 |
Molecular Genetics of the Assembly of Oxidative Phosphorylation Complexes
Summary: Mitochondrial DNA (mtDNA) is a high copy number genome that encodes a small number of proteins that have essential roles in cellular energy production. Mutations in mtDNA are an important cause of human disease. Using biochemical and genetic approaches, Eric Shoubridge and his colleagues are investigating the structural organization of mtDNA, the nature of the nuclear genetic factors that influence its transmission and segregation, and molecular mechanisms of mitochondrial disease.
Mitochondria are derived from α-proteobacteria, the result of an endosymbiotic fusion event that occurred more than a billion years ago. They are essential for a number of cellular processes in eukaryotes, such as iron-sulfur cluster and heme biosynthesis, and for aerobic energy production. Over the course of evolution, nearly all the genes that were present in the original endosymbiont were either transferred to the nucleus of the host cell or lost. The genes that remain in mammalian mitochondrial DNA (mtDNA) encode a handful of proteins that are essential subunits of the oxidative phosphorylation complexes as well as the tRNAs and rRNAs necessary for their translation. Defects in oxidative phosphorylation have been linked to a wide spectrum of multisystem disorders, with an estimated birth prevalence of 1 in 5,000. Most of these defects are caused by mutations in mtDNA.
The genetics of mtDNA is entirely distinct from Mendelian genetics because of the high ploidy of mtDNA and the fact that it is inherited maternally. Most cells contain hundreds or thousands of copies of mtDNA. In patients with mitochondrial disease, pathogenic mtDNAs usually coexist with wild-type mtDNAs (heteroplasmy). In general, the proportion of mutant mtDNAs must exceed a particular threshold to produce a biochemical phenotype. The rules that govern the transmission of mtDNA in the female germline and the segregation of mutant mtDNAs in different tissues during fetal and postnatal life are thus important determinants of whether an individual will express a pathogenic phenotype, what tissues will be involved, and how severe the disease. However, the factors affecting the transmission and segregation of pathogenic mtDNA mutants remain poorly understood.
Research in my laboratory is concerned with identifying and characterizing genes associated with mitochondrial disease, investigating the molecular mechanisms of mitochondrial disease, and studying the organization of mtDNA and the factors that control its transmission and segregation.
A Bottleneck for Transmission of mtDNA
To investigate the mechanisms of mtDNA transmission and segregation, it was necessary to construct animal models that segregated different mtDNA sequence variants, as there are no known naturally occurring mouse models of germline mtDNA heteroplasmy. We accomplished this with two common inbred laboratory mouse strains whose mtDNAs differ at about 100 sites, all presumably representing polymorphic sequence variants. We could demonstrate that segregation of these sequence variants in the female germline was rapid and stochastic, similar to the pattern of transmission of pathogenic mtDNAs in human pedigrees—the result of genetic drift and a bottleneck for the transmission of mtDNA that occurs during early oogenesis. We are currently investigating the exact shape and size of this genetic bottleneck and are attempting to uncover the mechanism by which it occurs. We know that mature oocytes contain at least 100,000 copies of mtDNA and that the early cleavage of the embryo, at least until the blastocyst stage, occurs without mtDNA replication. Exactly when mtDNA replication restarts after implantation of the embryo remains unknown, and we are investigating this using nucleotide analogues to label mtDNA in vivo. To determine precisely when the bottleneck occurs during germline maturation, we are tracking the evolution of genotypic variance in isolated primordial germ cells as they migrate and colonize the gonad. Using knockout mice, we are manipulating mtDNA copy number to study its influence on mtDNA transmission.
Nuclear Genetic Control of mtDNA Segregation
In the somatic tissues of our heteroplasmic mice, we uncovered an unexpected tissue-specific and age-related selection for different mtDNA haplotypes in the same animal, pointing to the existence of nuclear-encoded genes involved in the interaction between the nuclear and mitochondrial genomes. We were able to map this phenotype to three different quantitative trait loci (QTLs): Smdq1-3 (segregation of mitochondrial DNA quantitative trait) on chromosomes 5, 2, and 6. This is the first direct evidence for nuclear genetic control of mtDNA segregation. We are currently carrying out genetic crosses to narrow the intervals around the QTLs, with the aim of cloning the genes underlying this behavior. Investigation of the mechanism of mtDNA segregation in these animals suggested that it was based on some aspect of genome organization, and this has led us to investigate the structure of the mitochondrial nucleoid.
Structure of the Mitochondrial Nucleoid
Like nuclear DNA, mtDNA exists in a protein-mtDNA complex, the so-called mitochondrial nucleoid. However, there are no histones in mitochondria, and the proteins that contribute to mitochondrial chromatin structure have not been well characterized. Using mass spectrometry, we identified TFAM, a mitochondrial transcription factor in the HMG (high mobility group) box family, and single-stranded DNA binding protein as the most abundant proteins in purified nucleoids. Because of its known double-stranded DNA binding ability, we focused our initial studies on the interaction of TFAM with DNA. Surface plasmon resonance, gel shift, and atomic force microscopy analyses showed that TFAM binds to DNA cooperatively with nanomolar affinity as a homodimer. In solution, TFAM is capable of coordinating and fully compacting several DNA molecules to form spheroid structures that closely resemble nucleoids. To investigate in detail the mechanism by which TFAM organizes DNA, we used noncontact atomic force microscopy (ultra high vacuum), which achieves near cryo-electron microscope resolution, to observe the structural details of protein-DNA compaction intermediates. The formation of these complexes involves bending the DNA backbone and DNA loop formation, followed by filling in proximal available DNA sites until the DNA is compacted. These results demonstrate that TFAM alone is sufficient to organize mtDNA into nucleoid-like structures and provide a mechanism for their formation. We are now testing whether the TFAM-DNA structure can recruit other proteins that are integral to the structure of the nucleoid, and we are continuing to use mass spectrometry to identify additional nucleoid proteins.
To replicate or transcribe mtDNA, it would likely be necessary to modulate the high-affinity TFAM-DNA interaction. This is most probably accomplished by some form of posttranslational modification of TFAM, such as acetylation or phosphorylation, and we are currently attempting to identify potential modifications on purified TFAM using mass spectrometry. Sirt4, a mitochondrially targeted NAD+-dependent deacetylase, maps to Smdq-1 and is a candidate for this activity. We have constructed transgenic mice that overexpress Sirt4 from a strong, ubiquitous promoter and are now investigating the effects of such overexpression on mtDNA segregation in our heteroplasmic mouse model.
These investigations are also supported in part with grants from the National Institutes of Health and the Canadian Institutes of Health Research.
Last updated March 2007
|
|
|
|
