase (complex II), is
comprised entirely of nucleus-encoded subunits (Fig. 1).
Thus nuclear genes must specify most of the structural
and catalytic components directly involved in energy metabolism and, in addition, control mitochondrial transcription, translation, and DNA replication (27, 46, 185, 227).
Mammalian mitochondrial biogenesis is subject to
complex physiological control. Mitochondrial mass is induced in adult muscle in response to exercise (28) or
chronic electrical stimulation (232), presumably as an
adaptation to facilitate increased oxygen utilization. Calcium-dependent signaling has been linked to a transcriptional pathway of mitochondrial biogenesis in skeletal
muscle (155, 235). The proliferation of mitochondria occurs in the brown fat of rodents during adaptive thermogenesis coinciding with the activation and induction of
UCP-1, an uncoupling protein that dissipates the proton
gradient to produce heat as a response to cold exposure
(34, 175). During early mouse development, the mitochondrial DNA (mtDNA) content remains constant from fertilization to the blastocyst stage after which DNA replication and organelle division resumes (167). Subsequent
mitochondrial proliferation increases continuously throughout development, and tissue-specific factors are thought to
dictate the final adult complement of mtDNA (90). The
respiratory capacity of mitochondrial membranes is en-
TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA
hanced postnatally as an adaptation to oxygen exposure
outside the womb (217). These examples bear on a fundamental question in understanding the biology of eukaryotic cells, namely, what are the mechanisms of communication between physically separated nuclear and mitochondrial genetic systems in meeting cellular energy
demands?
B. Pathophysiology
In the last 20 years, mitochondrial dysfunction has
been recognized as an important contributor to an array
of human pathologies. Mitochondrial defects play a direct
role in certain well-defined neuromuscular diseases and
are also thought to contribute indirectly to many degenerative diseases. Since the identification of the first huPhysiol Rev • VOL
man pathological mutation in mtDNA, many such mutations have been catalogued. Mutations in mitochondrial
genes for respiratory proteins and translational RNAs,
particularly tRNAs, manifest themselves in a wide range
of clinical syndromes, most of which affect the neuromuscular system (55, 227). These mtDNA mutations are often
maternally inherited, and in some cases, patients with
certain mitochondrial myopathies exhibit excessive proliferation of abnormal mitochondria in muscle fibers, the
so-called ragged red fiber (226). In addition, a subset of
mitochondrial diseases exhibits a Mendelian inheritance
pattern typical of nuclear gene defects (199, 205). These
can affect respiratory protein subunits, assembly factors,
and gene products required for mtDNA maintenance and
stability.
In addition to single gene defects, dispersed lesions
in mtDNA that accumulate over time may play a role in
human pathologies including neurodegenerative disease
(189), diabetes (131), and ageing (57). It has been widely
speculated that free radical production by the mitochondrial respiratory chain contributes to the neuropathology
observed in dementia and other degenerative diseases.
Although there is good evidence for oxidative stress associated with neuropathology, it has been difficult to
prove whether this is a cause or a consequence of neuronal death (5). The accumulation of mutations in mtDNA is
also thought to contribute to human ageing. Documented
changes in mtDNA, including increased prevalence of
point mutations and deletions, have been observed in
aged individuals (43). This along with the known agerelated decrease in oxidative energy metabolism has led
to the hypothesis that mtDNA mutations impair the respiratory chain leading to a decline in oxidative phosphorylation (57).
Direct experimental support for this model comes
from recent studies in which a mtDNA polymerase that is
defective in proofreading function was substituted for the
normal polymerase in mice (213). Animals homozygous
for the defective allele had a mutator phenotype with
markedly increased levels of mtDNA point mutations and
deletions. Although the mutations far exceed those associated with normal aging, the mutator mice had a reduced
life span and a number of physiological changes related to
premature aging. There has also been considerable interest in mitochondrial dysfunction as a contributing factor
in the onset of type 2 diabetes (161). Insulin resistance in
healthy aged individuals with no family history has been
correlated with a decline in mitochondrial oxidative phosphorylation (163). The expression of a number of genes
involved in oxidative metabolism is reduced in diabetic
subjects as well as in those predisposed to diabetes because of family history (144, 162). Transcriptional activators and coactivators that regulate mitochondrial biogenesis have been suggested as potential contributors to this
phenomenon. In addition, mitochondrial functional insuf-
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FIG. 2. Schematic representation of the human mitochondrial genome. Genomic organization and structural features of human mtDNA
are depicted in a circular genomic map showing heavy (blue) and light
(black) strands assigned as such based on their buoyant densities.
Protein coding and rRNA genes are interspersed with 22 tRNA genes
(red bars denoted by the single-letter amino acid code). Duplicate tRNA
genes for leucine (L) and serine (S) are distinguished by their codon
recognition (parentheses). The D-loop regulatory region contains the
L- and H-strand promoters (LSP, HSP1, and HSP2), with arrows showing
the direction of transcription. The origin of H-strand replication (OH) is
within the D-loop, whereas the origin of L-strand replication (OL) is
displaced by approximately two-thirds of the genome within a cluster of
five tRNA genes (W, A, N, C, Y). Protein coding genes include the
following: cytochrome oxidase (COX) subunits 1, 2, and 3; NADH dehydrogenase (ND) subinits 1, 2, 3, 4, 4L, 5, and 6; ATP synthase (ATPS)
subunits 6 and 8; cytochrome b (Cyt b). ND6 and the 8 tRNA genes
transcribed from the L-strand as template are labeled on the inside of the
genomic map, whereas the remaining protein coding and RNA genes
transcribed from the H-strand as template are labeled on the outside.
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II. THE MITOCHONDRIAL GENETIC SYSTEM:
mtDNA STRUCTURE AND INHERITANCE
It is long established from both genetic and biochemical studies that the mitochondrial genetic apparatus has
features that are distinct from that of the nucleocytosolic
system. Early work pointed to a protein synthetic machinery in isolated mitochondria that could synthesize a small
number of proteins utilizing mitochondrial ribosomes,
rRNA, and tRNA (45). A major advance was the discovery
of mitochondrial DNA. Genetic studies in yeast led to the
observation that certain respiratory mutations displayed a
cytoplasmic inheritance pattern that resulted from the
random segregation of mtDNA molecules during mitosis.
This provided genetic evidence that mitochondria had
their own DNA before its existence was demonstrated
physically. This conclusion was supported by physical
evidence for the existence of mitochondrial DNA in yeast
and in other organisms (148). Since these early discoveries, much has been done to define the structure and gene
organization of mtDNA. The first complete mtDNA sequence was obtained from humans and sequences of mitochondrial genomes from many organisms have now
been catalogued (227). A striking result from this work is
that a similar complement of genes is conserved in mtDNAs
from all multicellular organisms.
In contrast to the nuclear genome, where repetitive
sequence families, introns, and vast intergenic regions
account for all but a few percent of the total DNA, the
mtDNA of mammals and other vertebrates exhibits striking economy of sequence organization. The vertebrate
mitochondrial genome exists as a closed circular molecule of ⬃16.5 kb whose entire protein coding capacity is
devoted to the synthesis of 13 proteins that function as
essential subunits for respiratory complexes I, III, IV, and
V (Figs. 1 and 2). The genes specifying complex II are
entirely nuclear. The mtDNA also encodes the 22 tRNAs
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and 2 ribosomal RNAs necessary for the translation of
these respiratory subunits within the mitochondrial matrix. Mitochondrial genes lack introns and are arranged
end on end with little or no intergenic regions. Some
respiratory protein genes overlap, and the adenine nucleotides of UAA termination codons are not encoded in the
mtDNA but rather are supplied by polyadenylation following RNA processing (152). Protein coding and rRNA genes
are interspersed with tRNA genes that are thought to
demarcate the cleavage sites of RNA processing. The only
substantial noncoding region is the D-loop, named after
the triple-stranded structure or displacement loop that is
formed by association of the nascent heavy (H)-strand in
this region (Fig. 2). The D-loop contains the origin of
heavy (H)-strand DNA replication and is also the site of
bidirectional transcription from opposing heavy (H) and
light (L) strand promoters (45). The designation of these
strands is assigned based on their relative migration upon
gradient centrifugation. Interestingly, the structural economy found in vertebrate mtDNA is not found in plants and
fungi where the mitochondrial genomes are much larger
and contain intergenic regions, introns, and multiple promoters and transcriptional units (48, 169).
The fact that mtDNA is a compartmentalized extrachromosomal element contributes to a mode of inheritance that differs from that of nuclear genes. Somatic
mammalian cells generally have 103-104 copies of mtDNA
with ⬃2–10 genomes per organelle (179). These genomes
replicate in a relaxed fashion that is independent of the
cell cycle that is defined by nuclear DNA replication (46,
25). Some mtDNA molecules undergo multiple rounds of
replication while others do not replicate. This, along with
random sampling during cell division, allows the segregation of sequence variants during mitosis (198).
In mammals, mtDNA is maternally inherited (for references, see Refs. 101, 198). In general, paternal mtDNA is
lost during the first few embryonic cell divisions and does
not contribute mtDNA to the offspring, although there are
reports of the presence of the paternal lineage in somatic
tissues (192). In one case, recombination between maternal and paternal genomes has been documented (109). In
addition, because mtDNA is a multicopy genome, an individual may harbor more than a single sequence, a condition referred to as heteroplasmy. A detrimental sequence variant may be tolerated in low copy because the
defective gene product(s) it encodes do not reach the
threshold for disrupting cellular function. However, sequence variants are known to segregate rapidly from heteroplasmy to homoplasmy in passing from one generation
to the next (12). This can result in offspring in which the
detrimental variant predominates, leading to a defective
mitochondrial phenotype. The molecular basis for this
rapid meiotic segregation has been ascribed to a bottleneck or sampling error in the female germ line. A massive
amplification of mtDNA occurs during oogenesis from
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ficiency has been found in the insulin-resistant offspring
of patients with type 2 diabetes (164). The fact that this
occurs in healthy individuals that are not diabetic suggests that an inherent defect in oxidative phosphorylation
may be a contributing factor. These associations of mitochondrial dysfunction with human degenerative disease
raise the basic question of how mammalian cells control
mitochondrial biogenesis. It has become increasingly apparent that transcriptional mechanisms contribute to the
biogenesis of mitochondria including the expression of
the respiratory apparatus. Transcriptional control operates through a subset of well-defined transcription factors
and transcriptional coactivators. These regulators exert
their influence on the expression of both nuclear and
mitochondrial genes required for the maintenance and
biogenesis of the organelle.
TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA
⬃103 copies in the primary oocyte to ⬃105 copies in the
mature oocyte (198). Replication of mtDNA is halted in
the mature oocyte, and the existing population of mtDNA
molecules is partitioned to the daughter cells during early
cell divisions until the copy number is diluted to approximately that found in somatic cells. Embryonic replication
does not resume until the blastocyst stage of development
(167).
III. TRANSCRIPTION OF mtDNA
A. Mitochondrial Transcriptional Units
ing a transcript that is processed to 1 mRNA and 8 of the
22 tRNAs (Fig. 3A). In addition, the preponderance of
evidence indicates that transcription from LSP is coupled
to H-strand replication (25). Mapping studies support the
conclusion that transcripts truncated in the vicinity of
CSB II, one of three evolutionarily conserved sequence
blocks (CSB I, II, and III), serve as primers for the initiation of H-strand replication (Fig. 3A). Notably, RNA with
its 5⬘-end mapped precisely to the LSP initiation site is
covalently linked to newly synthesized H-strand DNA
(38). In addition, the nascent H-strand DNA 5⬘-ends
closely match the 3⬘-ends of truncated transcripts initiated at PL (37). It is widely accepted that primer RNAs are
generated by cleavage of L-strand transcripts by mitochondrial RNA processing (MRP) endonuclease at specific sites that match the in vivo priming sites (Fig. 3A).
This ribonucleoprotein endonuclease also participates in
the processing of 5.8S rRNA precursors and contains a
nucleus-encoded RNA that is essential for catalysis (MRP
RNA) (158, 197). A recent alternative model, derived from
in vitro experiments using purified components, suggests
that termination events associated with CSB II can also
FIG. 3. Schematic representation of mtDNA transcription within the D-loop regulatory region. A: transcription initiation complexes are
assembled at bidirectional promoters within the D-loop. They are comprised of mitochondrial RNA polymerase (POLRMT), Tfam, a stimulatory
factor that unwinds DNA, and one of the two TFB isoforms (TFB1M or TFB2M) that function as dissociable specificity factors that contact both
the polymerase and Tfam. The TFBs are related to rRNA dimethyltransferases and thus may also function in RNA modification or processing.
Initiation occurring at LSP can traverse the entire template or be cleaved by MRP endonuclease (RNAse MRP) at discrete RNA 3 DNA transition
sites in the vicinity of the conserved sequence blocks (CSB I, II, III; gray shaded bars) demarcating the origin of heavy strand replication (OH). The
RNAse MRP cleavage sites correspond to the heterogeneous 5⬘-ends of the newly synthesized H-strand during mtDNA replication. Transcripts
initiating at HSP2 can also traverse the entire template, whereas those initiating at HSP1 terminate at a transcription terminator (TERM, yellow
shaded bar) localized to the tRNAL(UUR) gene to produce the 12S and 16S rRNAs. B: the transcription termination factor mTERF binds
simultaneously both HSP1 and TERM resulting in the looping out of the intervening 12S and 16S rDNA. This is thought to promote efficient recycling
of the transcription complexes resulting in a high rate of rDNA transcription. The mechanism likely contributes to maintaining a high ratio of rRNA
to mRNA required for operation of the mitochondrial translation machinery.
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In vertebrates, the D-loop regulatory region contains
bidirectional promoters for transcribing H- and L-strands
as well as the H-strand replication origin (OH) (Fig. 2).
The H- and L-strand transcriptional units differ from most
nuclear genes in that they are polygenic, specifying multiple RNAs (rRNA, tRNA, or mRNA). L-strand transcription is initiated at a single promoter (LSP), and RNA
synthesis can traverse the entire mtDNA template produc-
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scription and its potential functional implications for mitochondrial biogenesis await further characterization.
B. Tfam
The region of the D-loop that separates the LSP from
the two HSPs contains conserved nucleotide sequence
motifs that define the core promoter and mediate bidirectional transcription (212). In addition, the promoters
share an upstream enhancer that binds Tfam (previously
mtTF-1 and mtTFA), the first well-characterized transcription factor from vertebrate mitochondria (Fig. 3A). Tfam
was first identified as a high mobility group (HMG)-box
protein that stimulates transcription through specific
binding to recognition sites upstream from both LSP and
HSP (69). Structurally, it consists of two tandemly arranged HMG motifs and a COOH-terminal tail. Tfam resembles other HMG proteins, in that it can bend and
unwind DNA, properties potentially linked to its ability to
stimulate transcription upon binding DNA (159, 70). Tetrameric binding of Tfam to its recognition site is thought
to promote bidirectional transcription by facilitating symmetrical interactions with other transcriptional components (7). In addition to specific promoter recognition,
Tfam binds nonspecifically to apparently random sites on
mtDNA (70, 71). This property, along with its abundance
in mitochondria, suggests that it plays a role in the stabilization and maintenance of the mitochondrial chromosome. ABF2p, a related HMG-box factor from yeast, resembles Tfam and is required for both mtDNA maintenance and respiratory competence (54). Expression of
human Tfam in ABF2p-deficient yeast cells can rescue
both phenotypes (160). Thus, although highly divergent in
primary structure, the human and yeast proteins are functionally interchangeable in supporting mitochondrial respiratory function in yeast. However, despite this functional complementation, ABF2p lacks an activation domain and does not stimulate transcription from yeast
mitochondrial promoters in vitro (237, 50). The transcriptional activation function of Tfam resides in a COOHterminal activation domain that is required both for transcriptional competence and for specific binding to promoter recognition sites (50).
As discussed below, a Tfam knockout mouse displays embryonic lethality and a depletion of mtDNA confirming an essential role for the protein in mtDNA maintenance in mammals (113). Interestingly, Tfam levels correlate well with increased mtDNA in ragged-red muscle
fibers and decreased mtDNA levels in mtDNA-depleted
cells (168). This is consistent with the observation that
transgenic mice overexpressing human Tfam display increased mtDNA copy number (59). The mtDNA abundance measured in somatic tissues and in embryos is
proportional to the amount of Tfam expressed in each,
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account for the primer RNA 3⬘-ends that mark the major
transition sites between RNA and DNA synthesis (165). In
contrast to the LSP, H-strand transcription is initiated
from two closely spaced promoters, HSP1 and HSP2
(Figs. 2 and 3A). Transcription initiating at HSP2 results in
long polygenic transcripts that are processed to give 14
tRNAs, 12 mRNAs, and the 2 rRNAs. HSP1 on the other
hand appears specialized for the production of ribosomal
RNAs (Fig. 3A). As discussed below, termination events
coupled to initiation at HSP1 provide a mechanism for
controlling the relative abundance of rRNA to mRNA.
Transcription of mtDNA is completely dependent on
nucleus-encoded gene products. In yeast, transcription is
directed by a single subunit RNA polymerase (RPO41p),
which shares sequence similarities with the T7 and T3
bacteriophage polymerases (104, 134). Although RPO41p
can initiate transcription nonselectively from synthetic
DNA templates, the specificity factor Mtf1p (discussed
below) is required for promoter recognition and selective
initiation (104). Interestingly, RPO41p, through its interactions with mitochondrial promoters and nucleoside
triphosphates, may act as a sensor of ATP availability.
Mitochondrial transcript abundance is correlated with
respiration-coupled ATP synthesis through a requirement
for a high ATP concentration for transcription initiation.
This represents a potentially novel and elegant mechanism for linking energy production to the transcription of
respiratory subunits (4).
Although purification of the human polymerase to
homogeneity has been elusive, a human cDNA that encodes a protein with sequence similarity to yeast mitochondrial and T3/T7 bacteriophage polymerases was
identified in database screenings (211). As expected for a
bona fide mitochondrial polymerase, the 140-kDa protein
product, designated POLRMT, is localized to mitochondria
via an NH2-terminal targeting presequence. The utilization of
single subunit bacteriophage-related polymerases seems
nearly universal for mitochondrial transcription. A potential
link between mitochondrial transcription and translation
in mammalian cells is implicated by the finding of a direct
interaction between POLRMT and mitochondrial ribosomal
protein L12 (MRPL12) (231). MRPL12 stimulates transcription from both LSP and HSP promoters in an in vitro
transcription system. Moreover, it exists in a complex
with POLRMT in HeLa cell extracts, and its in vivo overexpression can enhance the steady-state levels of mtDNAencoded transcripts. It is not yet clear whether this occurs
through effects on transcription or RNA stability. Finally,
an alternative splice variant of POLRMT that is missing
the NH2-terminal 262 amino acids including the mitochondrial targeting presequence has been identified (108). Although it is currently unclear whether this protein functions as an independent nuclear polymerase, it has been
localized to the nucleus and linked to the expression of a
number of nuclear genes. Its precise role in nuclear tran-
TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA
C. mtTFB
In yeast, RPO41p associates with a 43-kDa specificity
factor MTF1p also known as sc-mtTFB (196). The primary
structure of sc-mtTFB bears some resemblance to prokaryotic sigma factors (99), but a crystal structure reveals
significant homology to rRNA methyltransferase (191).
Although individually neither RPO41p nor MTF1p can
interact with yeast mitochondrial promoters, together
they engage in specific promoter recognition (104). Evidence for a vertebrate factor that functions in analogous
fashion to the yeast sc-mtTFB came from biochemical
studies in Xenopus laevis (24, 24). The partially purified
factor bound DNA nonspecifically and stimulated transcription in vitro by purified mitochondrial RNA polymerase. Although purification and molecular cloning of the
vertebrate factor has been elusive, this impasse has been
resolved by the identification of a human mtTFB cDNA
(138). The encoded protein is localized to mitochondria
and stimulates transcription from an L-strand promoter
in vitro, properties consistent with it being a functional
homolog of sc-mtTFB. The human protein exhibits nonsequence-specific DNA binding and requires Tfam to stimulate transcription from mitochondrial templates in vitro.
Two isoforms of h-mtTFB, termed TFB1M and TFB2M,
have been identified independently (65). TFB1M is identical to the original mtTFB and has about 1/10 the transcriptional activity of TFB2M. As depicted in Figure 3A,
both proteins work together with Tfam and mtRNA polymerase to direct proper initiation from H- and L-strand
promoters in an in vitro system comprised of purified
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recombinant proteins (65). TFB1M has been detected in a
complex with Tfam and POLRMT in transcriptionally
competent mitochondrial extracts. Moreover, both TFB
isoforms make direct contact with the COOH-terminal
activation domain of Tfam (139). These observations support the notion that mtDNA transcription in vertebrate
systems is directed by three essential components (Fig.
3A). Interestingly, like the yeast factor, both TFBs are
related to rRNA methyltransferases. TFB1M can bind
S-adenosylmethionine and methylate rRNA at a stem-loop
structure that is conserved between bacterial and mitochondrial rRNAs (195). However, this activity is not required for transcriptional activation by the factor (139). It
has yet to be determined whether the proteins are bifunctional or whether they evolved a single function from an
ancestral methyltransferase.
Recent experiments suggest that the two TFB isoforms are not functionally identical. At early times following serum stimulation of quiescent fibroblasts, mRNA for
the TFB1M isoform is transiently downregulated relative
to that of the TFB2M isoform, suggesting that the latter is
favored in the transition to proliferative growth (73). In
Drosophila cultured cells, RNAi knockdown of the Drosophila B2 isoform results in reduced mtDNA transcription and copy number (136). This contrasts with RNAi
knockdown of the B1 isoform, which has no effect on
mtDNA transcription or replication but does result in
reduced mitochondrial translation (135). It is also notable
that overexpression of either TFB2M or Tfam in this
system increases mtDNA copy number, whereas overexpression of TFB1M fails to do so. The stimulatory effect of
Tfam is consistent with the observation that overexpression of human Tfam in mice increases mtDNA copy number (59). Thus both gain- and loss-of-function experiments
support a role for Tfam and TFB2M in mtDNA copy
number control. The inability of TFB1M to function in a
similar capacity in Drosophila cells is surprising in light of
the ability of the mammalian protein to bind the transcription activation domain of Tfam and stimulate transcription in vitro.
D. Transcription Termination and Mitochondrial
Gene Expression
Specific termination events play an important role in
governing the steady-state levels of mitochondrial transcripts. The high rate of rRNA synthesis relative to mRNA
has been ascribed to more frequent initiation of H-strand
transcription at HSP1. As shown in Figure 3A, HSP1 directs transcription from a site within the tRNAPhe gene.
Most transcripts initiated at HSP1 terminate downstream
from the 16S rRNA gene at a strong bidirectional terminator localized within the adjacent tRNALeu gene (44).
The 28-nucleotide terminator binds mTERF, a 34-kDa pro-
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suggesting that Tfam can function as a limiting determinant of mtDNA copy number. Because human Tfam is a
poor activator of mouse LSP and HSP, overexpression of
the human protein results in an increase in mtDNA without affecting respiratory capacity, suggesting that copy
number control by Tfam is independent of its transcriptional function. In fact, both endogenous Tfam and a
mutated derivative that lacks the COOH-terminal activation domain are equally competent in maintaining mtDNA
copy number in cultured cells (103). These observations
are reminiscent of Abf2p, which does not direct transcription but is required for mtDNA maintenance (54). Abf2p is
an abundantly expressed component of mitochondrial
nucleoids, protein-DNA complexes thought to be a basic
segregating unit of mtDNA (105). Tfam is also expressed
at levels high enough to coat mtDNA and has been recovered in vertebrate nucleoids in association with other
proteins required for mtDNA genomic integrity and expression (26, 102, 230). Thus both its in vivo and in vitro
properties implicate Tfam as an ideal target for regulatory
pathways that control both mtDNA maintenance and transcriptional expression.
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analysis of the cytochrome c control region revealed a
palindromic recognition site for a transcription factor
designated as nuclear respiratory factor 1 (NRF-1) (63)
(Fig. 4). Specific NRF-1 binding sites are present in the
promoters of several nuclear genes required for mitochondrial respiratory function (39, 64). The protein binds
its recognition site as a homodimer through a unique DNA
binding domain and functions as a positive regulator of
transcription (78, 222). This is consistent with the presence of a COOH-terminal transcriptional activation domain (Fig. 4) comprised of glutamine-containing clusters
of hydrophobic amino acid residues (79). NRF-1 exists as
a phosphoprotein in proliferating mammalian cells and
serine phosphorylation within a concise NH2-terminal domain (Fig. 4) enhances both its DNA binding (78) and
trans-activation functions (92). Although other mammalian NRF-1 isoforms have not been found, NRF-1 is related, through its DNA binding domain, to developmental
regulatory proteins in sea urchins (32) and Drosophila
(53). In addition, chicken (74), zebrafish (20), and mouse
(187) homologs of NRF-1 have been characterized.
NRF-1 has now been linked to the expression of
many genes required for mitochondrial respiratory function including the vast majority of nuclear genes that
encode subunits of the five respiratory complexes (for
recent compilations, see Refs. 106, 183, 184). In addition,
considerable evidence supports a potential integrative
function for NRF-1 in coordinating respiratory subunit
expression with that of the mitochondrial transcriptional
NRF-1
IV. NUCLEAR CONTROL OF
MITOCHONDRIAL FUNCTION
DNA binding
1
A. Nuclear Transcription Factors Governing
Respiratory Gene Expression
NLS
A
503
B
serine
phosphorylation
Isolation of cytochrome c genes in yeast and subsequently in mammalian cells opened the way to the molecular analysis of the control of nuclear genes governing
mitochondrial respiratory function. In yeast, transcriptional regulation of the two cytochrome c isoforms is
mediated through upstream enhancer elements within
their promoters (77). These enhancers bind specific transcriptional activators and repressors that direct dramatic
changes in gene expression in response to oxygen and
carbon source availability (169, 242). Although cytochromes c are highly conserved at the functional level
between yeast and mammals (186), the mammalian cytochrome c promoter contains recognition sites for transcription factors that bear no obvious relationship to
those identified in yeast (62). In particular, systematic
YGCGCAYGCGCR
RCGCGTRCGCGY
80
80
85
90
100
95
75
95
100
100
100
85
1. Cytochrome c and the identification of NRF-1
Physiol Rev • VOL
trans-activation
FIG. 4. Summary of nuclear respiratory factor (NRF)-1 functional
domains and DNA recognition site. NRF-1 has a central DNA binding
domain (stippled box) flanked by a nuclear localization signal (horizontally hatched box) and a bipartite transcriptional activation domain
(vertically hatched boxes). The protein is phosphorylated in exponentially growing cells at multiple serine residues within a concise NH2terminal domain (cross-hatched box). NRF-1 binds a GC-rich palindrome (shown below the diagram) as a homodimer and makes guanine
nucleotide contacts (filled circles) over a single turn of the DNA helix.
Numbers below the NRF-1 consensus binding site represent the percentage of the time the indicated nucleotide is present at that position in 20
functional NRF-1 sites. Y, pyrimidine nucleotide; R, purine nucleotide.
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tein that specifies site-specific transcription termination
in fractionated mitochondrial lysates (49). Although the
purified recombinant protein displays the expected binding specificity, it is not sufficient for termination, suggesting that an additional component(s) may be required (66).
Interestingly, in addition to binding the termination site,
mTERF stimulates transcription from HSP1, suggesting
that transcription initiation and termination are coupled
(13, 111). Recent experiments demonstrate that this is
indeed the case. Multiple lines of in vitro and in vivo
evidence establish that mTERF binds simultaneously
both the HSP1 initiation site and the terminator resulting
in the looping out of intervening rDNA (Fig. 3B). These
interactions are proposed to enhance the reinitiation rate
at HSP1 resulting in a higher rate of rRNA synthesis. This
mechanism likely accounts for the stimulatory effects of
mTERF on transcription (133). Additional complexity to
mTERF function is suggested by the observation of several mTERF-related genes in vertebrates (129). The protein products all have putative mitochondrial targeting
signals and in one case mitochondrial localization has
been confirmed (40). Interestingly, this isoform, designated as mTERFL, differs from mTERF in that it is downregulated upon serum induction of serum-starved cells.
This is reminiscent of the differential expression of
TFB1M and TFB2M in response to serum stimulation (73).
It is possible that functionally distinct transcription complexes arise from the differential expression of alternative
isoforms of these transcription initiation and termination
factors.
TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA
dase assembly factor (206). NRF-1 control over key components of the protein import and assembly machinery is
suggestive of a broader role for the factor in orchestrating
events in mitochondrial biogenesis beyond the transcriptional expression of the respiratory chain.
This hypothesis is reinforced by reports that associate elevations in NRF-1 mRNA or DNA binding activity
with generalized effects on mitochondrial biogenesis.
Both NRF-1 and its coactivator PGC-1␣ (see below) are
upregulated during the adaptive response of skeletal muscle to exercise training (15, 147). A similar effect which
mimics exercise-induced mitochondrial biogenesis occurs in cultured myotubules in response to increased
calcium (154). Treatment of rats with a creatine analog,
which induces muscle adaptations resembling those observed during exercise, leads to the activation of AMPactivated protein kinase. This coincides with increased
NRF-1 DNA binding activity, cytochrome c content, and
mitochondrial density (21). Both NRF-1 and Tfam mRNAs
increase in cells depleted of mtDNA, presumably as a
response to increased oxidative stress (141). NRF-1 and
NRF-2 (see below) along with Tfam are also upregulated
FIG. 5. Diagrammatic summary of the nuclear control of mitochondrial functions by NRF-1 and NRF-2 (GABP). NRFs contribute both directly
and indirectly to the expression of many genes required for the maintenance and function of the mitochondrial respiratory apparatus. NRFs act on
genes encoding cytochrome c, the majority of nuclear subunits of respiratory complexes I–V, and the rate-limiting heme biosynthetic enzyme
5-aminolevulinate synthase. In addition, NRFs promote the expression of key components of the mitochondrial transcription and translation
machinery that are necessary for the production of respiratory subunits encoded by mtDNA. These include Tfam, TFB1M, and TFB2M as well as
a number of mitochondrial ribosomal proteins and tRNA synthetases. Recent findings suggest that NRFs are also involved in the expression of key
components of the protein import and assembly machinery.
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machinery. As illustrated in Figure 5, NRF-1 binds and
activates the promoters of Tfam (224) and both TFB
isoform genes (73) whose products (as discussed above)
are major regulators of mitochondrial transcription.
NRF-1 has also been linked to the expression of 5-aminolevulinate synthase (30) and uroporphyrinogen III synthase (2), key enzymes of the heme biosynthetic pathway.
The former is the rate-limiting enzyme in the biosynthesis
of heme, a cofactor essential to the function of electron
carriers encoded by both genomes (Fig. 5). Moreover,
NRF-1 acts on genes whose functions are not restricted to
the bigenomic expression of the respiratory apparatus
(Fig. 5). The outer membrane multisubunit receptor complex designated as TOMM is required for the import of the
thousands of proteins that contribute to diverse mitochondrial functions (214). TOMM20 is a key receptor
subunit involved in the initial interaction with precursor
proteins targeted to mitochondria. NRF-1 activates
TOMM20 gene transcription through a specific promoter
recognition site and is bound to the TOMM20 promoter
region in vivo (23). Similarly, NRF-1 is implicated in the
expression of mouse COX17, a putative cytochrome oxi-
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RICHARD C. SCARPULLA
Physiol Rev • VOL
This along with derepression by release of E2F factors
from a subset of NRF-1 target genes may help promote
cell proliferation. Notably, NRF-1 has also been implicated in the transcriptional repression of E2F1 (58) and
the activation of E2F6 (107). These results are consistent
with a role for NRF-1 in regulating cell cycle progression
and may explain the early embryonic lethality of NRF-1
knockout embryos (96).
2. NRF-2 (GABP) an activator of cytochrome
oxidase expression
A second nuclear factor designated as NRF-2 was
identified based on its specific binding to essential cisacting elements in the cytochrome oxidase subunit IV
(COXIV) promoter (181, 182). NRF-2 binding sites contained of the GGAA core motif that is characteristic of the
ETS-domain family of transcription factors. Direct repeats of this sequence motif are interspersed with multiple transcription initiation sites within the mouse COXIV
promoter (223, 36). Human NRF-2 was purified to homogeneity from HeLa cell nuclear extracts and is comprised
of five subunits (Fig. 6). These include a DNA-binding ␣
subunit and four others (␤1, ␤2, ␥1, and ␥2,) that complex
with ␣ but alone do not bind DNA. Molecular cloning of
the five NRF-2 subunits (80) revealed that NRF-2 is the
human homolog of mouse GABP (112). The two additional human subunits, ␤1 and ␥1, are minor splice variants of GABP subunits ␤1 and ␤2 (80). The GABP ␤1
subunit, corresponding to NRF-2 ␤1 and ␤2 (80), has a
dimerization domain that facilitates cooperative binding
of a heterotetrameric complex to tandem binding sites
(210). In solution, GABP exists as a heterodimer but is
induced to form the heterotetramer ␣2␤2 by DNA containing two or more binding sites (41). The crystal structure
of the heterotetramer bound to DNA has been determined
(19). All of the non-DNA-binding subunits contain a transcriptional activation domain (Fig. 6). This domain resembles that found in NRF-1 and has been localized to a
region upstream from the homodimerization domain (79).
As observed for COXIV, COXVb transcription is also
activated by NRF-2 through directly repeated recognition
sites within the proximal promoter, suggesting that NRF-2
is a general activator of cytochrome oxidase subunit gene
expression (225). Several lines of evidence point to a
direct role for NRF-2 in the expression of all 10 nucleusencoded cytochrome oxidase subunits. NRF-2 has been
associated in vivo with multiple COX subunit promoters
by chromatin immunoprecipitation (157). Moreover, expression of a dominant negative NRF-2 allele reduced
COX expression, and an siRNA directed against NRF-2␣
reduced expression of all 10 nucleus-encoded COX subunits (156). These findings are consistent with an important regulatory function for NRF-2 in cytochrome oxidase
expression.
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in response to lipopolysaccharide-induced oxidative damage to mitochondria, presumably to enhance mtDNA levels and OXPHOS activity (203). Exogenous oxidants restore NRF-1 and normal cell growth to ␳o hepatoma cells
where reduced oxidant levels are associated with loss of
NRF-1 and growth delay. Interestingly, NRF-1 occupancy
of the Tfam promoter increases under pro-oxidant conditions, and the control of Tfam expression by NRF-1 is
blocked by inhibition of NRF-1 phosphorylation by Akt
(166). This suggests that redox regulation of NRF-1 target
genes such as Tfam is modulated by redox-regulated
phosphorylation events. Finally, both NRF-1 (DNA binding activity and mRNA) and Tfam (protein and mRNA) are
upregulated in the skeletal muscle of aged human subjects (120). This may be a compensatory response to
age-related reductions in energy metabolism. These associations are consistent with a general integrative role for
NRF-1 in nucleomitochondrial interactions.
It is important to note that NRF-1 targets are not
restricted to genes involved in mitochondrial function. An
initial search for NRF-1 binding sites in mammalian promoters revealed a number of primate and rodent genes
whose functions are not linked directly to mitochondrial
biogenesis (222). Among these are genes encoding metabolic enzymes, components of signaling pathways, and
gene products necessary for chromosome maintenance
and nucleic acid metabolism among others. Moreover,
NRF-1 is among seven identified transcription factors
whose recognition sites are most frequently found in the
proximal promoters of ubiquitously expressed genes (72).
Recently, chromatin immunoprecipitations (ChIP) coupled
with microarray assay (ChIP-on-chip) were used to identify
human promoters that are bound by NRF-1 in vivo (33).
Survey of ⬃13,000 human promoters by ChIP-on-chip
analysis (174) identified 691 genes whose promoters are
occupied by NRF-1 in living cells (33). As expected, a
majority of these genes are involved in mitochondrial
biogenesis and metabolism, including many that had not
been previously identified. These include a collection of
mitochondrial ribosomal protein and tRNA synthetase
genes. Notably, a significant subset of the NRF-1 target
genes was also bound by the growth regulatory transcription factor E2F, suggesting that NRF-1 participates in the
regulation of a subset of E2F-responsive genes. This subset was enriched in genes required for DNA replication,
mitosis, and cytokinesis. Although NRF-1 was bound to its
target promoters under conditions of transcriptional repression, an NRF-1 siRNA reduced expression of several
E2F target genes along with Tfam and cytochrome c,
confirming that it functions as a transcriptional activator.
NRF-1 exists in the dephosphorylated state in serumstarved cells and is phosphorylated upon serum addition
(78). Since phosphorylation enhances NRF-1 transcriptional activity (92), it is possible that phosphorylation
controls the functional state of the DNA bound factor.
TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA
621
DNA binding
ETS
(51.4 kDa)
transactivation
heterodimerization
1
homodimerization
(42.5 kDa)
VNLTGLVSSENSSKATDETG
2
(41.3 kDa)
1 (38.1kDa)
FIG. 6. Summary of NRF-2 (GABP) functional domains. NRF-2, the human homolog of mouse GABP, is comprised of 5 subunits. The ␣-subunit
contains the ETS domain (stippled box) and can bind its DNA recognition site in the absence of the other subunits. The ␤- and ␥-subunits contain
the trans-activation domain (bracketed) and can complex with ␣ through interactions between the ankrin repeats (arrows) and the ETS domain.
The ␤-subunits differ from the ␥-subunits in that they have an NH2-terminal homodimerization domain (cross-hatched box). This allows the
formation of a heterotetrameric complex (␣2␤2) that binds tandem NRF-2 recognition sites in respiratory gene promoters with high affinity. The
␣␥-complexes lacking the homodimerization domain are transcriptionally competent but bind DNA with much weaker affinity. The ␤1- and
␥1-subunits are alternative splice variants of ␤2 and ␥2 that contain an additional 20-amino acid insertion (solid box with sequence below) of
unknown function.
A strong correlation has been observed between
NRF-2␣ (GABP␣) and cytochrome oxidase expression in
the visual cortex (149). NRF-2␣ and cytochrome oxidase
were associated with metabolic state and neuronal activity in mature neurons, suggesting that NRF-2␣ is important in maintaining neuronal function. Moreover, the correlation was extended to NRF-2␣ mRNA, indicating that
NRF-2␣ may be regulated at the transcriptional level by
neuronal activity (82). Both NRF-2␣ and -␤ subunits are
enriched in the nucleus in response to neuronal stimulation (241), and both subunits are specifically translocated
to the nucleus in response to neuronal depolarization
(238). In fact, the neuronal factor heregulin directs the
threonine phosphorylation of GABP␣, stimulating both its
transcriptional activity and its mobilization to the nucleus
where it activates transcription of acetylcholine receptor
subunits (188, 204).
In addition to the COX promoters, functional NRF-2
sites have been identified in a number of other genes
related to respiratory chain expression (for a recent compilation, see Refs. 106, 183). These include genes for Tfam
(113, 173) as well as TFB1M and TFB2M (138, 65) (Fig. 7).
Three of the four human succinate dehydrogenase (complex II) subunit genes also have both NRF-1 and NRF-2
sites in their promoters (14, 60, 94). 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
promoters and the rodent Tfam (42) and TFB (172) promoters do not have obvious NRF-1 consensus sites. This
contrasts with the human Tfam (224) and TFB (73) promoters, which rely on both NRF-1 and NRF-2 for their
Physiol Rev • VOL
activities (Fig. 7). Both NRF-1 and NRF-2␣ occupy both
TFB1M and -2M promoters in vivo as determined by chromatin immunoprecipitation (73). Thus, like NRF-1, NRF-2
participates in coordinating the expression of essential
respiratory chain proteins with key components of the
mitochondrial transcription machinery. Such a mechanism may serve to ensure the coordinate bigenomic expression of respiratory subunits.
3. ERR␣
The estrogen-related receptor ERR␣ has been linked
to the regulation of oxidative metabolism. ERR␣, one of a
family of orphan nuclear receptors including ERR␤ and
ERR␥, resembles the estrogen receptor but does not bind
estrogen or other ligands. ERR␣ levels are high in oxidative tissues such as kidney, heart, and brown fat, and it
acts as a regulator of ␤-oxidation via its control of the
medium-chain acyl-coenzyme A dehydrogenase (MCAD)
promoter (97). The ␣- and ␥-isoforms increase postnatally
in the heart along with enzymes promoting mitochondrial
fatty acid uptake and oxidation (98). ERR␣ knockout
mice have reduced fat mass and are resistant to dietinduced obesity, suggesting defects in lipid metabolism
(132). Recent studies have also implicated ERR␣ in PGC-1␣induced mitochondrial biogenesis (143, 190). A computational approach coupled with PGC-1␣-induced RNA expression profiles identified both ERR␣ and GABP␣ in a
double positive-feedback loop with PGC-1␣ in directing
expression of a subset of mitochondria-related genes
(143). An inhibitor of ERR␣ attenuated PGC-1␣-mediated
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2 (36.9 kDa)
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RICHARD C. SCARPULLA
5. Other nuclear factors
FIG.
7. Arrangement of NRF-1 and NRF-2 recognition sites in human promoters specifying key components of the mitochondrial transcription and replication machinery. Shown are linear representations of
the 5⬘-flanking regions proximal to the putative transcription initiation
sites (arrows) of human genes encoding Tfam, TFB1M, TFB2M, and
POL␥2. Each promoter region bears a strong resemblance to a number
of respiratory subunit promoters in that they contain a single NRF-1 site
(blue bars) and two or more tandem NRF-2 sites (red bars). In addition,
each promoter has at least one Sp1 recognition site (gray bars).
target gene expression and total cellular respiration. In
this scheme, NRF-1 was downstream of the ERR␣-directed changes in expression. It should be noted that
PGC-1␣ was abundantly overexpressed from an adenovirus vector in many of the experiments utilized to validate
this model. This generally results in massive changes in
mitochondrial biogenesis that are not observed in genetic
knockouts of PGC-1␣ or ERR␣ (discussed below). Nevertheless, ERR␣ does mediate the effects of virally expressed PGC-1␣ on mitochondria-related genes such as
Tfam and ATP synthase ␤-subunit (190). It is also of
interest to note that PGC-1␣ functions through NRF-2
(GABP) in the control of a program of neuromuscular
gene expression in skeletal muscle (85). Thus these factors likely coordinate broad adaptive changes beyond
their initially observed functions in respiratory chain expression.
4. PPARs and fatty acid oxidation
A number of mitochondrial oxidative pathways feed
into the respiratory apparatus. Important among these is
the pathway for fatty acid oxidation, which consists of a
Physiol Rev • VOL
Additional nuclear factors have been implicated in the
expression of respiratory genes. The cytochrome c promoter
contains cis-elements that recognize transcription factors
of the ATF/CREB family (63, 75). These elements bind
CREB both in vitro and in vivo, and phosphorylation of
CREB and NRF-1 is linked to the serum induction
of cytochrome c in quiescent fibroblasts (92, 220). Although CREB sites are common to cAMP- and growthactivated genes (137), these elements are not a general
feature of nuclear respiratory promoters. Cytochrome c
appears to be limiting for mitochondrial respiration early
in the program of serum-induced entry to the cell cycle
(92). CREB activation of cytochrome c expression likely
contributes to its relatively rapid induction in response to
serum-stimulated growth. This may be a mechanism for
ramping up cellular respiration in preparation for cell
growth and division.
Maximal cytochrome c promoter activity also depends on synergy between functional recognition sites for
the general transcription factor Sp1 (63). Sp1 is also involved in the activation and/or repression of cytochrome
c1 (125) and adenine nucleotide translocase 2 genes (124),
both of which lack NRF sites (240). Muscle-specific COX
subunit genes, in contrast to their ubiquitously expressed
isoforms (193), depend on MEF-2 and/or E-box consensus
elements for their tissue-specific expression (228). The
initiator element transcription factor YY1 has been implicated in both positive and negative control of cytochrome
oxidase subunit gene expression (18, 194). Interestingly,
YY1 knockout mice resemble the NRF-1 and NRF-2␣ knockouts in that they exhibit peri-implantation lethality (56).
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series of enzymes within the mitochondrial matrix that
oxidize fatty acids to acetyl CoA. To date, the transcriptional expression of the genes encoding this pathway has
not been linked to the transcription factors that govern
the expression of the respiratory chain. Rather, PPAR␣
and -␦, members of the nuclear hormone receptor superfamily, have been implicated as transcriptional regulators
of fatty acid oxidation enzymes in tissues with high rates
of fat oxidation including heart, kidney, and skeletal muscle (17, 81, 97). Conversely, the PPARs have not been
associated with the expression of the respiratory apparatus. This suggests that higher order integration of diverse
transcription factors may be necessary to coordinate the
large number of genes required for mitochondrial biogenesis and function. It is notable that ERR␣ regulates the
expression, during adipocyte differentiation, of MCAD, a
key enzyme in fatty acid oxidation (219). Thus ERR␣,
through its control of both respiratory and fatty acid
oxidation genes, may participate in coordinating the expression of the two pathways in certain physiological
contexts.
TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA
6. Dual function factors
There are several reports suggesting that certain nuclear transcription factors or their derivatives have a dual
function in directing mitochondrial gene expression.
Binding sites for ligand-dependent trans-activators have
been observed in mtDNA (51, 234). In the best-studied
example, a 43-kDa protein, closely related to the c-Erb
A␣1 thyroid hormone receptor, has been localized to the
mitochondrial matrix and observed to bind the mitochondrial D-loop in vitro (234). This protein appears to be an
NH2-terminally truncated variant of the nuclear ␣1 thyroid
hormone receptor. The overexpressed variant gives a particulate cytosolic immunofluorescence and stimulates mitochondrial respiration as measured by rhodamine staining and cytochrome oxidase activity. The functional significance of this finding is supported by the observation of
a direct effect of thyroid hormone on mitochondrial RNA
synthesis (61). Similarly, the rat liver glucocorticoid receptor (GR) was found to translocate to the mitochondria
upon treatment of animals with dexamethasone (52). Six
glucocorticoid response elements (GREs) were found in
mtDNA and were shown to bind GR in vitro (51). Two of
the six elements are present in the D-loop, but the other
four are within the COXI and COXIII genes, far removed
from the in vivo sites of mtDNA transcription initiation.
I␬B-␣ and NF␬B have also been localized to mitochondria (29, 47). In one case, they were found in the
intermembrane space associated with the adenine nucleotide translocator and were released upon induction of
apoptosis (29). In the other, they were localized to the
Physiol Rev • VOL
matrix where they were thought to act as negative regulators of mitochondrial mRNA expression (47). These
seemingly contradictory results illustrate the difficulty in
assigning function based on subcellular localization. It is
important to note that the transcriptional activity and
specificity of these shared nuclear factors using mtRNA
polymerase and a defined mtDNA template has not been
demonstrated. Thus it remains inconclusive as to whether
these nuclear factors are true regulators of mitochondrial
transcription.
B. Nuclear Coactivators in
Mitochondrial Biogenesis
Most of the evidence supports a model whereby a
relatively small number of nuclear transcription factors
serve to coordinate the expression of nuclear and mitochondrial respiratory proteins. Of these, the NRFs, Sp1,
and ERR␣ have been consistently associated with the
majority of genes required for respiratory chain expression. These factors exert direct control over transcription
of the nucleus-encoded respiratory subunits and act indirectly on the mitochondrial subunits via their regulation
of mitochondrial transcription factors. In addition, other
mitochondrial oxidative pathways are controlled by unrelated factors as exemplified by PPAR␣ and the fatty acid
oxidation pathway (81). This raises the question of how
diverse transcription factors are integrated into a program
of mitochondrial biogenesis. The discovery of the PGC-1␣
family of transcriptional coactivators has provided a
mechanistic framework for understanding how nuclear
regulatory pathways are coupled to the biogenesis of
mitochondria.
1. PGC-1␣
PGC-1␣, the founding member of a family of transcriptional coactivators, was identified in a differentiated
brown fat cell line on the basis of its interaction with
PPAR␥, an important regulator of adipocyte differentiation (171). Although it apparently lacks histone-modifying
activities itself, it interacts, through a potent NH2-terminal
transcriptional activation domain, with cofactors containing intrinsic chromatin remodeling activities (Fig. 8). In
addition to PPAR␥, PGC-1␣ binds several nuclear hormone receptors and trans-activates the PPAR␥- and thyroid receptor ␤-dependent expression of the UCP-1 promoter. Nuclear hormone receptor coactivator signature
motifs (LXXLL) adjacent to the activation domain are
essential for coactivation through certain nuclear receptors, including PPAR␣ and thyroid receptor ␤ (Fig. 8).
Coupling of transcription and RNA processing by PGC-1␣
occurs through COOH-terminal arginine/serine rich (R/S)
and RNA recognition motifs reminiscent of those found in
RNA splicing factors (142). The dramatic induction of
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More recently, c-Myc has been linked to respiratory
gene expression and mitochondrial biogenesis. C-Myc can
induce cytochrome c and other NRF-1 target genes by
binding noncanonical Myc/MAX binding sites contained
within certain NRF-1 sites and can sensitize cells to apoptosis by dysregulating NRF-1 target genes (146). A dominant negative NRF-1 allele can block this c-Myc-induced
apoptosis without affecting c-Myc-induced cell proliferation. In addition, Myc null fibroblasts were found deficient
in mitochondrial content, and c-Myc expression in these
cells partially restored mitochondrial mass (121). This
along with the observation that many mitochondria-related genes including Tfam are c-Myc targets led to the
conclusion that c-Myc can regulate mitochondrial biogenesis. One caveat to this conclusion is that c-Myc is estimated to have an enormous number of genome binding
sites that even exceeds the number of Myc molecules in
proliferating cells. Thus it is thought that only a minority
of genes bound by Myc/MAX are actually regulated by
c-Myc (1). In addition, because of the extremely large
number of Myc target genes, it is possible that the observed effects on mitochondrial biogenesis result from
indirect effects of Myc on cell growth and metabolism.
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RICHARD C. SCARPULLA
PGC-1␣ mRNA in brown fat upon cold exposure supports
its involvement in thermogenic regulation (126, 170). An
important part of the thermogenic program is the induction of mitochondrial biogenesis, and PGC-1␣ is a potent
inducer of this process. Ectopic overexpression of the
coactivator in cultured myoblasts and other cells induces
respiratory subunit mRNAs and increases COXIV and cytochrome c protein levels as well as the steady-state level
of mtDNA (236).
As illustrated in Figure 9, NRF-1 has been identified
as an important target for the induction of mitochondrial
biogenesis by PGC-1␣. The coactivator binds NRF-1 and
can trans-activate NRF-1 target genes involved in mitochondrial respiration. Moreover, a dominant negative allele of NRF-1 blocks the effects of PGC-1␣ on mitochondrial biogenesis providing in vivo evidence for a NRF-1dependent pathway (236). PGC-1␣ induction of both
NRF-1 and NRF-2 transcripts may also contribute to the
biogenic program. As depicted in Figure 9, PGC-1␣ may
link nuclear regulatory events to the mitochondrial transcriptional machinery through its transcriptional activation of Tfam, TFB1M, and TFB2M expression. As with
respiratory subunit genes, the coactivator targets the
NRF-1 and NRF-2 recognition sites within Tfam and TFB
promoters leading to increased mRNA expression (73).
ERR␣ has also been associated with PGC-1␣-induced mitochondrial biogenesis (143, 190). ERR␣ and GABP␣
(NRF-2␣) recognition sites are conserved in the promoters of a number of oxidative phosphorylation genes, including cytochrome c and ␤-ATP synthase, and PGC-1␣
can drive expression through these sites (143, 190) (Fig. 9).
Interestingly, computer modeling indicates that PGC-1␣ inPhysiol Rev • VOL
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FIG. 8. Summary of PGC-1␣ functional domains. Depicted is a linear
representation of PGC-1␣ showing key functional domains. The NH2terminal activation domain (vertical hatched box) is the site of interaction with histone-modifying cofactors represented by SRC-1 and CBP/
p300. The approximate positions for interaction with transcription factors including nuclear hormone receptors, PPAR␥ and NRF-1, and
MEF2C are indicated above and below the map. Also illustrated is the
central proline-rich region (cross-hatched box) and the COOH-terminal
domain comprised of the arginine/serine-rich R/S domain (open box)
and the RNA recognition motif (horizontal hatched box) proposed to
interact with RNA processing factors. The region containing p38 MAPK
phosphorylation sites is bracketed below.
duction of ERR␣ and GABP␣ (NRF-2␣) is upstream from
NRF-1 in the program of respiratory gene expression. The
PGC-1␣ induction of mitochondrial biogenesis has been
confirmed in transgenic mice. PGC-1␣ expression is elevated in the postnatal mouse heart, and cardiac-specific
overexpression of PGC-1␣ leads to massive increases in
mitochondrial content in cardiac myocytes (116). This
overexpression is associated with edema and dilated cardiomyopathy and is likely unrelated to normal PGC-1␣
function in the heart.
In addition to its effects on the respiratory chain and
mitochondrial transcription, PGC-1␣ promotes mitochondrial oxidative functions by inducing the expression of
genes of the mitochondrial fatty acid oxidation and heme
biosynthetic pathways (Fig. 9). As we have seen, PPAR␣
directs the transcriptional expression of fatty acid oxidation enzymes and is enriched in brown fat and other
tissues with high oxidative energy demands. Ligand-dependent binding of PPAR␣ through the LXXLL motif of
PGC-1␣ is associated with the trans-activation of PPAR␣dependent promoters (97, 218). In addition, overexpression of PGC-1␣ induces MCAD gene expression through
its direct interaction with ERR␣ (98). PGC-1␣ can also
utilize both NRF-1 and FOXO1, a forkhead box family
member, to induce transcription of ␦-aminolevulinate synthase, the first and rate-limiting enzyme of the heme biosynthetic pathway (86). Thus, as summarized in Figure 9,
PGC-1␣ has the potential to integrate the activities of a
diverse collection of transcription factors that have been
implicated in the expression and function of the mitochondrial oxidative machinery.
Physiological expression of PGC-1␣ at the transcriptional level can be modulated through cAMP-dependent
signaling. In brown adipocytes, sympathetic production
of norepinephrine acts predominantly through ␤3-adrenergic receptors (34). These receptors are coupled to adenyl cyclase via the Gs subtype of G proteins. The thermogenic signaling cascade results in enhanced cyclase activity and the increased production of cAMP. As shown in
Figure 9, elevated cAMP levels lead to activation of CREB
through its phosphorylation by protein kinase A (PKA).
CREB or related CREB family members, such as activating transcription factor 2 (ATF-2), mediate both direct
and indirect effects on the thermogenic program including the induction of PGC-1␣ and UCP1 (34, 35). The
PGC-1␣ promoter has a potent cAMP response element
(CRE) that serves as a target for CREB-mediated transcriptional activation (93). In addition to its role in the
thermogenic program, the coactivator is also markedly
induced in mouse liver in response to fasting where it
activates the genes encoding gluconeogenic enzymes
(239). The induction of gluconeogenesis by PGC-1␣ in
fasted liver also involves the activation of CREB by serine
phosphorylation in response to the catecholamine and
glucagon-mediated elevation of cAMP. CREB induces the
TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA
625
transcriptional expression of PGC-1␣, which in turn serves
as a coactivator of gluconeogenic gene expression (93).
The cAMP-dependent pathway is one of several that
direct the induction of PGC-1␣ in a number of tissues
(126). PGC-1␣ along with Tfam and NRF-1 are induced via
cGMP-dependent signaling resulting from elevated levels
of nitric oxide (NO) (150) (Fig. 9). The NO induction of
PGC-1␣ is correlated with increased mitochondrial biogenesis in several cell lines. The induced mitochondrial
mass is accompanied by increased oxidative phosphorylation-coupled respiration consistent with an increase in
functional mitochondria (151). These results were obtained by pharmacological increases in either NO or
cGMP, raising the question of whether physiological fluctuations in endogenous NO can regulate mitochondrial
content (114). Interestingly, tissue mitochondria in mice
with a homozygous disruption of the gene encoding endothelial NO synthase (eNOS⫺/⫺) were somewhat smaller
and less densely packed than in wild-type mice (150, 151).
These changes were accompanied by reductions in energy expenditure and mRNA levels for PGC-1␣, Tfam, and
NRF-1, arguing that basal mitochondrial content is affected by the loss of eNOS-generated NO. Establishment
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of an NO-dependent pathway of mitochondrial biogenesis
awaits confirmation of these findings.
A number of reports link the expression of PGC-1␣ to
exercise-induced mitochondrial biogenesis in skeletal
muscle (3, 15, 76, 154, 178, 207–209, 233). In each case,
PGC-1␣ mRNA and/or protein increase as an adaptive
response to endurance exercise of varying intensity and
duration. These findings are satisfying in the context of
the long-standing relationship between endurance exercise and enhanced mitochondrial respiratory chain expression and function (95). Both NRF-1 and NRF-2 DNA
binding activities were elevated along with the expression
of several NRF target genes in rat skeletal muscle following an exercise regimen (15). Similar changes in the PGC1␣-NRF pathway were mimicked in cultured myotubules
in response to increased calcium levels, suggesting that
PGC-1␣ signaling contributes to adaptive changes in mitochondrial biogenesis in skeletal muscle cells (154). Exercise and other neuromuscular activity may also lead to
the activation of the p38 mitogen-activated protein kinase
(MAPK) pathway resulting in PGC-1␣ induction through
ATF2 and MEF2 (3). There is evidence that depletion of
ATP during exercise leads to elevated AMP/ATP ratios,
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FIG. 9. Illustration summarizing PGC-1␣-mediated pathways governing mitochondrial biogenesis and function. Depicted in the nucleus (shaded
sphere) are the key transcription factors (NRF-1, NRF-2, ERR␣, PPAR␣, and MEF-2) that are PGC-1␣ targets and act on nuclear genes governing
the indicated mitochondrial functions. Some of the physiological effector pathways mediating changes in the transcriptional expression or function
of PGC-1␣ are also shown. The CREB activation of PGC-1␣ gene transcription in response to cold (thermogenesis), fasting (gluconeogenesis), and
exercise has been well documented. The physiological mechanisms of PGC-1␣ induction by nitric oxide are not established but may involve the
production of endogenous nitric oxide by eNOS. A potential pathway of retrograde signaling through calcium is also included.
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RICHARD C. SCARPULLA
of an interaction between hepatic nuclear receptor 4␣
(HNF4␣) and forkhead transcription factor 01 (FOXO1),
which mediate the expression of gluconeogenic genes.
PGC-1␤ also exhibits a marked preference for promoting
the ligand-dependent activity of ER␣ while having a minimal effect on ER␤ (110). These examples illustrate that
differences in transcription factor interactions mediate
differences in functional specificity between members of
the PGC-1 family.
Despite their differential utilization of nuclear hormone receptors, PGC-1␤ binds NRF-1 and is a potent
coactivator of NRF-1 target genes leading to increased
mitochondrial gene expression (127). Forced expression
of PGC-1␤ in cultured muscle cells results in increased
mitochondrial biogenesis and oxygen consumption, although PGC-1␣ has been associated with higher proton
leak rates than PGC-1␤ (202). Thus, although PGC-1␣ and -␤
are functionally distinct, they both utilize NRF-1 and other
transcription factors to induce mitochondrial biogenesis.
This has been confirmed in transgenic mice in which
PGC-1␤ is abundantly overexpressed in skeletal muscle
(10). The coactivator promotes massive increases in mitochondrial content that are accompanied by marked elevations in the expression of respiratory mRNAs and
proteins derived from the expression of both nuclear and
mitochondrial genes. This mitochondrial biogenesis is associated with the induction of type IIX oxidative muscle
fibers and increased capacity for aerobic exercise. These
changes are presumed to result from activation of a specific program of gene expression by PGC-1␤ through transcription factor targets such as MEF2, ERR␣, and PPAR␣
among others. Notably, the induction of type IIX fibers
was absent in the soleus muscle despite high-level expression of PGC-1␤ in this tissue, suggesting that abundant
expression of the coactivator alone is not sufficient. It will
be important to confirm the regulation of muscle fiber
type by PGC-1␤ in loss of function experiments.
2. PGC-1␤
Two mammalian relatives of PGC-1␣ have been characterized. PGC-1␤, although somewhat larger than PGC1␣, shares sequence similarity with PGC-1␣ along its entire length (110, 127). Like PGC-1␣, PGC-1␤ has an NH2terminal activation domain, LXXLL coactivator signatures,
and a COOH-terminal RNA recognition motif. Although
PGC-1␤ lacks an R/S domain, it is not clear whether it
differs from PGC-1␣ in its ability to couple transcription
and RNA slicing. Steady-state tissue expression of PGC-1␤
mRNA parallels that of PGC-1␣ with the highest levels in
brown fat, heart, and skeletal muscle, tissues high in
mitochondrial content and oxidative energy production.
However, PGC-1␤ differs from PGC-1␣ in that it is not
induced in brown fat upon cold exposure, and it is a poor
inducer of gluconeogenic gene expression in hepatocytes
and liver (140). This most likely results from the absence
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3. PRC
A database search for sequence similarities to PGC-1␣
identified the partial sequence of a large cDNA with a
COOH-terminal RS domain and RNA recognition motif
(6). Molecular cloning of the full-length cDNA revealed
additional sequence similarities with PGC-1␣ including
an acidic NH2-terminal region, an LXXLL signature for
nuclear receptor coactivators, and a central prolinerich region (Fig. 10). Although significant sequence similarity with PGC-1␣ is confined to these distinct regions,
their spatial conservation is highly suggestive of related
function. Thus the protein encoded by this cDNA was
designated as PGC-1␣-related coactivator (PRC) (6).
PRC resembles both PGC-1␣ and -␤ in that it binds
NRF-1 both in vitro and in vivo and can utilize NRF-1 for
the trans-activation of NRF-1 target genes (6). PRC has a
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which enhances AMP-activated protein kinase (AMPK)
activity (Fig. 9). In addition, increased calcium activates
Ca2⫹/calmodulin-dependent protein kinase (CaMK), and
this along with AMPK results in the induction of PGC-1␣
and activation of NRFs during energy deprivation (153,
243). In fact, as seen in other instances of PGC-1␣ induction, CREB phosphorylation, in this case by CaMK, directs
CREB-activated PGC-1␣ transcription (Fig. 9). This pathway is also driven by calcineurin A through myocyte
enhancer factor 2 (MEF2), which is a potent trans-activator of PGC-1␣ transcription in muscle (87). This same
factor has been linked to the expression of muscle-specific COX subunits (228). Although it is not clear weather
the NRFs are direct targets of these kinases, the associated increase in NRF-1 DNA binding activity coincides
with elevated expression of cytochrome c and 5-aminolevulinate synthase and enhanced mitochondrial density
(21). It should be noted that PGC-1␣ mRNA is induced to
similar levels in wild-type and in both AMPK ␣1 and ␣2
knockout mice, arguing that these isoforms of AMPK are
not essential to exercise-induced gene expression by
PGC-1␣ (100). This study was based on patterns of mRNA
expression alone, and thus a definitive conclusion awaits
a systematic analysis of mitochondrial biogenesis in these
mice.
Finally, a recent study shows that NRF-1 and NRF-2
occupancy of cytochrome c and cytochrome oxidase subunit IV promoters, respectively, occurs prior to the exercise-induced increase PGC-1␣ protein levels (233). Exercise also leads to the activation of ATF-2 through p38
mitogen-activated protein kinase (p38 MAPK). This transcription factor, a member of the CREB/ATF family, binds
the PGC-1␣ promoter and induces its transcription, perhaps as a secondary phase of the adaptive response. It is
unclear whether direct phosphorylation of PGC-1␣ by p38
MAPK is required in this model.
TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA
potent NH2-terminal activation domain that is highly similar to that of PGC-1␣ and is required for NRF-1-dependent trans-activation. These properties along with its nuclear localization and in vivo interaction with NRF-1 attest
to a transcriptional function for PRC. As with PGC-1␣,
binding of NRF-1 occurs through the NRF-1 DNA binding
domain. In addition to cytochrome c and 5-ALAS promoters, PRC can trans-activate the human TFB1M and
TFB2M promoters in a manner indistinguishable from
PGC-1␣ (73). The NRF binding sites within the proximal
promoters of these genes serve as targets for trans-activation by both coactivators.
Despite these similarities, PRC mRNA is not enriched
in brown versus white fat and is only slightly elevated in
brown fat upon cold exposure, arguing against a major
role for PRC in adaptive thermogenesis (6). In addition,
PRC expression is not particularly enriched in highly
oxidative tissues with abundant mitochondria. However,
as illustrated in Figure 11, PRC is rapidly induced upon
serum treatment of quiescent fibroblasts and is expressed
more abundantly in proliferating cells compared with
growth-arrested cells. Moreover, serum induction of PRC
is accompanied by a pattern of gene expression that is
qualitatively similar to that observed in response to elevated PGC-1␣ (73). This includes increased expression of
Tfam, TFB1M and -2M mRNAs, as well as those for both
nuclear and mitochondrial respiratory subunits. It is of
interest in this context that PRC, NRF-1, and Tfam are
markedly upregulated in thyroid oncocytomas in conjunction with increases in cytochrome oxidase activity and
mtDNA content (180). These thyroid tumors are characterized by dense mitochondrial accumulation and are apparently devoid of PGC-1␣. Both PRC and PGC-1␣ mRNAs are
rapidly induced in human skeletal muscle in response to
endurance exercise (178). PRC mRNA expression is high
initially and persists for ⬃4 h but then modulates to a
basal level at 24 h after exercise. Thus PRC may direct
early adaptive changes in gene expression in response to
exercise.
Serum induction of PRC has the characteristics of
immediate early genes, those genes whose expression is
induced by serum growth factors in the absence of de
novo protein synthesis (91). Their induction is among the
very earliest events in the genetic program of cell growth.
FIG. 11. Diagram summarizing the putative role of PRC during the transition from quiescence to proliferative growth (G0 3 G1 transition). Cells
that exit the cell cycle either by contact inhibition or serum withdrawal (G0) have reduced levels of PRC mRNA and protein expression that
coincides with reduced occupancy of the NRF-1 and CREB-dependent cytochrome c promoter in vivo. G0 cells stimulated to grow by serum exhibit
a rapid and cycloheximide-independent induction of PRC mRNA during the transition from G0 to G1. Serum stimulation leads to increased PRC
protein expression and increased PRC occupancy of the cytochrome c promoter. This is accompanied by the rapid mitogenic phosphorylation of
CREB by the pp90RSK family of protein kinases and the phosphorylation of NRF-1 by as yet unidentified kinases. CREB phosphorylation is rapid and
transient, whereas NRF-1 phosphorylation is slower and persists in proliferating cells along with elevated PRC expression. The relationship, if any,
between PRC promoter occupancy and these phosphorylation events is not known.
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FIG. 10. Diagram comparing sequence motifs conserved between
PGC-1-related coactivator (PRC) and PGC-1␣. Dashed lines connect
sequence similarities between the activation domains (vertically
hatched box), the proline-rich region (cross-hatched box), the R/S
domain (open box), and the RNA recognition motif (horizontally
hatched box).
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RICHARD C. SCARPULLA
V. LESSONS FROM MOUSE GENE KNOCKOUTS
The complexity of the pathways of mitochondrial
biogenesis is illustrated by the phenotypes of knockout
mice. As summarized in Table 1, these studies reveal that
both essential and nonessential genes define the mitochondrial phenotype. The essential genes are generally
single copy and encode both nuclear and mitochondrial
transcription factors. These genes are not only required
for the maintenance of mitochondrial function but also
for proper embryogenesis and organismal viability. They
include Tfam, the NRFs, and other factors that are fundamental to the expression of nuclear and mitochondrial
genes. The nuclear transcription factors implicated in
mitochondrial biogenesis are not exclusive to this process
but rather integrate the expression of mitochondria with
that of other fundamental cellular functions. The nonessential genes are members of small gene families and
function as important transcriptional modulators of mitochondrial expression and function. These include the
transcription factors ERR␣ and PPAR␣ as well as the
PGC-1 family of coactivators that target key transcription
factors in specifying tissue-specific energetic properties, a
subset of which is mitochondrial content. Individually
they are not required for viability or for maintaining basal
functional levels of mitochondria (Table 1). However,
through their regulated expression and transcriptional
specificities, these regulatory factors play an important
role in relaying extracellular signals to the transcription
machinery.
A. Transcription Factor Knockouts
A targeted disruption of Tfam confirmed its in vivo
function in mitochondrial transcription and replication
(Table 1). A homozygous Tfam knockout results in
postimplantation lethality between embryonic days E8.5
and E10.5 (113). The E8.5 embryos are severely depleted
of mtDNA and are deficient in both cytochrome oxidase
1. Mouse knockouts of nucleus-encoded transcriptional regulators implicated in mitochondrial
biogenesis and function
TABLE
Gene Knockout
Viability
Mitochondrial Phenotype
Tfam (germ line)
PolgA
NRF-1
Embryonic lethal between days E8.5 and E11.5
Embryonic lethal between days E7.5 and E8.5
Embryonic lethal between days E3.5 and E6.5
NRF-2 (GABP)
YY1
PPAR␣
Preimplantation lethal
Embryonic lethal between days E3.5 and E8.5
Viable and fertile
ERR␣
Viable and fertile
PGC-1␣ (Lin et al.)
Viable and fertile, increased postnatal mortality
PGC-1␣ (Leone et al.)
Viable and fertile, normal mortality
PGC-1␤
Viable and fertile
mtDNA depletion, severe respiratory chain deficiency
mtDNA depletion, cytochrome oxidase deficiency
mtDNA depletion, loss of mitochondrial membrane potential,
blastocyst growth defect
Not determined
Not determined
Reduced constitutive expression of mitochondrial fatty acid
oxidation enzymes, defective fatty acid oxidation in
response to fasting
Normal food consumption and energy expenditure, reduced
lipogenesis, cold intolerance
Normal mitochondrial morphology, reduced O2 consumption
in hepatocytes, fasting hypoglycemia, cold intolerance,
defective cardiac ATP production
Increased body mass, modest reduction in skeletal muscle
mitochondria, normal gluconeogenesis
Normal energy intake and expenditure, reduction in skeletal
muscle and heart mitochondria
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Like other genes in this class, PRC mRNA is increased
rapidly by serum induction of quiescent fibroblasts in the
presence of the protein synthesis inhibitor cycloheximide
(220). The mRNA for PRC and other immediate early
genes is superinduced and also markedly stabilized by the
drug, probably because of a requirement for protein synthesis for mRNA turnover. Cytochrome c is also serum
induced in part through the NRF-1 and CREB sites within
its promoter (92). NRF-1 and CREB have been implicated
as regulators of cell growth. As discussed above, NRF-1
occupies the promoters of many genes required for DNA
replication, cytokinesis, and mitosis including a number
of those targeted by the E2F family of growth regulators
(33). CREB is well known as a target of mitogenic signaling pathways (137). Interestingly, PRC binds CREB
through the same binding sites used for NRF-1, and both
factors exist in a complex with PRC in cell extracts (220).
In addition, NRF-1 and CREB both occupy the cytochrome c promoter in vivo, and PRC occupancy of the
cytochrome c promoter increases upon serum induction
of quiescent fibroblasts. This is consistent with a model
whereby PRC targets these transcription factors in response to mitogenic signals. This is supported by the
observation that expression of the PRC NRF-1/CREB
binding domain in trans leads to dominant negative inhibition of cell growth on galactose as a carbon source
(220). Growth on galactose requires mitochondrial respiration, suggesting that PRC may link the expression of
genes required for both cell growth and mitochondrial
respiration.
TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA
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companied by defects in the development of the central
nervous system (20).
As observed for NRF-1, GABP␣ (NRF-2␣) homozygous null mice also exhibit a peri-implantation lethal phenotype (176). Although no determination of the state of
mtDNA or mitochondrial function was made in the mutant embryos, the early lethality may result from a combination of mitochondrial and nonmitochondrial defects.
Homozygous knockouts of other Ets family transcription
factors also exhibit early embryonic lethality, suggesting
that members of the family are unable to compensate for
one another during embryonic development. It is surprising in light of the NRF knockouts that mice homozygous
null for ERR␣ are viable and fertile and display no defects
in food intake or energy expenditure (Table 1), although
they do have reduced fat mass and are resistant to high-fat
diet-induced obesity (132). The ERR␣ knockouts show a
modest reduction in cytochrome c mRNA, but no changes
in the global expression of other respiratory subunit
genes have been noted. The absence of a profound mitochondrial deficiency in these mice may be explained by
the fact that ERR␣ is one of a small family of related
factors whose members may compensate for the loss of a
single isoform. In contrast, Tfam and NRF-1 are the products of unique genes. Recent work demonstrates that the
ERR␣ knockout mice are deficient in adaptive thermogenesis because of decreased mitochondrial mass in brown
adipose tissue (221). Although PGC-1␣ and UCP-1 were
induced normally by cold exposure in the ERR␣ knockouts, the decrease in mitochondria was accompanied by
reductions in respiratory gene expression, mtDNA copy
number, and the oxidative capacity of the tissue. The
remaining mitochondria were morphologically and functionally normal. The results argue that ERR␣ has a specific function in differentiation-induced mitochondrial
biogenesis in brown adipose tissue without affecting
basal levels of mitochondrial maintenance.
A similar theme is echoed in mice with a targeted
disruption of PPAR␣ (Table 1). The PPAR␣ knockouts are
also viable and fertile with no obvious phenotypic abnormalities (115). However, they do have a markedly blunted
response to peroxisome proliferators, demonstrating a
crucial role for PPAR␣ in the pleiotropic response to
these agents. In addition, the mice display lower constitutive expression of several mitochondrial fatty acid metabolizing enzymes (8) and, in contrast to wild-type mice,
the PPAR␣ knockouts were resistant to the induction of
these mitochondrial enzymes in both liver and heart in
response to fasting (119). Thus, although not required for
normal development, viability, or mitochondrial maintenance, PPAR␣ plays a crucial role in orchestrating regulatory responses necessary for optimal mitochondrial oxidative functions.
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and succinate dehydrogenase activities. In addition, tissue-specific Tfam knockout animals show a strong correlation between Tfam and mtDNA abundance (122, 201).
These studies establish an essential role for Tfam in the
maintenance of mtDNA copy number in vivo. Interestingly, a homozygous knockout of mitochondrial DNA
polymerase ␥ also results in developmental arrest at E7.5–
E8.5, severe depletion of mtDNA, and loss of cytochrome
oxidase activity (83). These similarities in mitochondrial
phenotype between Tfam and DNA polymerase ␥ knockouts (Table 1) illustrate that intact mtDNA transcription
and replication machinery is required for mtDNA maintenance and for development beyond late gastrulation and
early organogenesis. It is also notable that overexpressing
Twinkle, a mtDNA helicase, in transgenic mice leads to
increased mtDNA copy number (215). In humans, dominant mutations in Twinkle result in the accumulation of
multiple mtDNA deletions in several somatic tissues.
These results reinforce the conclusion that mtDNA transcription and replication factors are essential to the expression of the mitochondrial respiratory chain in mammalian tissues.
Mouse gene knockouts of several of the nuclear transcription factors implicated in mitochondrial biogenesis
have also been analyzed (Table 1). A homozygous NRF-1
mouse knockout results in early embryonic lethality between embryonic days E3.5 and E6.5 (96). Although they
are morphologically normal, the NRF-1 null blastocysts
degenerate rapidly in culture and have severely reduced
mtDNA levels accompanied by a deficiency in maintaining
a mitochondrial membrane potential. The mtDNA depletion in the blastocyst does not result from a defect in
mtDNA amplification during oogenesis, arguing that the
mtDNA depletion occurs between fertilization and the
blastocyst stage. The phenotype is consistent with the loss
of an NRF-1-dependent pathway of mtDNA maintenance.
However, depletion of mtDNA alone does not explain the
peri-implantation lethality of the homozygous NRF-1 nulls
because Tfam knockout embryos are also depleted of
mtDNA but survive to between embryonic days E8.5 and
E10.5 (113). Thus the earlier lethality of NRF-1 nulls may
result from the decreased expression of NRF-1 target
genes that are required for other cell growth and developmental functions. Among these may be the subset of
genes identified as targets of both NRF-1 and the E2F
family by ChiP on chip assay (33). It is of interest in this
context that loss-of-function mutations in NRF-1 have a
dramatic negative effect on both Drosophila and zebrafish development. Partial loss-of-function mutations in
EWG, a NRF-1 relative in Drosophila, result in aberrant
intersegmental axonal projection pathways in the embryo
(53). Total loss-of-function mutations in EWG are embryonic lethal. The NRF-1 gene in zebrafish is expressed in
the eye and central nervous system of developing embryos. Its disruption gives a larval-lethal phenotype ac-
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B. Coactivator Knockouts
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As we have seen, gain-of-function experiments have
established that overexpression of either PGC-1␣ or
PGC-1␤ can orchestrate massive increases in mitochondrial biogenesis both in cultured cells and in mouse tissues (10, 116, 236). These changes have been ascribed to
their ability to induce the expression of key transcription
factors (NRF-1, NRF-2, ERR␣) and to interact specifically
with these factors in mediating the activation of genes
that promote the biogenesis of mitochondria. Thus it is
surprising that mice with a targeted disruption of PGC-1␣
are viable and show no changes in mitochondrial abundance or morphology in liver or brown fat (128). They do
show reduced oxygen consumption in isolated hepatocytes and reduced expression of several mRNAs linked to
mitochondrial function. The only tissues to display obvious morphological abnormalities were brown adipose tissue and the striatum of the brain. Defects in the striatum
have been associated with movement disorders, and the
PGC-1␣ null mice were markedly hyperactive. This hyperactivity correlated with a loss of axons in the striatum as
well as with reductions in nucleus-encoded mRNAs for
respiratory- and brain-specific genes. Subsequent experiments with the PGC-1␣ null mice demonstrated that
PGC-1␣ is not required for mitochondrial biogenesis (9).
However, the mice are deficient in the expression of
genes necessary for mitochondrial function and in oxidative enzyme activities in heart and skeletal muscle. These
changes coincide with reduced cardiac ATP production
and a defect in work output in response to physiological
stimuli. Recently, a skeletal muscle-specific PGC-1␣ knockout mouse was characterized (84). These mice clearly
show multiple muscle defects including reduced exercise
tolerance and abnormalities in the maintenance of normal
muscle fiber composition. The results illustrate that the
true tissue-specific functions of PGC family coactivators
may be masked by pleiotropic phenotypes in mice harboring a generalized gene disruption in all of their tissues.
The phenotype of an independently constructed
PGC-1␣ null mouse confirmed that the coactivator is not
required for normal development or for the global biogenesis or maintenance of mitochondria (118) (Table 1).
However, these mice had reduced mitochondrial density
in slow-twitch skeletal muscle and modestly reduced respiratory capacity in both liver and skeletal muscle. There
are also a number of significant differences with the previously described knockout including the absence of a
defect in gluconeogenesis and a disparity in cardiac phenotype. These have been ascribed to differences in genetic background or targeting strategies (68).
Recently, a targeted disruption of the mouse PGC-1␤
gene was constructed by removal of exons containing the
nuclear hormone receptor coactivator signature motifs
(LXXLL) and introduction of a premature stop codon
(117). Mice with a homozygous disruption of the gene
were viable and fertile and exhibited no gross changes in
whole body substrate utilization or total energy input or
output (Table 1). They did however have a higher overall
metabolic rate associated with a lean phenotype compared with wild-type littermates. Closer examination revealed a number of tissue alterations in mitochondrial
gene expression and respiratory function. There were
clear reductions in some genes associated with mitochondrial electron transport chain function in brown and
white fat, skeletal muscle, and heart. These included respiratory subunits encoded by both nuclear and mitochondrial genes. Although diminished mRNA levels were
not always accompanied by similar changes in the encoded protein, mitochondrial volume was reduced in both
heart and skeletal muscle. In these cases, the accompanying decreases in electron transport and oxidative phosphorylation activities resulted from reduced numbers of
functionally normal mitochondria. In brown fat, mitochondrial content was normal, but the knockout mice
exhibited a blunted response to norepinephrine-induced
thermogenesis. These phenotypes clearly point to an important modulatory role for PGC-1␤ in maintaining optimal tissue energetics.
The absence of a dramatic mitochondrial biogenesis
defect in either the PGC-1␣ or PGC-1␤ null mice may
result from mechanisms that compensate for the individual loss of these coactivators. Since the PGC-1 family
members share several functional motifs and can all
trans-activate NRF target genes, the remaining family
members may substitute functionally for the ablated
member. This is seen in the brown fat of the PGC-1␤
knockout where PGC-1␣ levels are markedly upregulated,
possibly resulting in the maintenance of normal mitochondrial content in this tissue (117). This issue was
addressed by knocking down the expression of PGC-1␤
by shRNA in a preadipocyte cell line derived from PGC-1␣
knockout mice (216). Although PGC-1␣ appears not to be
required for adipocyte differentiation, its loss is correlated with an inability to induce cAMP-dependent expression of genes necessary for thermogenesis, including
UCP-1 and cytochrome c. UCP-1, however, is induced by
insulin and retinoic acid in the absence of PGC-1␣. Reduced expression of PGC-1␤ in the PGC-1␣ null background led to a failure to establish and maintain differentiated levels of mitochondrial density and function in
mature brown adipocytes. Mitochondrial content is reduced to a level present in undifferentiated preadipocytes. Thus, although neither coactivator is required to
direct the program of adipocyte differentiation, one or the
other is essential to achieve a differentiated mitochondrial phenotype. This is reminiscent of the case of ERR␣,
suggesting that the PGC-ERR␣ regulatory pathway directs
differentiation-specific changes in mitochondrial content
in brown fat and possibly other tissues. It remains an
TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA
open question as to whether PRC can support basal levels
of mitochondrial biogenesis in the absence of PGC-1␣ and
-␤. It is of interest in this context that expression of a
dominant negative allele of PRC from a lentivirus vector
inhibits respiratory growth in cultured cells (220). Clearly,
the PGC-1 coactivators exhibit highly specialized regulatory functions but, individually, do not appear to be the
sole determinants of mitochondrial content in the majority of tissues.
VI. RETROGRADE PATHWAYS
The regulated expression of the PGC-1 family of inducible coactivators provides a mechanism for modulating mitochondrial biogenesis and function in response to
a variety of extracellular signals. In addition, pathways
exist for the communication of the functional state of
mitochondria back to the nuclear transcriptional machinery. This phenomenon of retrograde signaling is well studied at the transcriptional level in the yeast Saccharomyces
cerevisiae (130). In yeast cells that are depleted of mitochondrial DNA (␳0 cells), the resulting respiratory deficiency reduces the supply of glutamate, which is derived
from the ␣-ketoglutarate of the citric acid cycle. Cells
compensate by increasing production of peroxisomes, the
sites of fatty acid oxidation in yeast. This generates
acetyl-CoA, which fuels the citric acid cycle. The nuclear
CIT2 gene encoding peroxisomal citrate synthase is
markedly induced in ␳0 cells. This enzyme is part of the
glyoxylate cycle, and its induction in response to a defect
in mitochondrial respiration allows cells to generate citrate (200). Positive transcriptional control of peroxisomal citrate synthase and other peroxisomal proteins in ␳0
cells is mediated by basic helix-loop-helix-leucine zipper
transcription factors, Rtg1p and Rtg3p (177). A third factor Rtg2p facilitates the translocation of Rtg1p and Rtg3p
from the cytoplasm to the nucleus where they bind to
their target genes as a heterodimer and drive transcription. Rtg2p has an ATP binding site and is thought to
function as a sensor of the functional state of mitochondria in part through its interaction with the phosphoprotein Mks1p. The phosphorylated state of Mks1p dictates
its interaction with Rtg2p or negative regulators creating
a molecular switch for control of the RTG pathway (67).
B. Mammalian Retrograde Regulation
The Rtg pathway has not been identified in vertebrate
cells, but there are a number of examples where the
expression of nuclear genes is altered by deficiencies in
mitochondrial function (31). Defective mitochondria that
Physiol Rev • VOL
result from certain mitochondrial disease mutations proliferate in diseased muscle fibers giving rise to ragged red
fibers (145, 226). Specific nuclear genes involved in ATP
production also display elevated expression in cells with
mtDNA mutations (89). An altered pattern of nuclear gene
expression, involving proteins of the mitochondrial inner
membrane, intermediate filaments, and ribosomes, occurs
in human cells upon depletion of mtDNA (123). Likewise,
chicken cells either depleted of mtDNA or treated with
the mitochondrial protein synthesis inhibitor chloramphenicol have increased levels of mRNAs for elongation
factor 1␣, ␤-actin, v-myc, and GAPDH (229). Although the
mechanistic bases for these phenomena have not been
elucidated, they are thought to represent nuclear responses to deficiencies in ATP production.
Recent studies suggest that retrograde signaling in
animal cells may be mediated by calcium (22). Inhibition
of mitochondrial respiratory function either by depletion
of mtDNA or by metabolic inhibitors results in a stress
response that coincides with elevated cytosolic calcium
levels. The response includes increased expression of
calcium-responsive transcription factors and cytochrome
oxidase subunit Vb. Calcium signaling has also been associated with a PGC-1-dependent pathway of mitochondrial biogenesis in skeletal muscle (155, 235). Constitutive
expression of calcium/calmodulin-dependent protein kinase IV (CaMKIV) in the skeletal muscle of transgenic
mice leads to increased mtDNA copy number and respiratory enzymes as well as elevated PGC-1␣ levels. Thus
calcium may be an important link between the relay of
extracellular signals to the nucleus and the bidirectional
communication between nucleus and mitochondria. This
notion is reinforced by the observation that CREB is
found in its transcriptionally active, phosphorylated state
in cells exhibiting mitochondrial insufficiency resulting
either from the loss of mtDNA or by pathological mutation in mtDNA (11). This coincides with the activation of
CaMKIV, which, as discussed, can lead to CREB phosphorylation in response to calcium. It remains to be determined
whether calcium signaling through CREB and the PGC-1
family coactivators represents a physiologically meaningful
pathway of retrograde regulation in vertebrate cells.
VII. CONCLUSION
Much progress has been made in uncovering the
transcriptional mechanisms that govern the function and
biogenesis of mitochondria. The identification and characterization of important regulatory factors has led to a
framework for understanding the continuity between signaling events affecting cellular energetics and the expression of both nuclear and mitochondrial genes that dictate
the mitochondrial phenotype. The preponderance of evidence suggests that nucleus-encoded transcription fac-
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A. The RTG Pathway in Yeast
631
632
RICHARD C. SCARPULLA
Physiol Rev • VOL
tions of PRC and PGC-1␤ should clarify this issue. It will
also be of interest to determine whether retrograde signaling is mediated by any of these coactivators.
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence:
R. C. Scarpulla, Dept. of Cell and Molecular Biology, Northwestern Medical School, 303 East Chicago Ave., Chicago, IL 60611.
GRANTS
Work in the author’s laboratory was supported by National
Institute of General Medical Sciences Grant GM-32525–25.
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