Associate Professor Dan Mishmar explains the significance of research into mitochondrial DNA and the direction of his work at the Ben-Gurion University of the Negev in Israel

Mitochondria have their own distinct genome. What accounts for the sequence variations among individuals around the globe?

Unlike the nuclear genome, mitochondrial DNA (mtDNA) resides in multiple copies per cell, ranging from ~100,000 copies in the ovum to ~100 copies in the sperm. Mutations that occur in our body’s cells result in increased intracellular sequence variation; if such mutations occur in the ovum (but not the sperm) they will be transferred to the next generation, and will either be fixed or lost during cell divisions depending on a bottleneck that occurs during the maturation of the ova. Therefore, cells that divide a lot, such as those in the haematopoietic system, may lose much of their intracellular variation as compared to slow-dividing post-mitotic cells, such as neurons and skeletal muscle cells. It is estimated that mtDNA has an order

Mitochondrial DNA mutations

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Mechanisms of disease and evolutionA team of Israeli life scientists is intensively investigating mitochondrial DNA and its relationship to two phenomena determined by the same principles – evolution and disease

of magnitude higher mutation rate than the nuclear genome on the evolutionary timescale. To these pieces of evidence, one could add evolutionary forces such as Darwinian selection that shape variation both within the cell and between individuals. We recently exemplified all this through the study of intracellular mtDNA variation in human identical twins.

Can you elucidate your hypothesis surrounding the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region?

MtDNA is inherited solely through the maternal lineage and therefore genetic variation in the population stems from the accumulation of mutations during the course of evolution. Since mtDNA does not undergo recombination, one cannot assess the functionality of individual mtDNA mutations independent of their linked genetic background. Moreover, there is currently no available technology to mutate the human mtDNA sequence at will in the cell.

Therefore, to assess the functionality of individual mutations we sought an in vitro assay to overcome the aforementioned obstacles. Since many of the evolutionary variants lie within or adjacent to mtDNA promoters, we chose in vitro transcription as an assay to assess the functionality of mtDNA variants. This led us to discover that certain ancient variants in the vicinity of mtDNA promoters affect in vitro transcription, the binding capacity of mitochondrial transcription factor A (TFAM) and the replication efficiency of mtDNA

(mtDNA copy number in cells). With this in mind, we asked ourselves whether the mtDNA transcription machinery is confined to the mtDNA promoter or if there are additional undiscovered transcriptional regulatory elements in other human mtDNA regions. This question led us to screen for alternative transcription regulatory elements throughout the mtDNA sequence in a hypothesis-free manner, which yielded the exciting preliminary identification of novel transcription factors that bind and regulate human mtDNA.

You and your collaborators screened thousands of complete human mtDNA sequences for natural variation in experimentally established protein and RNA-coding genes, and regulatory regions. Have your investigations uncovered new information on mutations?

We recently published a comprehensive analysis of more than 9,800 whole human mtDNA sequences representing all major human populations. In that study, we aimed at identifying the mutations that were retained in the human phylogeny in certain ‘branches’ of the phylogenetic tree, ie. mutations that define genetic backgrounds. We also identified mutations that independently recurred in distant mtDNA lineages.

Strikingly, we found mutations with a high functional potential both among the large repertoire of lineage-defining and recurrent ‘nodal’ mutations. This raises a question: if those mutations are found in the general

population and they are similar to disease-causing mutations, how are we still healthy? The answer is still open, but our hypothesis is that these mutations were likely compensated by changes elsewhere in the genome, including the nucleus. If this is true, certain combinations of mutations involving these functional variants will be beneficial and others will alter susceptibility to diseases. Our preliminary results indicate just that.

What do your team members contribute to your investigations? Does being based at Ben-Gurion University offer advantages in terms of collaboration?

I am blessed by a group of outstanding PhD and MSc students and a very capable technician who not only perform the experiments but contribute intellectually to the design and experimental testing of our hypotheses. For disease association studies, especially in relation to Type 2 diabetes, we have a wonderful ongoing collaboration with the Israeli Diabetes Research Group (IDRG), to which I am most grateful for medical information and DNA samples of hundreds of patients and controls. In the frame of other projects we have excellent local, European and US collaborators.

Ben-Gurion University is a dynamic and relatively young research institute which is eager to provide a vivid working environment for researchers, lively discussions with colleagues and cutting-edge research facilities. This atmosphere is essential for my work.

MITOCHONDRIA ARE THE powerhouse of mammalian life. Of the many different organelles in a human cell, mitochondria are responsible for providing us with most of our energy; being the hosts of oxidative phosphorylation (OXPHOS), a key process in aerobic respiration. They are also assigned many other essential tasks, such as playing a role in apoptosis, the programmed death of cells. This process is vital to embryo development and to our existence, and its malfunction leads to uncontrolled cellular growth found in cancer.

Beyond fulfilling their share of the cellular division of labour, mitochondria stand out from other organelles in one vital respect – they are the only organelles in animals, apart from the nucleus, to contain their own DNA. This mitochondrial DNA (mtDNA) contains genes that are vital for the functioning of the mitochondrion, such as programming for enzymes involved in OXPHOS, as well as being involved in the creation of transfer

RNA (tRNA) and ribosomal RNA (rRNA) involved in uniting amino acids to form proteins.

mtDNA exists in a double-stranded ring and has a very high mutation rate due to a poor repair mechanism and high number of replication cycles. It thus evolves rapidly, making it useful in phylogeny – the study of evolutionary relationships by comparing DNA sequences between species. As it is inherited from the mother only, mtDNA allows maternal lineage to be traced, and is thus affected purely by mutation accumulation rather than by recombination. Because there are many mitochondria within a cell, mutations in mtDNA permit variation to occur both between and within cells. Associate Professor Dan Mishmar, an expert in mtDNA from the Ben-Gurion University of the Negev in Israel, explains: “This is the beauty of mitochondrial genetics: it exhibits inter-individual variation, but unlike the nucleus it also displays intracellular sequence variation that may accumulate over the

lifetime of the individual. It plays a role not only in the generation of population genetic variants but also alters mitochondrial function during the ageing process and plays a role in age-related disorders such as Parkinson’s disease”.

EVOLVING DISEASE

Genetic variation in mtDNA was, until recently, considered a mere academic curiosity. However, the work of Mishmar and colleagues is part of a momentous shift which emphasises the significance of mtDNA mutations on the human phenotype. For the team, understanding mtDNA is vital to an understanding of human complex diseases, as well as major evolutionary transitions and the emergence of new species. Mutations in mtDNA, just as in nuclear DNA (nDNA), have the potential to cause disease. The researchers are driven by their unique perspective that sees disease through the lens of evolution, understanding disease-causing mutations and

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genetic variations as being governed by the same evolutionary principles.

Much of their work concerns complex diseases, which are determined by multiple genes in multiple locations as well as by environmental factors, as Mishmar underlines: “Complex diseases could be caused by combinations of variants, each of which contributes only little to the disease phenotype”. This active gene-environment interface draws the forces of natural selection into play. A simple example of the tight relationship between evolution and disease would be the positive selection of efficient energy-producing genotypes in an ancestral age when food was scarce. This same adaptation, upon encountering the abundance of food in the modern world, has led to the rise of obesity and diabetes.

Whilst it could be assumed that evolutionary processes would negatively select disease-causing mutations in mtDNA, there is evidence that in some contexts they could present an evolutionary advantage. The group, hypothesising that recurrent nodal mutations are retained due to positive selection pressures, have investigated the functions of these variants. By looking at the recurrence of mtDNA ancient variants in a different genetic context, they have shown that the genetic landscape in which a mutation occurs can have a strong influence on its effect on the development of mitochondrial disorders such as the legal blindness caused by Leber’s hereditary optic neuropathy (LHON).

TOGETHER FOREVER

Mishmar and his team have also investigated the interdependence of mitochondria and the nucleus. It is generally believed that mitochondria originated from a process of endosymbiosis; they were originally independent bacteria which became absorbed into the cell. This may partially

explain the roots of mtDNA. Over the billions of years that have passed since this event, the nucleus and mitochondria have become symbiotic in their functioning.

This interdependence is highlighted in the experimental generation of cytoplasmic hybrids – cybrids – in which mitochondria from one species are present within a nucleus from another. Such hybrid cells often display reduced mitochondrial functionality, providing evidence that mtDNA and nDNA must thus remain compatible despite the rapid mutation rate of mtDNA, ie. the mitochondria and nucleus coevolve.

TWO SIDES OF THE SAME COIN

Mishmar’s team has focused on protein subunits of complex I that are encoded by the mtDNA and nDNA. The researchers have provided evidence of how these subunits encoded by nDNA and mtDNA directly interact. They have also uncovered evidence that mtDNA is not only regulated by specific mitochondrial regulatory factors but is integrated into the general cellular regulatory system; editing occurs in transcripts of mtDNA just as it does in the nucleus. In addition to these important results, Mishmar’s group has explored beyond the known mtDNA transcription factors, discovering that several nDNA transcription factors are also used in mitochondria. These are significant contributions to our still hazy knowledge of mitochondrial transcription and translation.

The tight relationship between nDNA and mtDNA has also been shown to be pivotal to disease susceptibility. Mishmar’s group was one of the first to show that the significance of mtDNA in determining susceptibility to Type 2 diabetes depends on the combination of mtDNA variants and as yet unknown variants in nDNA, rather than being dependent on variants in mtDNA or in nDNA alone, as Mishmar elaborates: “The latter finding exemplified the importance of functional cooperation between the nuclear and mitochondrial genomes not only during evolution but also for human health”.

THE MITOCHONDRIAL FUTURE

Since the functional impact of a genetic variant is likely to be only slight, the team is now searching for experimental methods sensitive enough to determine this. They are also currently working to unravel the mysteries of mtDNA transcription, identifying factors and their binding sites involved in the regulation of this process. In addition, the researchers are pioneering methods of identifying the signatures of natural selection both for variants between individuals and for the intracellular mtDNA variants.

Mishmar’s work has the potential to shed light on the functioning of the mitochondrion as an organelle vital to life, as well as to unpick the intricacies of its high degree of interdependency with the nucleus. Through this new knowledge, it is hoped that further insights will be gleaned into both evolutionary processes and susceptibility to a host of diseases, paving the way towards preventative medicines.

Mishmar’s work has the potential

to shed light on the functioning of

the mitochondrion as an organelle

vital to life as well as to pick apart

the intricacies of its high degree of

interdependency with the nucleus

MtDNAOBJECTIVES

To provide evidence for the hypothesis that mitochondrial genetic variability and evolutionary dynamics play a role in major evolutionary transitions including the emergence of new species and also in the tendency of humans to develop complex disease phenotypes.

KEY COLLABORATORS/PARTNERS

The Israeli Diabetes Research Group (IDRG)

Dr Raz Zarivach; Professor Amir Aharoni; Professor Amos Bouskila; Professor Ofer Ovadia, Ben-Gurion University of the Negev, Israel

Professor Eran Meshorer, Hebrew University, Israel

Dr Leo Nijtmans, Radboud University, The Netherlands

FUNDING

Israeli Science Foundation

Israeli Ministry of Health

Israeli Cancer Association

CONTACT

Associate Professor Dan Mishmar Group leader

Ben-Gurion University of the Negev Department of Life Sciences Building 40, Room 005 Beer Sheva, 84105 Israel

T +972 8 646 1355 E dmishmar@bgu.ac.il

www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1000474

ASSOCIATE PROFESSOR DAN MISHMAR obtained a BA in Archaeology from Hebrew University in 1992. He was then supervised by Professor Batsheva Kerem whilst completing a PhD in Human Genetics also at Hebrew University. His postdoctoral training mentor was Professor Douglas C Wallace. Since 2004, he has been Principle Investigator (Senior Lecturer) at the Ben-Gurion University of the Negev, Israel, before becoming Associate Professor in 2011.

Electron microscope image of mitochondria in human cells.

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