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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

I. Introduction 612

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A. Overview 612
B. Pathophysiology 613
II. The Mitochondrial Genetic System: mtDNA Structure and Inheritance 614
III. Transcription of mtDNA 615
A. Mitochondrial transcriptional units 615
B. Tfam 616
C. mtTFB 617
D. Transcription termination and mitochondrial gene expression 617
IV. Nuclear Control of Mitochondrial Function 618
A. Nuclear transcription factors governing respiratory gene expression 618
B. Nuclear coactivators in mitochondrial biogenesis 623
V. Lessons From Mouse Gene Knockouts 628
A. Transcription factor knockouts 628
B. Coactivator knockouts 630
VI. Retrograde Pathways 631
A. The RTG pathway in yeast 631
B. Mammalian retrograde regulation 631
VII. Conclusion 631

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 machin-
ery, 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 transcrip-
tional 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.

www.prv.org 0031-9333/08 $18.00 Copyright 2008 the American Physiological Society 611
612 RICHARD C. SCARPULLA

I. INTRODUCTION molecular architecture and biological functions of the


organelle. Because of this limited coding capacity, mito-
chondria are genetically semiautonomous in that they rely
A. Overview
heavily on the expression of nuclear genes for all of their
biological functions. For example, the majority of protein
Mitochondria are ubiquitous membrane-bound or-
subunits that comprise the five inner membrane com-
ganelles that are a defining feature of the eukaryotic cell.
The organelle is comprised of a soluble matrix sur- plexes of the electron transport chain and oxidative phos-
rounded by a double membrane, an ion impermeable phorylation system are nucleus encoded (Fig. 1). The
inner membrane, and a permeable outer membrane. Early largest of these, complex I, the NADH dehydrogenase, has
biochemists recognized the importance of mitochondria 39 of its 46 subunits specified by nuclear genes, whereas
as the sites of aerobic oxidation of metabolic fuels. It is the smallest, the succinate dehydrogenase (complex II), is
now well established that they contribute to many impor- comprised entirely of nucleus-encoded subunits (Fig. 1).
tant functions including pyruvate and fatty acid oxidation, Thus nuclear genes must specify most of the structural
and catalytic components directly involved in energy me-

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nitrogen metabolism, and heme biosynthesis among oth-
ers. Most notably, the mitochondrion is the site of the tabolism and, in addition, control mitochondrial transcrip-
electron transport chain and oxidative phosphorylation tion, translation, and DNA replication (27, 46, 185, 227).
system that provides the bulk of cellular energy in the Mammalian mitochondrial biogenesis is subject to
form of ATP (16, 88). Most of the chemical bond energy complex physiological control. Mitochondrial mass is in-
from the oxidation of fats and carbohydrates is converted duced in adult muscle in response to exercise (28) or
to the reducing power of NADH and FADH2 within the chronic electrical stimulation (232), presumably as an
mitochondrial matrix. The respiratory apparatus consists adaptation to facilitate increased oxygen utilization. Cal-
of a series of electrogenic proton pumps that convert this cium-dependent signaling has been linked to a transcrip-
reducing potential to an electrochemical proton gradient tional pathway of mitochondrial biogenesis in skeletal
across the inner membrane (Fig. 1). The electrochemical muscle (155, 235). The proliferation of mitochondria oc-
potential of this gradient is converted via the ATP syn- curs in the brown fat of rodents during adaptive thermo-
thase to the high-energy phosphate bonds of ATP. In genesis coinciding with the activation and induction of
addition, the gradient can be dissipated in specialized UCP-1, an uncoupling protein that dissipates the proton
brown adipocytes by uncoupling proteins to generate gradient to produce heat as a response to cold exposure
heat. (34, 175). During early mouse development, the mitochon-
Mitochondria and their chloroplast cousins are drial DNA (mtDNA) content remains constant from fertil-
unique among eukaryotic extranuclear organelles in that ization to the blastocyst stage after which DNA replica-
they contain their own genetic system. In vertebrates, this tion and organelle division resumes (167). Subsequent
system is based on a mitochondrial genome consisting of mitochondrial proliferation increases continuously through-
a circular double-stranded DNA (mtDNA) (Fig. 2). The out development, and tissue-specific factors are thought to
gene complement of mtDNA comprises but a small frac- dictate the final adult complement of mtDNA (90). The
tion of the number of genes necessary to account for the respiratory capacity of mitochondrial membranes is en-

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 (IV) 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 elec-
tron 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 cy-
tosolic 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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TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA 613

man pathological mutation in mtDNA, many such muta-


tions 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 neuromus-
cular system (55, 227). These mtDNA mutations are often
maternally inherited, and in some cases, patients with
certain mitochondrial myopathies exhibit excessive pro-
liferation 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

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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 mitochon-
drial respiratory chain contributes to the neuropathology
observed in dementia and other degenerative diseases.
FIG. 2. Schematic representation of the human mitochondrial ge- Although there is good evidence for oxidative stress as-
nome. Genomic organization and structural features of human mtDNA sociated with neuropathology, it has been difficult to
are depicted in a circular genomic map showing heavy (blue) and light
(black) strands assigned as such based on their buoyant densities. prove whether this is a cause or a consequence of neuro-
Protein coding and rRNA genes are interspersed with 22 tRNA genes nal death (5). The accumulation of mutations in mtDNA is
(red bars denoted by the single-letter amino acid code). Duplicate tRNA also thought to contribute to human ageing. Documented
genes for leucine (L) and serine (S) are distinguished by their codon
recognition (parentheses). The D-loop regulatory region contains the changes in mtDNA, including increased prevalence of
L- and H-strand promoters (LSP, HSP1, and HSP2), with arrows showing point mutations and deletions, have been observed in
the direction of transcription. The origin of H-strand replication (OH) is aged individuals (43). This along with the known age-
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 related decrease in oxidative energy metabolism has led
five tRNA genes (W, A, N, C, Y). Protein coding genes include the to the hypothesis that mtDNA mutations impair the respi-
following: cytochrome oxidase (COX) subunits 1, 2, and 3; NADH dehy- ratory chain leading to a decline in oxidative phosphory-
drogenase (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 lation (57).
transcribed from the L-strand as template are labeled on the inside of the Direct experimental support for this model comes
genomic map, whereas the remaining protein coding and RNA genes from recent studies in which a mtDNA polymerase that is
transcribed from the H-strand as template are labeled on the outside.
defective in proofreading function was substituted for the
normal polymerase in mice (213). Animals homozygous
for the defective allele had a mutator phenotype with
hanced postnatally as an adaptation to oxygen exposure
markedly increased levels of mtDNA point mutations and
outside the womb (217). These examples bear on a fun-
deletions. Although the mutations far exceed those asso-
damental question in understanding the biology of eu-
ciated with normal aging, the mutator mice had a reduced
karyotic cells, namely, what are the mechanisms of com-
life span and a number of physiological changes related to
munication between physically separated nuclear and mi-
premature aging. There has also been considerable inter-
tochondrial genetic systems in meeting cellular energy
est in mitochondrial dysfunction as a contributing factor
demands?
in the onset of type 2 diabetes (161). Insulin resistance in
healthy aged individuals with no family history has been
B. Pathophysiology correlated with a decline in mitochondrial oxidative phos-
phorylation (163). The expression of a number of genes
In the last 20 years, mitochondrial dysfunction has involved in oxidative metabolism is reduced in diabetic
been recognized as an important contributor to an array subjects as well as in those predisposed to diabetes be-
of human pathologies. Mitochondrial defects play a direct cause of family history (144, 162). Transcriptional activa-
role in certain well-defined neuromuscular diseases and tors and coactivators that regulate mitochondrial biogen-
are also thought to contribute indirectly to many degen- esis have been suggested as potential contributors to this
erative diseases. Since the identification of the first hu- phenomenon. In addition, mitochondrial functional insuf-

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614 RICHARD C. SCARPULLA

ficiency has been found in the insulin-resistant offspring and 2 ribosomal RNAs necessary for the translation of
of patients with type 2 diabetes (164). The fact that this these respiratory subunits within the mitochondrial ma-
occurs in healthy individuals that are not diabetic sug- trix. Mitochondrial genes lack introns and are arranged
gests that an inherent defect in oxidative phosphorylation end on end with little or no intergenic regions. Some
may be a contributing factor. These associations of mito- respiratory protein genes overlap, and the adenine nucle-
chondrial dysfunction with human degenerative disease otides of UAA termination codons are not encoded in the
raise the basic question of how mammalian cells control mtDNA but rather are supplied by polyadenylation follow-
mitochondrial biogenesis. It has become increasingly ap- ing RNA processing (152). Protein coding and rRNA genes
parent that transcriptional mechanisms contribute to the are interspersed with tRNA genes that are thought to
biogenesis of mitochondria including the expression of demarcate the cleavage sites of RNA processing. The only
the respiratory apparatus. Transcriptional control oper- substantial noncoding region is the D-loop, named after
ates through a subset of well-defined transcription factors the triple-stranded structure or displacement loop that is
and transcriptional coactivators. These regulators exert formed by association of the nascent heavy (H)-strand in
their influence on the expression of both nuclear and this region (Fig. 2). The D-loop contains the origin of

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mitochondrial genes required for the maintenance and heavy (H)-strand DNA replication and is also the site of
biogenesis of the organelle. 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
II. THE MITOCHONDRIAL GENETIC SYSTEM: gradient centrifugation. Interestingly, the structural econ-
mtDNA STRUCTURE AND INHERITANCE omy found in vertebrate mtDNA is not found in plants and
fungi where the mitochondrial genomes are much larger
It is long established from both genetic and biochem- and contain intergenic regions, introns, and multiple pro-
ical studies that the mitochondrial genetic apparatus has moters and transcriptional units (48, 169).
features that are distinct from that of the nucleocytosolic The fact that mtDNA is a compartmentalized extra-
system. Early work pointed to a protein synthetic machin- chromosomal element contributes to a mode of inheri-
ery in isolated mitochondria that could synthesize a small tance that differs from that of nuclear genes. Somatic
number of proteins utilizing mitochondrial ribosomes, mammalian cells generally have 103-104 copies of mtDNA
rRNA, and tRNA (45). A major advance was the discovery with 210 genomes per organelle (179). These genomes
of mitochondrial DNA. Genetic studies in yeast led to the replicate in a relaxed fashion that is independent of the
observation that certain respiratory mutations displayed a cell cycle that is defined by nuclear DNA replication (46,
cytoplasmic inheritance pattern that resulted from the 25). Some mtDNA molecules undergo multiple rounds of
random segregation of mtDNA molecules during mitosis. replication while others do not replicate. This, along with
This provided genetic evidence that mitochondria had random sampling during cell division, allows the segrega-
their own DNA before its existence was demonstrated tion of sequence variants during mitosis (198).
physically. This conclusion was supported by physical In mammals, mtDNA is maternally inherited (for ref-
evidence for the existence of mitochondrial DNA in yeast erences, see Refs. 101, 198). In general, paternal mtDNA is
and in other organisms (148). Since these early discover- lost during the first few embryonic cell divisions and does
ies, much has been done to define the structure and gene not contribute mtDNA to the offspring, although there are
organization of mtDNA. The first complete mtDNA se- reports of the presence of the paternal lineage in somatic
quence was obtained from humans and sequences of mi- tissues (192). In one case, recombination between mater-
tochondrial genomes from many organisms have now nal and paternal genomes has been documented (109). In
been catalogued (227). A striking result from this work is addition, because mtDNA is a multicopy genome, an in-
that a similar complement of genes is conserved in mtDNAs dividual may harbor more than a single sequence, a con-
from all multicellular organisms. dition referred to as heteroplasmy. A detrimental se-
In contrast to the nuclear genome, where repetitive quence variant may be tolerated in low copy because the
sequence families, introns, and vast intergenic regions defective gene product(s) it encodes do not reach the
account for all but a few percent of the total DNA, the threshold for disrupting cellular function. However, se-
mtDNA of mammals and other vertebrates exhibits strik- quence variants are known to segregate rapidly from het-
ing economy of sequence organization. The vertebrate eroplasmy to homoplasmy in passing from one generation
mitochondrial genome exists as a closed circular mole- to the next (12). This can result in offspring in which the
cule of 16.5 kb whose entire protein coding capacity is detrimental variant predominates, leading to a defective
devoted to the synthesis of 13 proteins that function as mitochondrial phenotype. The molecular basis for this
essential subunits for respiratory complexes I, III, IV, and rapid meiotic segregation has been ascribed to a bottle-
V (Figs. 1 and 2). The genes specifying complex II are neck or sampling error in the female germ line. A massive
entirely nuclear. The mtDNA also encodes the 22 tRNAs amplification of mtDNA occurs during oogenesis from

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TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA 615

103 copies in the primary oocyte to 105 copies in the ing a transcript that is processed to 1 mRNA and 8 of the
mature oocyte (198). Replication of mtDNA is halted in 22 tRNAs (Fig. 3A). In addition, the preponderance of
the mature oocyte, and the existing population of mtDNA evidence indicates that transcription from LSP is coupled
molecules is partitioned to the daughter cells during early to H-strand replication (25). Mapping studies support the
cell divisions until the copy number is diluted to approx- conclusion that transcripts truncated in the vicinity of
imately that found in somatic cells. Embryonic replication CSB II, one of three evolutionarily conserved sequence
does not resume until the blastocyst stage of development blocks (CSB I, II, and III), serve as primers for the initia-
(167). tion 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
III. TRANSCRIPTION OF mtDNA
(38). In addition, the nascent H-strand DNA 5-ends
closely match the 3-ends of truncated transcripts initi-
A. Mitochondrial Transcriptional Units ated at PL (37). It is widely accepted that primer RNAs are
generated by cleavage of L-strand transcripts by mito-

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In vertebrates, the D-loop regulatory region contains chondrial RNA processing (MRP) endonuclease at spe-
bidirectional promoters for transcribing H- and L-strands cific sites that match the in vivo priming sites (Fig. 3A).
as well as the H-strand replication origin (OH) (Fig. 2). This ribonucleoprotein endonuclease also participates in
The H- and L-strand transcriptional units differ from most the processing of 5.8S rRNA precursors and contains a
nuclear genes in that they are polygenic, specifying mul- nucleus-encoded RNA that is essential for catalysis (MRP
tiple RNAs (rRNA, tRNA, or mRNA). L-strand transcrip- RNA) (158, 197). A recent alternative model, derived from
tion is initiated at a single promoter (LSP), and RNA in vitro experiments using purified components, suggests
synthesis can traverse the entire mtDNA template produc- 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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616 RICHARD C. SCARPULLA

account for the primer RNA 3-ends that mark the major scription and its potential functional implications for mi-
transition sites between RNA and DNA synthesis (165). In tochondrial biogenesis await further characterization.
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 B. Tfam
long polygenic transcripts that are processed to give 14
tRNAs, 12 mRNAs, and the 2 rRNAs. HSP1 on the other The region of the D-loop that separates the LSP from
hand appears specialized for the production of ribosomal the two HSPs contains conserved nucleotide sequence
RNAs (Fig. 3A). As discussed below, termination events motifs that define the core promoter and mediate bidirec-
coupled to initiation at HSP1 provide a mechanism for tional transcription (212). In addition, the promoters
controlling the relative abundance of rRNA to mRNA. share an upstream enhancer that binds Tfam (previously
Transcription of mtDNA is completely dependent on mtTF-1 and mtTFA), the first well-characterized transcrip-
nucleus-encoded gene products. In yeast, transcription is tion factor from vertebrate mitochondria (Fig. 3A). Tfam
directed by a single subunit RNA polymerase (RPO41p), was first identified as a high mobility group (HMG)-box

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which shares sequence similarities with the T7 and T3 protein that stimulates transcription through specific
bacteriophage polymerases (104, 134). Although RPO41p binding to recognition sites upstream from both LSP and
can initiate transcription nonselectively from synthetic HSP (69). Structurally, it consists of two tandemly ar-
DNA templates, the specificity factor Mtf1p (discussed ranged HMG motifs and a COOH-terminal tail. Tfam re-
below) is required for promoter recognition and selective sembles other HMG proteins, in that it can bend and
initiation (104). Interestingly, RPO41p, through its inter- unwind DNA, properties potentially linked to its ability to
actions with mitochondrial promoters and nucleoside stimulate transcription upon binding DNA (159, 70). Tet-
triphosphates, may act as a sensor of ATP availability. rameric binding of Tfam to its recognition site is thought
Mitochondrial transcript abundance is correlated with to promote bidirectional transcription by facilitating sym-
respiration-coupled ATP synthesis through a requirement metrical interactions with other transcriptional compo-
for a high ATP concentration for transcription initiation. nents (7). In addition to specific promoter recognition,
This represents a potentially novel and elegant mecha- Tfam binds nonspecifically to apparently random sites on
nism for linking energy production to the transcription of mtDNA (70, 71). This property, along with its abundance
respiratory subunits (4). in mitochondria, suggests that it plays a role in the stabi-
Although purification of the human polymerase to lization and maintenance of the mitochondrial chromo-
homogeneity has been elusive, a human cDNA that en- some. ABF2p, a related HMG-box factor from yeast, re-
codes a protein with sequence similarity to yeast mito- sembles Tfam and is required for both mtDNA mainte-
chondrial and T3/T7 bacteriophage polymerases was nance and respiratory competence (54). Expression of
identified in database screenings (211). As expected for a human Tfam in ABF2p-deficient yeast cells can rescue
bona fide mitochondrial polymerase, the 140-kDa protein both phenotypes (160). Thus, although highly divergent in
product, designated POLRMT, is localized to mitochondria primary structure, the human and yeast proteins are func-
via an NH2-terminal targeting presequence. The utilization of tionally interchangeable in supporting mitochondrial re-
single subunit bacteriophage-related polymerases seems spiratory function in yeast. However, despite this func-
nearly universal for mitochondrial transcription. A potential tional complementation, ABF2p lacks an activation do-
link between mitochondrial transcription and translation main and does not stimulate transcription from yeast
in mammalian cells is implicated by the finding of a direct mitochondrial promoters in vitro (237, 50). The transcrip-
interaction between POLRMT and mitochondrial ribosomal tional activation function of Tfam resides in a COOH-
protein L12 (MRPL12) (231). MRPL12 stimulates transcrip- terminal activation domain that is required both for tran-
tion from both LSP and HSP promoters in an in vitro scriptional competence and for specific binding to pro-
transcription system. Moreover, it exists in a complex moter recognition sites (50).
with POLRMT in HeLa cell extracts, and its in vivo over- As discussed below, a Tfam knockout mouse dis-
expression can enhance the steady-state levels of mtDNA- plays embryonic lethality and a depletion of mtDNA con-
encoded transcripts. It is not yet clear whether this occurs firming an essential role for the protein in mtDNA main-
through effects on transcription or RNA stability. Finally, tenance in mammals (113). Interestingly, Tfam levels cor-
an alternative splice variant of POLRMT that is missing relate well with increased mtDNA in ragged-red muscle
the NH2-terminal 262 amino acids including the mitochon- fibers and decreased mtDNA levels in mtDNA-depleted
drial targeting presequence has been identified (108). Al- cells (168). This is consistent with the observation that
though it is currently unclear whether this protein func- transgenic mice overexpressing human Tfam display in-
tions as an independent nuclear polymerase, it has been creased mtDNA copy number (59). The mtDNA abun-
localized to the nucleus and linked to the expression of a dance measured in somatic tissues and in embryos is
number of nuclear genes. Its precise role in nuclear tran- proportional to the amount of Tfam expressed in each,

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TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA 617

suggesting that Tfam can function as a limiting determi- recombinant proteins (65). TFB1M has been detected in a
nant of mtDNA copy number. Because human Tfam is a complex with Tfam and POLRMT in transcriptionally
poor activator of mouse LSP and HSP, overexpression of competent mitochondrial extracts. Moreover, both TFB
the human protein results in an increase in mtDNA with- isoforms make direct contact with the COOH-terminal
out affecting respiratory capacity, suggesting that copy activation domain of Tfam (139). These observations sup-
number control by Tfam is independent of its transcrip- port the notion that mtDNA transcription in vertebrate
tional function. In fact, both endogenous Tfam and a systems is directed by three essential components (Fig.
mutated derivative that lacks the COOH-terminal activa- 3A). Interestingly, like the yeast factor, both TFBs are
tion domain are equally competent in maintaining mtDNA related to rRNA methyltransferases. TFB1M can bind
copy number in cultured cells (103). These observations S-adenosylmethionine and methylate rRNA at a stem-loop
are reminiscent of Abf2p, which does not direct transcrip- structure that is conserved between bacterial and mito-
tion but is required for mtDNA maintenance (54). Abf2p is chondrial rRNAs (195). However, this activity is not re-
an abundantly expressed component of mitochondrial quired for transcriptional activation by the factor (139). It
nucleoids, protein-DNA complexes thought to be a basic has yet to be determined whether the proteins are bifunc-

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segregating unit of mtDNA (105). Tfam is also expressed tional or whether they evolved a single function from an
at levels high enough to coat mtDNA and has been recov- ancestral methyltransferase.
ered in vertebrate nucleoids in association with other Recent experiments suggest that the two TFB iso-
proteins required for mtDNA genomic integrity and expres- forms are not functionally identical. At early times follow-
sion (26, 102, 230). Thus both its in vivo and in vitro ing serum stimulation of quiescent fibroblasts, mRNA for
properties implicate Tfam as an ideal target for regulatory the TFB1M isoform is transiently downregulated relative
pathways that control both mtDNA maintenance and tran- to that of the TFB2M isoform, suggesting that the latter is
scriptional expression. favored in the transition to proliferative growth (73). In
Drosophila cultured cells, RNAi knockdown of the Dro-
sophila B2 isoform results in reduced mtDNA transcrip-
C. mtTFB tion and copy number (136). This contrasts with RNAi
knockdown of the B1 isoform, which has no effect on
In yeast, RPO41p associates with a 43-kDa specificity mtDNA transcription or replication but does result in
factor MTF1p also known as sc-mtTFB (196). The primary reduced mitochondrial translation (135). It is also notable
structure of sc-mtTFB bears some resemblance to pro- that overexpression of either TFB2M or Tfam in this
karyotic sigma factors (99), but a crystal structure reveals system increases mtDNA copy number, whereas overex-
significant homology to rRNA methyltransferase (191). pression of TFB1M fails to do so. The stimulatory effect of
Although individually neither RPO41p nor MTF1p can Tfam is consistent with the observation that overexpres-
interact with yeast mitochondrial promoters, together sion of human Tfam in mice increases mtDNA copy num-
they engage in specific promoter recognition (104). Evi- ber (59). Thus both gain- and loss-of-function experiments
dence for a vertebrate factor that functions in analogous support a role for Tfam and TFB2M in mtDNA copy
fashion to the yeast sc-mtTFB came from biochemical number control. The inability of TFB1M to function in a
studies in Xenopus laevis (24, 24). The partially purified similar capacity in Drosophila cells is surprising in light of
factor bound DNA nonspecifically and stimulated tran- the ability of the mammalian protein to bind the transcrip-
scription in vitro by purified mitochondrial RNA polymer- tion activation domain of Tfam and stimulate transcrip-
ase. Although purification and molecular cloning of the tion in vitro.
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 D. Transcription Termination and Mitochondrial
and stimulates transcription from an L-strand promoter Gene Expression
in vitro, properties consistent with it being a functional
homolog of sc-mtTFB. The human protein exhibits non- Specific termination events play an important role in
sequence-specific DNA binding and requires Tfam to stim- governing the steady-state levels of mitochondrial tran-
ulate transcription from mitochondrial templates in vitro. scripts. The high rate of rRNA synthesis relative to mRNA
Two isoforms of h-mtTFB, termed TFB1M and TFB2M, has been ascribed to more frequent initiation of H-strand
have been identified independently (65). TFB1M is iden- transcription at HSP1. As shown in Figure 3A, HSP1 di-
tical to the original mtTFB and has about 1/10 the tran- rects transcription from a site within the tRNAPhe gene.
scriptional activity of TFB2M. As depicted in Figure 3A, Most transcripts initiated at HSP1 terminate downstream
both proteins work together with Tfam and mtRNA poly- from the 16S rRNA gene at a strong bidirectional termi-
merase to direct proper initiation from H- and L-strand nator localized within the adjacent tRNALeu gene (44).
promoters in an in vitro system comprised of purified The 28-nucleotide terminator binds mTERF, a 34-kDa pro-

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618 RICHARD C. SCARPULLA

tein that specifies site-specific transcription termination analysis of the cytochrome c control region revealed a
in fractionated mitochondrial lysates (49). Although the palindromic recognition site for a transcription factor
purified recombinant protein displays the expected bind- designated as nuclear respiratory factor 1 (NRF-1) (63)
ing specificity, it is not sufficient for termination, suggest- (Fig. 4). Specific NRF-1 binding sites are present in the
ing that an additional component(s) may be required (66). promoters of several nuclear genes required for mito-
Interestingly, in addition to binding the termination site, chondrial respiratory function (39, 64). The protein binds
mTERF stimulates transcription from HSP1, suggesting its recognition site as a homodimer through a unique DNA
that transcription initiation and termination are coupled binding domain and functions as a positive regulator of
(13, 111). Recent experiments demonstrate that this is transcription (78, 222). This is consistent with the pres-
indeed the case. Multiple lines of in vitro and in vivo ence of a COOH-terminal transcriptional activation do-
evidence establish that mTERF binds simultaneously main (Fig. 4) comprised of glutamine-containing clusters
both the HSP1 initiation site and the terminator resulting of hydrophobic amino acid residues (79). NRF-1 exists as
in the looping out of intervening rDNA (Fig. 3B). These a phosphoprotein in proliferating mammalian cells and
interactions are proposed to enhance the reinitiation rate serine phosphorylation within a concise NH2-terminal do-

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at HSP1 resulting in a higher rate of rRNA synthesis. This main (Fig. 4) enhances both its DNA binding (78) and
mechanism likely accounts for the stimulatory effects of trans-activation functions (92). Although other mamma-
mTERF on transcription (133). Additional complexity to lian NRF-1 isoforms have not been found, NRF-1 is re-
mTERF function is suggested by the observation of sev- lated, through its DNA binding domain, to developmental
eral mTERF-related genes in vertebrates (129). The pro- regulatory proteins in sea urchins (32) and Drosophila
tein products all have putative mitochondrial targeting (53). In addition, chicken (74), zebrafish (20), and mouse
signals and in one case mitochondrial localization has (187) homologs of NRF-1 have been characterized.
been confirmed (40). Interestingly, this isoform, desig- NRF-1 has now been linked to the expression of
nated as mTERFL, differs from mTERF in that it is down- many genes required for mitochondrial respiratory func-
regulated upon serum induction of serum-starved cells. tion including the vast majority of nuclear genes that
This is reminiscent of the differential expression of encode subunits of the five respiratory complexes (for
TFB1M and TFB2M in response to serum stimulation (73). recent compilations, see Refs. 106, 183, 184). In addition,
It is possible that functionally distinct transcription com- considerable evidence supports a potential integrative
plexes arise from the differential expression of alternative function for NRF-1 in coordinating respiratory subunit
isoforms of these transcription initiation and termination expression with that of the mitochondrial transcriptional
factors.

IV. NUCLEAR CONTROL OF NRF-1


MITOCHONDRIAL FUNCTION
1 DNA binding trans-activation 503

A. Nuclear Transcription Factors Governing


NLS A B
Respiratory Gene Expression
serine
1. Cytochrome c and the identification of NRF-1 phosphorylation

Isolation of cytochrome c genes in yeast and subse-


quently in mammalian cells opened the way to the molec- YGCGCAYGCGCR
ular analysis of the control of nuclear genes governing RCGCGTRCGCGY
mitochondrial respiratory function. In yeast, transcrip-
100
100
100

100
85
90

95
75

85
80
80

95

tional regulation of the two cytochrome c isoforms is


mediated through upstream enhancer elements within
FIG. 4. Summary of nuclear respiratory factor (NRF)-1 functional
their promoters (77). These enhancers bind specific tran-
domains and DNA recognition site. NRF-1 has a central DNA binding
scriptional activators and repressors that direct dramatic domain (stippled box) flanked by a nuclear localization signal (horizon-
changes in gene expression in response to oxygen and tally hatched box) and a bipartite transcriptional activation domain
carbon source availability (169, 242). Although cyto- (vertically hatched boxes). The protein is phosphorylated in exponen-
tially growing cells at multiple serine residues within a concise NH2-
chromes c are highly conserved at the functional level terminal domain (cross-hatched box). NRF-1 binds a GC-rich palin-
between yeast and mammals (186), the mammalian cyto- drome (shown below the diagram) as a homodimer and makes guanine
chrome c promoter contains recognition sites for tran- nucleotide contacts (filled circles) over a single turn of the DNA helix.
Numbers below the NRF-1 consensus binding site represent the percent-
scription factors that bear no obvious relationship to age of the time the indicated nucleotide is present at that position in 20
those identified in yeast (62). In particular, systematic functional NRF-1 sites. Y, pyrimidine nucleotide; R, purine nucleotide.

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TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA 619

machinery. As illustrated in Figure 5, NRF-1 binds and dase assembly factor (206). NRF-1 control over key com-
activates the promoters of Tfam (224) and both TFB ponents of the protein import and assembly machinery is
isoform genes (73) whose products (as discussed above) suggestive of a broader role for the factor in orchestrating
are major regulators of mitochondrial transcription. events in mitochondrial biogenesis beyond the transcrip-
NRF-1 has also been linked to the expression of 5-amin- tional expression of the respiratory chain.
olevulinate synthase (30) and uroporphyrinogen III syn- This hypothesis is reinforced by reports that associ-
thase (2), key enzymes of the heme biosynthetic pathway. ate elevations in NRF-1 mRNA or DNA binding activity
The former is the rate-limiting enzyme in the biosynthesis with generalized effects on mitochondrial biogenesis.
of heme, a cofactor essential to the function of electron Both NRF-1 and its coactivator PGC-1 (see below) are
carriers encoded by both genomes (Fig. 5). Moreover, upregulated during the adaptive response of skeletal mus-
NRF-1 acts on genes whose functions are not restricted to cle to exercise training (15, 147). A similar effect which
the bigenomic expression of the respiratory apparatus mimics exercise-induced mitochondrial biogenesis oc-
(Fig. 5). The outer membrane multisubunit receptor com- curs in cultured myotubules in response to increased
plex designated as TOMM is required for the import of the calcium (154). Treatment of rats with a creatine analog,

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thousands of proteins that contribute to diverse mito- which induces muscle adaptations resembling those ob-
chondrial functions (214). TOMM20 is a key receptor served during exercise, leads to the activation of AMP-
subunit involved in the initial interaction with precursor activated protein kinase. This coincides with increased
proteins targeted to mitochondria. NRF-1 activates NRF-1 DNA binding activity, cytochrome c content, and
TOMM20 gene transcription through a specific promoter mitochondrial density (21). Both NRF-1 and Tfam mRNAs
recognition site and is bound to the TOMM20 promoter increase in cells depleted of mtDNA, presumably as a
region in vivo (23). Similarly, NRF-1 is implicated in the response to increased oxidative stress (141). NRF-1 and
expression of mouse COX17, a putative cytochrome oxi- 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 IV, 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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620 RICHARD C. SCARPULLA

in response to lipopolysaccharide-induced oxidative dam- This along with derepression by release of E2F factors
age to mitochondria, presumably to enhance mtDNA lev- from a subset of NRF-1 target genes may help promote
els and OXPHOS activity (203). Exogenous oxidants re- cell proliferation. Notably, NRF-1 has also been impli-
store NRF-1 and normal cell growth to o hepatoma cells cated in the transcriptional repression of E2F1 (58) and
where reduced oxidant levels are associated with loss of the activation of E2F6 (107). These results are consistent
NRF-1 and growth delay. Interestingly, NRF-1 occupancy with a role for NRF-1 in regulating cell cycle progression
of the Tfam promoter increases under pro-oxidant condi- and may explain the early embryonic lethality of NRF-1
tions, and the control of Tfam expression by NRF-1 is knockout embryos (96).
blocked by inhibition of NRF-1 phosphorylation by Akt
(166). This suggests that redox regulation of NRF-1 target 2. NRF-2 (GABP) an activator of cytochrome
genes such as Tfam is modulated by redox-regulated oxidase expression
phosphorylation events. Finally, both NRF-1 (DNA bind-
ing activity and mRNA) and Tfam (protein and mRNA) are A second nuclear factor designated as NRF-2 was
upregulated in the skeletal muscle of aged human sub- identified based on its specific binding to essential cis-

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jects (120). This may be a compensatory response to acting elements in the cytochrome oxidase subunit IV
age-related reductions in energy metabolism. These asso- (COXIV) promoter (181, 182). NRF-2 binding sites con-
ciations are consistent with a general integrative role for tained of the GGAA core motif that is characteristic of the
NRF-1 in nucleomitochondrial interactions. ETS-domain family of transcription factors. Direct re-
It is important to note that NRF-1 targets are not peats of this sequence motif are interspersed with multi-
restricted to genes involved in mitochondrial function. An ple transcription initiation sites within the mouse COXIV
initial search for NRF-1 binding sites in mammalian pro- promoter (223, 36). Human NRF-2 was purified to homo-
moters revealed a number of primate and rodent genes geneity from HeLa cell nuclear extracts and is comprised
whose functions are not linked directly to mitochondrial of five subunits (Fig. 6). These include a DNA-binding
biogenesis (222). Among these are genes encoding meta- subunit and four others (1, 2, 1, and 2,) that complex
bolic enzymes, components of signaling pathways, and with but alone do not bind DNA. Molecular cloning of
gene products necessary for chromosome maintenance the five NRF-2 subunits (80) revealed that NRF-2 is the
and nucleic acid metabolism among others. Moreover, human homolog of mouse GABP (112). The two addi-
NRF-1 is among seven identified transcription factors tional human subunits, 1 and 1, are minor splice vari-
whose recognition sites are most frequently found in the ants of GABP subunits 1 and 2 (80). The GABP 1
proximal promoters of ubiquitously expressed genes (72). subunit, corresponding to NRF-2 1 and 2 (80), has a
Recently, chromatin immunoprecipitations (ChIP) coupled dimerization domain that facilitates cooperative binding
with microarray assay (ChIP-on-chip) were used to identify of a heterotetrameric complex to tandem binding sites
human promoters that are bound by NRF-1 in vivo (33). (210). In solution, GABP exists as a heterodimer but is
Survey of 13,000 human promoters by ChIP-on-chip induced to form the heterotetramer 22 by DNA contain-
analysis (174) identified 691 genes whose promoters are ing two or more binding sites (41). The crystal structure
occupied by NRF-1 in living cells (33). As expected, a of the heterotetramer bound to DNA has been determined
majority of these genes are involved in mitochondrial (19). All of the non-DNA-binding subunits contain a tran-
biogenesis and metabolism, including many that had not scriptional activation domain (Fig. 6). This domain resem-
been previously identified. These include a collection of bles that found in NRF-1 and has been localized to a
mitochondrial ribosomal protein and tRNA synthetase region upstream from the homodimerization domain (79).
genes. Notably, a significant subset of the NRF-1 target As observed for COXIV, COXVb transcription is also
genes was also bound by the growth regulatory transcrip- activated by NRF-2 through directly repeated recognition
tion factor E2F, suggesting that NRF-1 participates in the sites within the proximal promoter, suggesting that NRF-2
regulation of a subset of E2F-responsive genes. This sub- is a general activator of cytochrome oxidase subunit gene
set was enriched in genes required for DNA replication, expression (225). Several lines of evidence point to a
mitosis, and cytokinesis. Although NRF-1 was bound to its direct role for NRF-2 in the expression of all 10 nucleus-
target promoters under conditions of transcriptional re- encoded cytochrome oxidase subunits. NRF-2 has been
pression, an NRF-1 siRNA reduced expression of several associated in vivo with multiple COX subunit promoters
E2F target genes along with Tfam and cytochrome c, by chromatin immunoprecipitation (157). Moreover, ex-
confirming that it functions as a transcriptional activator. pression of a dominant negative NRF-2 allele reduced
NRF-1 exists in the dephosphorylated state in serum- COX expression, and an siRNA directed against NRF-2
starved cells and is phosphorylated upon serum addition reduced expression of all 10 nucleus-encoded COX sub-
(78). Since phosphorylation enhances NRF-1 transcrip- units (156). These findings are consistent with an impor-
tional activity (92), it is possible that phosphorylation tant regulatory function for NRF-2 in cytochrome oxidase
controls the functional state of the DNA bound factor. expression.

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TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA 621

DNA binding

(51.4 kDa) ETS


hetero- trans- homo-
dimerization activation dimerization

(42.5 kDa)
1
VNLTGLVSSENSSKATDETG
(41.3 kDa)
2

1 (38.1kDa)

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2 (36.9 kDa)

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 (22) 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 activities (Fig. 7). Both NRF-1 and NRF-2 occupy both
NRF-2 (GABP) and cytochrome oxidase expression in TFB1M and -2M promoters in vivo as determined by chro-
the visual cortex (149). NRF-2 and cytochrome oxidase matin immunoprecipitation (73). Thus, like NRF-1, NRF-2
were associated with metabolic state and neuronal activ- participates in coordinating the expression of essential
ity in mature neurons, suggesting that NRF-2 is impor- respiratory chain proteins with key components of the
tant in maintaining neuronal function. Moreover, the cor- mitochondrial transcription machinery. Such a mecha-
relation was extended to NRF-2 mRNA, indicating that nism may serve to ensure the coordinate bigenomic ex-
NRF-2 may be regulated at the transcriptional level by pression of respiratory subunits.
neuronal activity (82). Both NRF-2 and - subunits are
enriched in the nucleus in response to neuronal stimula- 3. ERR
tion (241), and both subunits are specifically translocated
to the nucleus in response to neuronal depolarization The estrogen-related receptor ERR has been linked
(238). In fact, the neuronal factor heregulin directs the to the regulation of oxidative metabolism. ERR, one of a
threonine phosphorylation of GABP, stimulating both its family of orphan nuclear receptors including ERR and
transcriptional activity and its mobilization to the nucleus ERR, resembles the estrogen receptor but does not bind
where it activates transcription of acetylcholine receptor estrogen or other ligands. ERR levels are high in oxida-
subunits (188, 204). tive tissues such as kidney, heart, and brown fat, and it
In addition to the COX promoters, functional NRF-2 acts as a regulator of -oxidation via its control of the
sites have been identified in a number of other genes medium-chain acyl-coenzyme A dehydrogenase (MCAD)
related to respiratory chain expression (for a recent com- promoter (97). The - and -isoforms increase postnatally
pilation, see Refs. 106, 183). These include genes for Tfam in the heart along with enzymes promoting mitochondrial
(113, 173) as well as TFB1M and TFB2M (138, 65) (Fig. 7). fatty acid uptake and oxidation (98). ERR knockout
Three of the four human succinate dehydrogenase (com- mice have reduced fat mass and are resistant to diet-
plex II) subunit genes also have both NRF-1 and NRF-2 induced obesity, suggesting defects in lipid metabolism
sites in their promoters (14, 60, 94). In many cases, NRF-1 (132). Recent studies have also implicated ERR in PGC-1-
sites are also present in NRF-2-dependent promoters, but induced mitochondrial biogenesis (143, 190). A computa-
this is not a general rule. For example, several COX tional approach coupled with PGC-1-induced RNA ex-
promoters and the rodent Tfam (42) and TFB (172) pro- pression profiles identified both ERR and GABP in a
moters do not have obvious NRF-1 consensus sites. This double positive-feedback loop with PGC-1 in directing
contrasts with the human Tfam (224) and TFB (73) pro- expression of a subset of mitochondria-related genes
moters, which rely on both NRF-1 and NRF-2 for their (143). An inhibitor of ERR attenuated PGC-1-mediated

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622 RICHARD C. SCARPULLA

series of enzymes within the mitochondrial matrix that


oxidize fatty acids to acetyl CoA. To date, the transcrip-
tional 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 super-
family, have been implicated as transcriptional regulators
of fatty acid oxidation enzymes in tissues with high rates
of fat oxidation including heart, kidney, and skeletal mus-
cle (17, 81, 97). Conversely, the PPARs have not been
associated with the expression of the respiratory appara-
tus. This suggests that higher order integration of diverse
transcription factors may be necessary to coordinate the
large number of genes required for mitochondrial biogen-

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esis 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 ex-
pression of the two pathways in certain physiological
contexts.

5. Other nuclear factors


FIG. 7. Arrangement of NRF-1 and NRF-2 recognition sites in hu-
man promoters specifying key components of the mitochondrial tran- Additional nuclear factors have been implicated in the
scription and replication machinery. Shown are linear representations of
the 5-flanking regions proximal to the putative transcription initiation expression of respiratory genes. The cytochrome c promoter
sites (arrows) of human genes encoding Tfam, TFB1M, TFB2M, and contains cis-elements that recognize transcription factors
POL2. Each promoter region bears a strong resemblance to a number of the ATF/CREB family (63, 75). These elements bind
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, CREB both in vitro and in vivo, and phosphorylation of
each promoter has at least one Sp1 recognition site (gray bars). CREB and NRF-1 is linked to the serum induction
of cytochrome c in quiescent fibroblasts (92, 220). Al-
though CREB sites are common to cAMP- and growth-
target gene expression and total cellular respiration. In activated genes (137), these elements are not a general
this scheme, NRF-1 was downstream of the ERR-di- feature of nuclear respiratory promoters. Cytochrome c
rected changes in expression. It should be noted that appears to be limiting for mitochondrial respiration early
PGC-1 was abundantly overexpressed from an adenovi- in the program of serum-induced entry to the cell cycle
rus vector in many of the experiments utilized to validate (92). CREB activation of cytochrome c expression likely
this model. This generally results in massive changes in contributes to its relatively rapid induction in response to
mitochondrial biogenesis that are not observed in genetic serum-stimulated growth. This may be a mechanism for
knockouts of PGC-1 or ERR (discussed below). Never- ramping up cellular respiration in preparation for cell
theless, ERR does mediate the effects of virally ex- growth and division.
pressed PGC-1 on mitochondria-related genes such as Maximal cytochrome c promoter activity also de-
Tfam and ATP synthase -subunit (190). It is also of pends on synergy between functional recognition sites for
interest to note that PGC-1 functions through NRF-2 the general transcription factor Sp1 (63). Sp1 is also in-
(GABP) in the control of a program of neuromuscular volved in the activation and/or repression of cytochrome
gene expression in skeletal muscle (85). Thus these fac- c1 (125) and adenine nucleotide translocase 2 genes (124),
tors likely coordinate broad adaptive changes beyond both of which lack NRF sites (240). Muscle-specific COX
their initially observed functions in respiratory chain ex- subunit genes, in contrast to their ubiquitously expressed
pression. isoforms (193), depend on MEF-2 and/or E-box consensus
elements for their tissue-specific expression (228). The
4. PPARs and fatty acid oxidation initiator element transcription factor YY1 has been impli-
cated in both positive and negative control of cytochrome
A number of mitochondrial oxidative pathways feed oxidase subunit gene expression (18, 194). Interestingly,
into the respiratory apparatus. Important among these is YY1 knockout mice resemble the NRF-1 and NRF-2 knock-
the pathway for fatty acid oxidation, which consists of a outs in that they exhibit peri-implantation lethality (56).

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TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA 623

More recently, c-Myc has been linked to respiratory matrix where they were thought to act as negative regu-
gene expression and mitochondrial biogenesis. C-Myc can lators of mitochondrial mRNA expression (47). These
induce cytochrome c and other NRF-1 target genes by seemingly contradictory results illustrate the difficulty in
binding noncanonical Myc/MAX binding sites contained assigning function based on subcellular localization. It is
within certain NRF-1 sites and can sensitize cells to apop- important to note that the transcriptional activity and
tosis by dysregulating NRF-1 target genes (146). A domi- specificity of these shared nuclear factors using mtRNA
nant negative NRF-1 allele can block this c-Myc-induced polymerase and a defined mtDNA template has not been
apoptosis without affecting c-Myc-induced cell prolifera- demonstrated. Thus it remains inconclusive as to whether
tion. In addition, Myc null fibroblasts were found deficient these nuclear factors are true regulators of mitochondrial
in mitochondrial content, and c-Myc expression in these transcription.
cells partially restored mitochondrial mass (121). This
along with the observation that many mitochondria-re-
B. Nuclear Coactivators in
lated genes including Tfam are c-Myc targets led to the
Mitochondrial Biogenesis
conclusion that c-Myc can regulate mitochondrial biogen-

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esis. One caveat to this conclusion is that c-Myc is esti-
Most of the evidence supports a model whereby a
mated to have an enormous number of genome binding
relatively small number of nuclear transcription factors
sites that even exceeds the number of Myc molecules in
serve to coordinate the expression of nuclear and mito-
proliferating cells. Thus it is thought that only a minority
chondrial respiratory proteins. Of these, the NRFs, Sp1,
of genes bound by Myc/MAX are actually regulated by
and ERR have been consistently associated with the
c-Myc (1). In addition, because of the extremely large
majority of genes required for respiratory chain expres-
number of Myc target genes, it is possible that the ob-
sion. These factors exert direct control over transcription
served effects on mitochondrial biogenesis result from
of the nucleus-encoded respiratory subunits and act indi-
indirect effects of Myc on cell growth and metabolism.
rectly on the mitochondrial subunits via their regulation
of mitochondrial transcription factors. In addition, other
6. Dual function factors mitochondrial oxidative pathways are controlled by unre-
lated factors as exemplified by PPAR and the fatty acid
There are several reports suggesting that certain nu-
oxidation pathway (81). This raises the question of how
clear transcription factors or their derivatives have a dual
diverse transcription factors are integrated into a program
function in directing mitochondrial gene expression.
of mitochondrial biogenesis. The discovery of the PGC-1
Binding sites for ligand-dependent trans-activators have
family of transcriptional coactivators has provided a
been observed in mtDNA (51, 234). In the best-studied
mechanistic framework for understanding how nuclear
example, a 43-kDa protein, closely related to the c-Erb
regulatory pathways are coupled to the biogenesis of
A1 thyroid hormone receptor, has been localized to the
mitochondria.
mitochondrial matrix and observed to bind the mitochon-
drial D-loop in vitro (234). This protein appears to be an
1. PGC-1
NH2-terminally truncated variant of the nuclear 1 thyroid
hormone receptor. The overexpressed variant gives a par- PGC-1, the founding member of a family of tran-
ticulate cytosolic immunofluorescence and stimulates mi- scriptional coactivators, was identified in a differentiated
tochondrial respiration as measured by rhodamine stain- brown fat cell line on the basis of its interaction with
ing and cytochrome oxidase activity. The functional sig- PPAR, an important regulator of adipocyte differentia-
nificance of this finding is supported by the observation of tion (171). Although it apparently lacks histone-modifying
a direct effect of thyroid hormone on mitochondrial RNA activities itself, it interacts, through a potent NH2-terminal
synthesis (61). Similarly, the rat liver glucocorticoid re- transcriptional activation domain, with cofactors contain-
ceptor (GR) was found to translocate to the mitochondria ing intrinsic chromatin remodeling activities (Fig. 8). In
upon treatment of animals with dexamethasone (52). Six addition to PPAR, PGC-1 binds several nuclear hor-
glucocorticoid response elements (GREs) were found in mone receptors and trans-activates the PPAR- and thy-
mtDNA and were shown to bind GR in vitro (51). Two of roid receptor -dependent expression of the UCP-1 pro-
the six elements are present in the D-loop, but the other moter. Nuclear hormone receptor coactivator signature
four are within the COXI and COXIII genes, far removed motifs (LXXLL) adjacent to the activation domain are
from the in vivo sites of mtDNA transcription initiation. essential for coactivation through certain nuclear recep-
IB- and NFB have also been localized to mito- tors, including PPAR and thyroid receptor (Fig. 8).
chondria (29, 47). In one case, they were found in the Coupling of transcription and RNA processing by PGC-1
intermembrane space associated with the adenine nucle- occurs through COOH-terminal arginine/serine rich (R/S)
otide translocator and were released upon induction of and RNA recognition motifs reminiscent of those found in
apoptosis (29). In the other, they were localized to the RNA splicing factors (142). The dramatic induction of

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624 RICHARD C. SCARPULLA

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 ele-
vated 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 car-
diomyopathy 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 mitochon-
FIG. 8. Summary of PGC-1 functional domains. Depicted is a linear
drial oxidative functions by inducing the expression of
representation of PGC-1 showing key functional domains. The NH2- genes of the mitochondrial fatty acid oxidation and heme

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terminal activation domain (vertical hatched box) is the site of interac- biosynthetic pathways (Fig. 9). As we have seen, PPAR
tion with histone-modifying cofactors represented by SRC-1 and CBP/
p300. The approximate positions for interaction with transcription fac-
directs the transcriptional expression of fatty acid oxida-
tors including nuclear hormone receptors, PPAR and NRF-1, and tion enzymes and is enriched in brown fat and other
MEF2C are indicated above and below the map. Also illustrated is the tissues with high oxidative energy demands. Ligand-de-
central proline-rich region (cross-hatched box) and the COOH-terminal
domain comprised of the arginine/serine-rich R/S domain (open box)
pendent binding of PPAR through the LXXLL motif of
and the RNA recognition motif (horizontal hatched box) proposed to PGC-1 is associated with the trans-activation of PPAR-
interact with RNA processing factors. The region containing p38 MAPK dependent promoters (97, 218). In addition, overexpres-
phosphorylation sites is bracketed below.
sion of PGC-1 induces MCAD gene expression through
its direct interaction with ERR (98). PGC-1 can also
PGC-1 mRNA in brown fat upon cold exposure supports utilize both NRF-1 and FOXO1, a forkhead box family
its involvement in thermogenic regulation (126, 170). An member, to induce transcription of -aminolevulinate syn-
important part of the thermogenic program is the induc- thase, the first and rate-limiting enzyme of the heme bio-
tion of mitochondrial biogenesis, and PGC-1 is a potent synthetic pathway (86). Thus, as summarized in Figure 9,
inducer of this process. Ectopic overexpression of the PGC-1 has the potential to integrate the activities of a
coactivator in cultured myoblasts and other cells induces diverse collection of transcription factors that have been
respiratory subunit mRNAs and increases COXIV and cy- implicated in the expression and function of the mito-
tochrome c protein levels as well as the steady-state level chondrial oxidative machinery.
of mtDNA (236). Physiological expression of PGC-1 at the transcrip-
As illustrated in Figure 9, NRF-1 has been identified tional level can be modulated through cAMP-dependent
as an important target for the induction of mitochondrial signaling. In brown adipocytes, sympathetic production
biogenesis by PGC-1. The coactivator binds NRF-1 and of norepinephrine acts predominantly through 3-adren-
can trans-activate NRF-1 target genes involved in mito- ergic receptors (34). These receptors are coupled to ade-
chondrial respiration. Moreover, a dominant negative al- nyl cyclase via the Gs subtype of G proteins. The thermo-
lele of NRF-1 blocks the effects of PGC-1 on mitochon- genic signaling cascade results in enhanced cyclase activ-
drial biogenesis providing in vivo evidence for a NRF-1- ity and the increased production of cAMP. As shown in
dependent pathway (236). PGC-1 induction of both Figure 9, elevated cAMP levels lead to activation of CREB
NRF-1 and NRF-2 transcripts may also contribute to the through its phosphorylation by protein kinase A (PKA).
biogenic program. As depicted in Figure 9, PGC-1 may CREB or related CREB family members, such as activat-
link nuclear regulatory events to the mitochondrial tran- ing transcription factor 2 (ATF-2), mediate both direct
scriptional machinery through its transcriptional activa- and indirect effects on the thermogenic program includ-
tion of Tfam, TFB1M, and TFB2M expression. As with ing the induction of PGC-1 and UCP1 (34, 35). The
respiratory subunit genes, the coactivator targets the PGC-1 promoter has a potent cAMP response element
NRF-1 and NRF-2 recognition sites within Tfam and TFB (CRE) that serves as a target for CREB-mediated tran-
promoters leading to increased mRNA expression (73). scriptional activation (93). In addition to its role in the
ERR has also been associated with PGC-1-induced mi- thermogenic program, the coactivator is also markedly
tochondrial biogenesis (143, 190). ERR and GABP induced in mouse liver in response to fasting where it
(NRF-2) recognition sites are conserved in the promot- activates the genes encoding gluconeogenic enzymes
ers of a number of oxidative phosphorylation genes, in- (239). The induction of gluconeogenesis by PGC-1 in
cluding cytochrome c and -ATP synthase, and PGC-1 fasted liver also involves the activation of CREB by serine
can drive expression through these sites (143, 190) (Fig. 9). phosphorylation in response to the catecholamine and
Interestingly, computer modeling indicates that PGC-1 in- glucagon-mediated elevation of cAMP. CREB induces the

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TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA 625

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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.

transcriptional expression of PGC-1, which in turn serves of an NO-dependent pathway of mitochondrial biogenesis
as a coactivator of gluconeogenic gene expression (93). awaits confirmation of these findings.
The cAMP-dependent pathway is one of several that A number of reports link the expression of PGC-1 to
direct the induction of PGC-1 in a number of tissues exercise-induced mitochondrial biogenesis in skeletal
(126). PGC-1 along with Tfam and NRF-1 are induced via muscle (3, 15, 76, 154, 178, 207209, 233). In each case,
cGMP-dependent signaling resulting from elevated levels PGC-1 mRNA and/or protein increase as an adaptive
of nitric oxide (NO) (150) (Fig. 9). The NO induction of response to endurance exercise of varying intensity and
PGC-1 is correlated with increased mitochondrial bio- duration. These findings are satisfying in the context of
genesis in several cell lines. The induced mitochondrial the long-standing relationship between endurance exer-
mass is accompanied by increased oxidative phosphory- cise and enhanced mitochondrial respiratory chain ex-
lation-coupled respiration consistent with an increase in pression and function (95). Both NRF-1 and NRF-2 DNA
functional mitochondria (151). These results were ob- binding activities were elevated along with the expression
tained by pharmacological increases in either NO or of several NRF target genes in rat skeletal muscle follow-
cGMP, raising the question of whether physiological fluc- ing an exercise regimen (15). Similar changes in the PGC-
tuations in endogenous NO can regulate mitochondrial 1-NRF pathway were mimicked in cultured myotubules
content (114). Interestingly, tissue mitochondria in mice in response to increased calcium levels, suggesting that
with a homozygous disruption of the gene encoding en- PGC-1 signaling contributes to adaptive changes in mi-
dothelial NO synthase (eNOS/) were somewhat smaller tochondrial biogenesis in skeletal muscle cells (154). Ex-
and less densely packed than in wild-type mice (150, 151). ercise and other neuromuscular activity may also lead to
These changes were accompanied by reductions in en- the activation of the p38 mitogen-activated protein kinase
ergy expenditure and mRNA levels for PGC-1, Tfam, and (MAPK) pathway resulting in PGC-1 induction through
NRF-1, arguing that basal mitochondrial content is af- ATF2 and MEF2 (3). There is evidence that depletion of
fected by the loss of eNOS-generated NO. Establishment ATP during exercise leads to elevated AMP/ATP ratios,

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626 RICHARD C. SCARPULLA

which enhances AMP-activated protein kinase (AMPK) of an interaction between hepatic nuclear receptor 4
activity (Fig. 9). In addition, increased calcium activates (HNF4) and forkhead transcription factor 01 (FOXO1),
Ca2/calmodulin-dependent protein kinase (CaMK), and which mediate the expression of gluconeogenic genes.
this along with AMPK results in the induction of PGC-1 PGC-1 also exhibits a marked preference for promoting
and activation of NRFs during energy deprivation (153, the ligand-dependent activity of ER while having a min-
243). In fact, as seen in other instances of PGC-1 induc- imal effect on ER (110). These examples illustrate that
tion, CREB phosphorylation, in this case by CaMK, directs differences in transcription factor interactions mediate
CREB-activated PGC-1 transcription (Fig. 9). This path- differences in functional specificity between members of
way is also driven by calcineurin A through myocyte the PGC-1 family.
enhancer factor 2 (MEF2), which is a potent trans-acti- Despite their differential utilization of nuclear hor-
vator of PGC-1 transcription in muscle (87). This same mone receptors, PGC-1 binds NRF-1 and is a potent
factor has been linked to the expression of muscle-spe- coactivator of NRF-1 target genes leading to increased
cific COX subunits (228). Although it is not clear weather mitochondrial gene expression (127). Forced expression
the NRFs are direct targets of these kinases, the associ- of PGC-1 in cultured muscle cells results in increased

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ated increase in NRF-1 DNA binding activity coincides mitochondrial biogenesis and oxygen consumption, al-
with elevated expression of cytochrome c and 5-aminole- though PGC-1 has been associated with higher proton
vulinate synthase and enhanced mitochondrial density leak rates than PGC-1 (202). Thus, although PGC-1 and -
(21). It should be noted that PGC-1 mRNA is induced to are functionally distinct, they both utilize NRF-1 and other
similar levels in wild-type and in both AMPK 1 and 2 transcription factors to induce mitochondrial biogenesis.
knockout mice, arguing that these isoforms of AMPK are This has been confirmed in transgenic mice in which
not essential to exercise-induced gene expression by PGC-1 is abundantly overexpressed in skeletal muscle
PGC-1 (100). This study was based on patterns of mRNA (10). The coactivator promotes massive increases in mi-
expression alone, and thus a definitive conclusion awaits tochondrial content that are accompanied by marked el-
a systematic analysis of mitochondrial biogenesis in these evations in the expression of respiratory mRNAs and
mice. proteins derived from the expression of both nuclear and
Finally, a recent study shows that NRF-1 and NRF-2 mitochondrial genes. This mitochondrial biogenesis is as-
occupancy of cytochrome c and cytochrome oxidase sub- sociated with the induction of type IIX oxidative muscle
unit IV promoters, respectively, occurs prior to the exer- fibers and increased capacity for aerobic exercise. These
cise-induced increase PGC-1 protein levels (233). Exer- changes are presumed to result from activation of a spe-
cise also leads to the activation of ATF-2 through p38 cific program of gene expression by PGC-1 through tran-
mitogen-activated protein kinase (p38 MAPK). This tran- scription factor targets such as MEF2, ERR, and PPAR
scription factor, a member of the CREB/ATF family, binds among others. Notably, the induction of type IIX fibers
the PGC-1 promoter and induces its transcription, per- was absent in the soleus muscle despite high-level expres-
haps as a secondary phase of the adaptive response. It is sion of PGC-1 in this tissue, suggesting that abundant
unclear whether direct phosphorylation of PGC-1 by p38 expression of the coactivator alone is not sufficient. It will
MAPK is required in this model. be important to confirm the regulation of muscle fiber
type by PGC-1 in loss of function experiments.
2. PGC-1
3. PRC
Two mammalian relatives of PGC-1 have been char-
acterized. PGC-1, although somewhat larger than PGC- A database search for sequence similarities to PGC-1
1, shares sequence similarity with PGC-1 along its en- identified the partial sequence of a large cDNA with a
tire length (110, 127). Like PGC-1, PGC-1 has an NH2- COOH-terminal RS domain and RNA recognition motif
terminal activation domain, LXXLL coactivator signatures, (6). Molecular cloning of the full-length cDNA revealed
and a COOH-terminal RNA recognition motif. Although additional sequence similarities with PGC-1 including
PGC-1 lacks an R/S domain, it is not clear whether it an acidic NH2-terminal region, an LXXLL signature for
differs from PGC-1 in its ability to couple transcription nuclear receptor coactivators, and a central proline-
and RNA slicing. Steady-state tissue expression of PGC-1 rich region (Fig. 10). Although significant sequence sim-
mRNA parallels that of PGC-1 with the highest levels in ilarity with PGC-1 is confined to these distinct regions,
brown fat, heart, and skeletal muscle, tissues high in their spatial conservation is highly suggestive of related
mitochondrial content and oxidative energy production. function. Thus the protein encoded by this cDNA was
However, PGC-1 differs from PGC-1 in that it is not designated as PGC-1-related coactivator (PRC) (6).
induced in brown fat upon cold exposure, and it is a poor PRC resembles both PGC-1 and - in that it binds
inducer of gluconeogenic gene expression in hepatocytes NRF-1 both in vitro and in vivo and can utilize NRF-1 for
and liver (140). This most likely results from the absence the trans-activation of NRF-1 target genes (6). PRC has a

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TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA 627

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 ele-
vated PGC-1 (73). This includes increased expression of
Tfam, TFB1M and -2M mRNAs, as well as those for both

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FIG. 10. Diagram comparing sequence motifs conserved between nuclear and mitochondrial respiratory subunits. It is of
PGC-1-related coactivator (PRC) and PGC-1. Dashed lines connect
sequence similarities between the activation domains (vertically
interest in this context that PRC, NRF-1, and Tfam are
hatched box), the proline-rich region (cross-hatched box), the R/S markedly upregulated in thyroid oncocytomas in conjunc-
domain (open box), and the RNA recognition motif (horizontally tion with increases in cytochrome oxidase activity and
hatched box).
mtDNA content (180). These thyroid tumors are character-
ized by dense mitochondrial accumulation and are appar-
potent NH2-terminal activation domain that is highly sim- ently devoid of PGC-1. Both PRC and PGC-1 mRNAs are
ilar to that of PGC-1 and is required for NRF-1-depen- rapidly induced in human skeletal muscle in response to
dent trans-activation. These properties along with its nu- endurance exercise (178). PRC mRNA expression is high
clear localization and in vivo interaction with NRF-1 attest initially and persists for 4 h but then modulates to a
to a transcriptional function for PRC. As with PGC-1, basal level at 24 h after exercise. Thus PRC may direct
binding of NRF-1 occurs through the NRF-1 DNA binding early adaptive changes in gene expression in response to
domain. In addition to cytochrome c and 5-ALAS promot- exercise.
ers, PRC can trans-activate the human TFB1M and Serum induction of PRC has the characteristics of
TFB2M promoters in a manner indistinguishable from immediate early genes, those genes whose expression is
PGC-1 (73). The NRF binding sites within the proximal induced by serum growth factors in the absence of de
promoters of these genes serve as targets for trans-acti- novo protein synthesis (91). Their induction is among the
vation by both coactivators. 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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628 RICHARD C. SCARPULLA

Like other genes in this class, PRC mRNA is increased mice. As summarized in Table 1, these studies reveal that
rapidly by serum induction of quiescent fibroblasts in the both essential and nonessential genes define the mito-
presence of the protein synthesis inhibitor cycloheximide chondrial phenotype. The essential genes are generally
(220). The mRNA for PRC and other immediate early single copy and encode both nuclear and mitochondrial
genes is superinduced and also markedly stabilized by the transcription factors. These genes are not only required
drug, probably because of a requirement for protein syn- for the maintenance of mitochondrial function but also
thesis for mRNA turnover. Cytochrome c is also serum for proper embryogenesis and organismal viability. They
induced in part through the NRF-1 and CREB sites within include Tfam, the NRFs, and other factors that are fun-
its promoter (92). NRF-1 and CREB have been implicated damental to the expression of nuclear and mitochondrial
as regulators of cell growth. As discussed above, NRF-1 genes. The nuclear transcription factors implicated in
occupies the promoters of many genes required for DNA mitochondrial biogenesis are not exclusive to this process
replication, cytokinesis, and mitosis including a number but rather integrate the expression of mitochondria with
of those targeted by the E2F family of growth regulators that of other fundamental cellular functions. The nones-
(33). CREB is well known as a target of mitogenic signal- sential genes are members of small gene families and

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ing pathways (137). Interestingly, PRC binds CREB function as important transcriptional modulators of mito-
through the same binding sites used for NRF-1, and both chondrial expression and function. These include the
factors exist in a complex with PRC in cell extracts (220). transcription factors ERR and PPAR as well as the
In addition, NRF-1 and CREB both occupy the cyto- PGC-1 family of coactivators that target key transcription
chrome c promoter in vivo, and PRC occupancy of the factors in specifying tissue-specific energetic properties, a
cytochrome c promoter increases upon serum induction subset of which is mitochondrial content. Individually
of quiescent fibroblasts. This is consistent with a model they are not required for viability or for maintaining basal
whereby PRC targets these transcription factors in re- functional levels of mitochondria (Table 1). However,
sponse to mitogenic signals. This is supported by the through their regulated expression and transcriptional
observation that expression of the PRC NRF-1/CREB specificities, these regulatory factors play an important
binding domain in trans leads to dominant negative inhi- role in relaying extracellular signals to the transcription
bition of cell growth on galactose as a carbon source machinery.
(220). Growth on galactose requires mitochondrial respi-
ration, suggesting that PRC may link the expression of
A. Transcription Factor Knockouts
genes required for both cell growth and mitochondrial
respiration.
A targeted disruption of Tfam confirmed its in vivo
function in mitochondrial transcription and replication
V. LESSONS FROM MOUSE GENE KNOCKOUTS (Table 1). A homozygous Tfam knockout results in
postimplantation lethality between embryonic days E8.5
The complexity of the pathways of mitochondrial and E10.5 (113). The E8.5 embryos are severely depleted
biogenesis is illustrated by the phenotypes of knockout of mtDNA and are deficient in both cytochrome oxidase

TABLE1. Mouse knockouts of nucleus-encoded transcriptional regulators implicated in mitochondrial


biogenesis and function

Gene Knockout Viability Mitochondrial Phenotype

Tfam (germ line) Embryonic lethal between days E8.5 and E11.5 mtDNA depletion, severe respiratory chain deficiency
PolgA Embryonic lethal between days E7.5 and E8.5 mtDNA depletion, cytochrome oxidase deficiency
NRF-1 Embryonic lethal between days E3.5 and E6.5 mtDNA depletion, loss of mitochondrial membrane potential,
blastocyst growth defect
NRF-2 (GABP) Preimplantation lethal Not determined
YY1 Embryonic lethal between days E3.5 and E8.5 Not determined
PPAR Viable and fertile Reduced constitutive expression of mitochondrial fatty acid
oxidation enzymes, defective fatty acid oxidation in
response to fasting
ERR Viable and fertile Normal food consumption and energy expenditure, reduced
lipogenesis, cold intolerance
PGC-1 (Lin et al.) Viable and fertile, increased postnatal mortality Normal mitochondrial morphology, reduced O2 consumption
in hepatocytes, fasting hypoglycemia, cold intolerance,
defective cardiac ATP production
PGC-1 (Leone et al.) Viable and fertile, normal mortality Increased body mass, modest reduction in skeletal muscle
mitochondria, normal gluconeogenesis
PGC-1 Viable and fertile Normal energy intake and expenditure, reduction in skeletal
muscle and heart mitochondria

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TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA 629

and succinate dehydrogenase activities. In addition, tis- companied by defects in the development of the central
sue-specific Tfam knockout animals show a strong corre- nervous system (20).
lation between Tfam and mtDNA abundance (122, 201). As observed for NRF-1, GABP (NRF-2) homozy-
These studies establish an essential role for Tfam in the gous null mice also exhibit a peri-implantation lethal phe-
maintenance of mtDNA copy number in vivo. Interest- notype (176). Although no determination of the state of
ingly, a homozygous knockout of mitochondrial DNA mtDNA or mitochondrial function was made in the mu-
polymerase also results in developmental arrest at E7.5 tant embryos, the early lethality may result from a com-
E8.5, severe depletion of mtDNA, and loss of cytochrome bination of mitochondrial and nonmitochondrial defects.
oxidase activity (83). These similarities in mitochondrial Homozygous knockouts of other Ets family transcription
phenotype between Tfam and DNA polymerase knock- factors also exhibit early embryonic lethality, suggesting
outs (Table 1) illustrate that intact mtDNA transcription that members of the family are unable to compensate for
and replication machinery is required for mtDNA mainte-
one another during embryonic development. It is surpris-
nance and for development beyond late gastrulation and
ing in light of the NRF knockouts that mice homozygous
early organogenesis. It is also notable that overexpressing
null for ERR are viable and fertile and display no defects

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Twinkle, a mtDNA helicase, in transgenic mice leads to
in food intake or energy expenditure (Table 1), although
increased mtDNA copy number (215). In humans, domi-
they do have reduced fat mass and are resistant to high-fat
nant mutations in Twinkle result in the accumulation of
multiple mtDNA deletions in several somatic tissues. diet-induced obesity (132). The ERR knockouts show a
These results reinforce the conclusion that mtDNA tran- modest reduction in cytochrome c mRNA, but no changes
scription and replication factors are essential to the ex- in the global expression of other respiratory subunit
pression of the mitochondrial respiratory chain in mam- genes have been noted. The absence of a profound mito-
malian tissues. chondrial deficiency in these mice may be explained by
Mouse gene knockouts of several of the nuclear tran- the fact that ERR is one of a small family of related
scription factors implicated in mitochondrial biogenesis factors whose members may compensate for the loss of a
have also been analyzed (Table 1). A homozygous NRF-1 single isoform. In contrast, Tfam and NRF-1 are the prod-
mouse knockout results in early embryonic lethality be- ucts of unique genes. Recent work demonstrates that the
tween embryonic days E3.5 and E6.5 (96). Although they ERR knockout mice are deficient in adaptive thermogen-
are morphologically normal, the NRF-1 null blastocysts esis because of decreased mitochondrial mass in brown
degenerate rapidly in culture and have severely reduced adipose tissue (221). Although PGC-1 and UCP-1 were
mtDNA levels accompanied by a deficiency in maintaining induced normally by cold exposure in the ERR knock-
a mitochondrial membrane potential. The mtDNA deple- outs, the decrease in mitochondria was accompanied by
tion in the blastocyst does not result from a defect in reductions in respiratory gene expression, mtDNA copy
mtDNA amplification during oogenesis, arguing that the number, and the oxidative capacity of the tissue. The
mtDNA depletion occurs between fertilization and the remaining mitochondria were morphologically and func-
blastocyst stage. The phenotype is consistent with the loss tionally normal. The results argue that ERR has a spe-
of an NRF-1-dependent pathway of mtDNA maintenance. cific function in differentiation-induced mitochondrial
However, depletion of mtDNA alone does not explain the biogenesis in brown adipose tissue without affecting
peri-implantation lethality of the homozygous NRF-1 nulls basal levels of mitochondrial maintenance.
because Tfam knockout embryos are also depleted of
A similar theme is echoed in mice with a targeted
mtDNA but survive to between embryonic days E8.5 and
disruption of PPAR (Table 1). The PPAR knockouts are
E10.5 (113). Thus the earlier lethality of NRF-1 nulls may
also viable and fertile with no obvious phenotypic abnor-
result from the decreased expression of NRF-1 target
malities (115). However, they do have a markedly blunted
genes that are required for other cell growth and devel-
opmental functions. Among these may be the subset of response to peroxisome proliferators, demonstrating a
genes identified as targets of both NRF-1 and the E2F crucial role for PPAR in the pleiotropic response to
family by ChiP on chip assay (33). It is of interest in this these agents. In addition, the mice display lower consti-
context that loss-of-function mutations in NRF-1 have a tutive expression of several mitochondrial fatty acid me-
dramatic negative effect on both Drosophila and ze- tabolizing enzymes (8) and, in contrast to wild-type mice,
brafish development. Partial loss-of-function mutations in the PPAR knockouts were resistant to the induction of
EWG, a NRF-1 relative in Drosophila, result in aberrant these mitochondrial enzymes in both liver and heart in
intersegmental axonal projection pathways in the embryo response to fasting (119). Thus, although not required for
(53). Total loss-of-function mutations in EWG are embry- normal development, viability, or mitochondrial mainte-
onic lethal. The NRF-1 gene in zebrafish is expressed in nance, PPAR plays a crucial role in orchestrating regu-
the eye and central nervous system of developing em- latory responses necessary for optimal mitochondrial ox-
bryos. Its disruption gives a larval-lethal phenotype ac- idative functions.

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630 RICHARD C. SCARPULLA

B. Coactivator Knockouts (117). Mice with a homozygous disruption of the gene


were viable and fertile and exhibited no gross changes in
As we have seen, gain-of-function experiments have whole body substrate utilization or total energy input or
established that overexpression of either PGC-1 or output (Table 1). They did however have a higher overall
PGC-1 can orchestrate massive increases in mitochon- metabolic rate associated with a lean phenotype com-
drial biogenesis both in cultured cells and in mouse tis- pared with wild-type littermates. Closer examination re-
sues (10, 116, 236). These changes have been ascribed to vealed a number of tissue alterations in mitochondrial
their ability to induce the expression of key transcription gene expression and respiratory function. There were
factors (NRF-1, NRF-2, ERR) and to interact specifically clear reductions in some genes associated with mitochon-
with these factors in mediating the activation of genes drial electron transport chain function in brown and
that promote the biogenesis of mitochondria. Thus it is white fat, skeletal muscle, and heart. These included re-
surprising that mice with a targeted disruption of PGC-1 spiratory subunits encoded by both nuclear and mito-
are viable and show no changes in mitochondrial abun- chondrial genes. Although diminished mRNA levels were
dance or morphology in liver or brown fat (128). They do not always accompanied by similar changes in the en-

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show reduced oxygen consumption in isolated hepato- coded protein, mitochondrial volume was reduced in both
cytes and reduced expression of several mRNAs linked to heart and skeletal muscle. In these cases, the accompa-
mitochondrial function. The only tissues to display obvi- nying decreases in electron transport and oxidative phos-
ous morphological abnormalities were brown adipose tis- phorylation activities resulted from reduced numbers of
sue and the striatum of the brain. Defects in the striatum functionally normal mitochondria. In brown fat, mito-
have been associated with movement disorders, and the chondrial content was normal, but the knockout mice
PGC-1 null mice were markedly hyperactive. This hyper- exhibited a blunted response to norepinephrine-induced
activity correlated with a loss of axons in the striatum as thermogenesis. These phenotypes clearly point to an im-
well as with reductions in nucleus-encoded mRNAs for portant modulatory role for PGC-1 in maintaining opti-
respiratory- and brain-specific genes. Subsequent experi- mal tissue energetics.
ments with the PGC-1 null mice demonstrated that The absence of a dramatic mitochondrial biogenesis
PGC-1 is not required for mitochondrial biogenesis (9). defect in either the PGC-1 or PGC-1 null mice may
However, the mice are deficient in the expression of result from mechanisms that compensate for the individ-
genes necessary for mitochondrial function and in oxida- ual loss of these coactivators. Since the PGC-1 family
tive enzyme activities in heart and skeletal muscle. These members share several functional motifs and can all
changes coincide with reduced cardiac ATP production trans-activate NRF target genes, the remaining family
and a defect in work output in response to physiological members may substitute functionally for the ablated
stimuli. Recently, a skeletal muscle-specific PGC-1 knock- member. This is seen in the brown fat of the PGC-1
out mouse was characterized (84). These mice clearly knockout where PGC-1 levels are markedly upregulated,
show multiple muscle defects including reduced exercise possibly resulting in the maintenance of normal mito-
tolerance and abnormalities in the maintenance of normal chondrial content in this tissue (117). This issue was
muscle fiber composition. The results illustrate that the addressed by knocking down the expression of PGC-1
true tissue-specific functions of PGC family coactivators by shRNA in a preadipocyte cell line derived from PGC-1
may be masked by pleiotropic phenotypes in mice har- knockout mice (216). Although PGC-1 appears not to be
boring a generalized gene disruption in all of their tissues. required for adipocyte differentiation, its loss is corre-
The phenotype of an independently constructed lated with an inability to induce cAMP-dependent expres-
PGC-1 null mouse confirmed that the coactivator is not sion of genes necessary for thermogenesis, including
required for normal development or for the global biogen- UCP-1 and cytochrome c. UCP-1, however, is induced by
esis or maintenance of mitochondria (118) (Table 1). insulin and retinoic acid in the absence of PGC-1. Re-
However, these mice had reduced mitochondrial density duced expression of PGC-1 in the PGC-1 null back-
in slow-twitch skeletal muscle and modestly reduced re- ground led to a failure to establish and maintain differen-
spiratory capacity in both liver and skeletal muscle. There tiated levels of mitochondrial density and function in
are also a number of significant differences with the pre- mature brown adipocytes. Mitochondrial content is re-
viously described knockout including the absence of a duced to a level present in undifferentiated preadipo-
defect in gluconeogenesis and a disparity in cardiac phe- cytes. Thus, although neither coactivator is required to
notype. These have been ascribed to differences in ge- direct the program of adipocyte differentiation, one or the
netic background or targeting strategies (68). other is essential to achieve a differentiated mitochon-
Recently, a targeted disruption of the mouse PGC-1 drial phenotype. This is reminiscent of the case of ERR,
gene was constructed by removal of exons containing the suggesting that the PGC-ERR regulatory pathway directs
nuclear hormone receptor coactivator signature motifs differentiation-specific changes in mitochondrial content
(LXXLL) and introduction of a premature stop codon in brown fat and possibly other tissues. It remains an

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TRANSCRIPTIONAL PARADIGMS IN MAMMALIAN MITOCHONDRIA 631

open question as to whether PRC can support basal levels result from certain mitochondrial disease mutations pro-
of mitochondrial biogenesis in the absence of PGC-1 and liferate in diseased muscle fibers giving rise to ragged red
-. It is of interest in this context that expression of a fibers (145, 226). Specific nuclear genes involved in ATP
dominant negative allele of PRC from a lentivirus vector production also display elevated expression in cells with
inhibits respiratory growth in cultured cells (220). Clearly, mtDNA mutations (89). An altered pattern of nuclear gene
the PGC-1 coactivators exhibit highly specialized regula- expression, involving proteins of the mitochondrial inner
tory functions but, individually, do not appear to be the membrane, intermediate filaments, and ribosomes, occurs
sole determinants of mitochondrial content in the major- in human cells upon depletion of mtDNA (123). Likewise,
ity of tissues. chicken cells either depleted of mtDNA or treated with
the mitochondrial protein synthesis inhibitor chloram-
phenicol have increased levels of mRNAs for elongation
VI. RETROGRADE PATHWAYS factor 1, -actin, v-myc, and GAPDH (229). Although the
mechanistic bases for these phenomena have not been
A. The RTG Pathway in Yeast elucidated, they are thought to represent nuclear re-

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sponses to deficiencies in ATP production.
The regulated expression of the PGC-1 family of in- Recent studies suggest that retrograde signaling in
ducible coactivators provides a mechanism for modulat- animal cells may be mediated by calcium (22). Inhibition
ing mitochondrial biogenesis and function in response to of mitochondrial respiratory function either by depletion
a variety of extracellular signals. In addition, pathways of mtDNA or by metabolic inhibitors results in a stress
exist for the communication of the functional state of response that coincides with elevated cytosolic calcium
mitochondria back to the nuclear transcriptional machin- levels. The response includes increased expression of
ery. This phenomenon of retrograde signaling is well stud- calcium-responsive transcription factors and cytochrome
ied at the transcriptional level in the yeast Saccharomyces oxidase subunit Vb. Calcium signaling has also been as-
cerevisiae (130). In yeast cells that are depleted of mito- sociated with a PGC-1-dependent pathway of mitochon-
chondrial DNA (0 cells), the resulting respiratory defi- drial biogenesis in skeletal muscle (155, 235). Constitutive
ciency reduces the supply of glutamate, which is derived expression of calcium/calmodulin-dependent protein ki-
from the -ketoglutarate of the citric acid cycle. Cells nase IV (CaMKIV) in the skeletal muscle of transgenic
compensate by increasing production of peroxisomes, the mice leads to increased mtDNA copy number and respi-
sites of fatty acid oxidation in yeast. This generates ratory enzymes as well as elevated PGC-1 levels. Thus
acetyl-CoA, which fuels the citric acid cycle. The nuclear calcium may be an important link between the relay of
CIT2 gene encoding peroxisomal citrate synthase is extracellular signals to the nucleus and the bidirectional
markedly induced in 0 cells. This enzyme is part of the communication between nucleus and mitochondria. This
glyoxylate cycle, and its induction in response to a defect notion is reinforced by the observation that CREB is
in mitochondrial respiration allows cells to generate ci- found in its transcriptionally active, phosphorylated state
trate (200). Positive transcriptional control of peroxiso- in cells exhibiting mitochondrial insufficiency resulting
mal citrate synthase and other peroxisomal proteins in 0 either from the loss of mtDNA or by pathological muta-
cells is mediated by basic helix-loop-helix-leucine zipper tion in mtDNA (11). This coincides with the activation of
transcription factors, Rtg1p and Rtg3p (177). A third fac- CaMKIV, which, as discussed, can lead to CREB phosphor-
tor Rtg2p facilitates the translocation of Rtg1p and Rtg3p ylation in response to calcium. It remains to be determined
from the cytoplasm to the nucleus where they bind to whether calcium signaling through CREB and the PGC-1
their target genes as a heterodimer and drive transcrip- family coactivators represents a physiologically meaningful
tion. Rtg2p has an ATP binding site and is thought to pathway of retrograde regulation in vertebrate cells.
function as a sensor of the functional state of mitochon-
dria in part through its interaction with the phosphopro-
VII. CONCLUSION
tein 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). Much progress has been made in uncovering the
transcriptional mechanisms that govern the function and
biogenesis of mitochondria. The identification and char-
B. Mammalian Retrograde Regulation acterization of important regulatory factors has led to a
framework for understanding the continuity between sig-
The Rtg pathway has not been identified in vertebrate naling events affecting cellular energetics and the expres-
cells, but there are a number of examples where the sion of both nuclear and mitochondrial genes that dictate
expression of nuclear genes is altered by deficiencies in the mitochondrial phenotype. The preponderance of evi-
mitochondrial function (31). Defective mitochondria that dence suggests that nucleus-encoded transcription fac-

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632 RICHARD C. SCARPULLA

tors acting on both nuclear and mitochondrial genes serve tions of PRC and PGC-1 should clarify this issue. It will
to coordinate the expression of the gene products re- also be of interest to determine whether retrograde sig-
quired for maintenance of the respiratory apparatus as naling is mediated by any of these coactivators.
well as other essential mitochondrial functions. A subset
ACKNOWLEDGMENTS
of these is also required for proper embryonic develop-
ment and organismal viability. Because the organelle is Address for reprint requests and other correspondence:
intimately associated with a myriad of cellular functions, R. C. Scarpulla, Dept. of Cell and Molecular Biology, Northwest-
it is not surprising to find that the factors governing ern Medical School, 303 East Chicago Ave., Chicago, IL 60611.
mitochondrial expression are engaged in other cellular GRANTS
activities as well. Thus they likely play a fundamental
Work in the authors laboratory was supported by National
integrative role in coordinating mitochondrial functions Institute of General Medical Sciences Grant GM-3252525.
with cellular events necessary for growth and develop-
ment. This likely accounts for the embryonic lethality
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