Periodic Reporting for period 4 - Mito-recombine (Homologous recombination and its application in manipulating animal mitochondrial DNA)
Okres sprawozdawczy: 2023-05-01 do 2024-10-31
Mitochondria, the key providers of cellular energy, are semi-autonomous organelles that house their own genome (mtDNA). Despite carrying a small number of genes, mtDNA has a big impact on health and disease. In humans, over 350 mtDNA mutations have been linked to a spectrum of incurable mitochondrial diseases that affect 1 in 4,300 individuals. Accumulation of mtDNA mutations is also known to contribute to mitochondrial dysfunction and various age-related conditions. Animal mitochondria possess the ability to repair DNA damages, which plays an important role in safeguarding mtDNA integrity during ageing. Nevertheless, very little is known about mtDNA repair compared to the vast literature on nuclear repair pathways. In addition to our limited knowledge of mtDNA maintenance, the lack of a robust method to genetically modify mtDNA in nearly all species leaves us little power to study other aspects of mtDNA biology and model disease progression caused by pathogenic mutations. Given the essential functions of mtDNA and its link to incurable diseases, there is increasing pressure for the development of genetic tools to modify mtDNA.
My previous work showed that there is recombination-based repair inside Drosophila mitochondria (Ma & O’Farrell, 2015). Furthermore, I established a system to induce recombination at specific sites and isolate recombinant mitochondrial genomes. This ERC project aims to characterise how recombination is carried out to repair mtDNA damages. We also proposed to develop tools based on recombination to genetically modify mtDNA for functional studies. There are three objectives:
1) Identify key components of the mitochondrial recombination machinery to advance our understanding of mtDNA repair and maintenance during evolution and ageing.
2) Developing a recombination toolkit to map trait-linked mtDNA sequences: this objective will link genotype to phenotype (forward genetics) to expand our knowledge of how mtDNA impacts health and disease. It also makes the study of genetic interactions between mitochondrial genes possible.
3) Build a site-directed mutagenesis system by establishing methods to deliver DNA into animal mitochondria: This work will resolve the long frustration of not having an efficient transformation system for mtDNA and enable the isolation of mutants of interest for functional studies (reverse genetics).
The proposed ERC work will enhance our knowledge of mtDNA maintenance and our ability to manipulate animal mtDNA. This makes both forward and reverse genetic studies for mitochondrial genomes possible, and thus gives us the power to dissect the complex roles mtDNA plays in development, ageing, disease and evolution.
My previous work showed that there is recombination-based repair inside Drosophila mitochondria (Ma & O’Farrell, 2015). Furthermore, I established a system to induce recombination at specific sites and isolate recombinant mitochondrial genomes. This ERC project aims to characterise how recombination is carried out to repair mtDNA damages. We also proposed to develop tools based on recombination to genetically modify mtDNA for functional studies. There are three objectives:
1) Identify key components of the mitochondrial recombination machinery to advance our understanding of mtDNA repair and maintenance during evolution and ageing.
2) Developing a recombination toolkit to map trait-linked mtDNA sequences: this objective will link genotype to phenotype (forward genetics) to expand our knowledge of how mtDNA impacts health and disease. It also makes the study of genetic interactions between mitochondrial genes possible.
3) Build a site-directed mutagenesis system by establishing methods to deliver DNA into animal mitochondria: This work will resolve the long frustration of not having an efficient transformation system for mtDNA and enable the isolation of mutants of interest for functional studies (reverse genetics).
The proposed ERC work will enhance our knowledge of mtDNA maintenance and our ability to manipulate animal mtDNA. This makes both forward and reverse genetic studies for mitochondrial genomes possible, and thus gives us the power to dissect the complex roles mtDNA plays in development, ageing, disease and evolution.
Aim I: Identifying components of the mtDNA recombination machinery
Recent studies showed that repair mechanisms inside animal mitochondria are more diverse and effective than previously thought, but the existence of recombination-based repair remains controversial. My previous work showed that in Drosophila, double-strand breaks on one mitochondrial genotype could be repaired based on the homologous sequence of other co-existing genotypes, indicating that there is recombination-based repair machinery inside mitochondria. To identify proteins mediating such a repair, in the first three years, we performed a candidate screen and identified REC as a helicase that drives mtDNA recombination and repair (Klucnika et al., JCB 2022). During this period, Michael Claxton focused on a genome-wide CRISPR screen to probe mtDNA damage response in human cells. So far, we have established RPE-Cas9-Tet3G cell lines expressing mitochondrially-targetted restriction enzymes upon Dox induction to cause DSBs in mitochondria. For the remaining period of this grant, we will transduce these cells with pooled sgRNA to identify proteins essential for cell survival upon mtDNA DSB damage.
Aim II: Developing a recombination toolkit to edit & map mtDNA
Probing interactions between mitochondrial genes has been challenging due to the multi-copy nature of mtDNA and a lack of genetic tools to edit and map mtDNA. Previously, my group established a system to isolate recombinant mitochondrial genomes in Drosophila. Using this toolkit, we found that sequence polymorphisms in one mitochondrial gene (mt:CoIII) could rescue the lethality caused by a detrimental mutation in another mitochondrial gene (mt:CoI). Through lipidomics profiling and biochemical and phenotypic analyses, we revealed that the two residues co-regulate cardiolipin binding to affect respiratory complex stability and activity. This work (Chiang et al, Nat Commun 2024) unwraps the complex intra-genomic interplays underlying mtDNA-linked disorders and how they influence disease expression.
Aim III: Establishing methods for delivery of exogenous DNA into mitochondria
We tried multiple methods to deliver exogenous DNA into fly mitochondria. However, none of these approaches worked. We will continue to explore other methods proposed in the application. In the meantime, staff recruited to work on this part of the project investigated mtDNA maintenance during late spermatogenesis in parallel. This work identified POLDIP2 as an essential player in eliminating paternal mtDNA in late spermatids to guarantee the maternal inheritance of mitochondrial genomes (Wang*, Meerod*, Cortes-Silva* et al) has been accepted in principle by EMBO J.
Recent studies showed that repair mechanisms inside animal mitochondria are more diverse and effective than previously thought, but the existence of recombination-based repair remains controversial. My previous work showed that in Drosophila, double-strand breaks on one mitochondrial genotype could be repaired based on the homologous sequence of other co-existing genotypes, indicating that there is recombination-based repair machinery inside mitochondria. To identify proteins mediating such a repair, in the first three years, we performed a candidate screen and identified REC as a helicase that drives mtDNA recombination and repair (Klucnika et al., JCB 2022). During this period, Michael Claxton focused on a genome-wide CRISPR screen to probe mtDNA damage response in human cells. So far, we have established RPE-Cas9-Tet3G cell lines expressing mitochondrially-targetted restriction enzymes upon Dox induction to cause DSBs in mitochondria. For the remaining period of this grant, we will transduce these cells with pooled sgRNA to identify proteins essential for cell survival upon mtDNA DSB damage.
Aim II: Developing a recombination toolkit to edit & map mtDNA
Probing interactions between mitochondrial genes has been challenging due to the multi-copy nature of mtDNA and a lack of genetic tools to edit and map mtDNA. Previously, my group established a system to isolate recombinant mitochondrial genomes in Drosophila. Using this toolkit, we found that sequence polymorphisms in one mitochondrial gene (mt:CoIII) could rescue the lethality caused by a detrimental mutation in another mitochondrial gene (mt:CoI). Through lipidomics profiling and biochemical and phenotypic analyses, we revealed that the two residues co-regulate cardiolipin binding to affect respiratory complex stability and activity. This work (Chiang et al, Nat Commun 2024) unwraps the complex intra-genomic interplays underlying mtDNA-linked disorders and how they influence disease expression.
Aim III: Establishing methods for delivery of exogenous DNA into mitochondria
We tried multiple methods to deliver exogenous DNA into fly mitochondria. However, none of these approaches worked. We will continue to explore other methods proposed in the application. In the meantime, staff recruited to work on this part of the project investigated mtDNA maintenance during late spermatogenesis in parallel. This work identified POLDIP2 as an essential player in eliminating paternal mtDNA in late spermatids to guarantee the maternal inheritance of mitochondrial genomes (Wang*, Meerod*, Cortes-Silva* et al) has been accepted in principle by EMBO J.
Since the beginning of this grant, we have made three important discoveries. First, identifying nuclear recombination proteins localising to mitochondria to safeguard mtDNA during development and ageing is a very exciting discovery (Klucnika et al, 2024). To date, very little is known about repair machinery inside mitochondria. In particular, the existence and significance of recombination-based repair remain controversial and completely uncharacterized for mtDNA. Our work thus provides new insights into this under-explored area and future avenues for more detailed characterisation of repair proteins in maintaining animal mtDNA. We also expect to identify and characterise more mtDNA repair proteins by our ongoing candidate screen.
Second, we believe our work showing genetic polymorphisms in one mitochondrial gene can modulate the pathogenic expression of mtDNA mutations in another mitochondrial gene represents the first example of gene interaction inside mitochondria. This work (Chiang et al, Nat Commun 2024) demonstrates the power of our mtDNA recombination system in uncovering multifaceted intra-genome interactions underlying complex mtDNA-linked disorders. Our system opens a new avenue to dissect the molecular basis of how natural mtDNA sequence variations modulate the pathogenicity of a given mtDNA mutation, which could aid disease diagnosis and treatment. Mapping interactions between mitochondrial genes has not been possible until this study.
Finally, the grant also contributed to two other publications that probe mtDNA and mitochondrial dynamics during spermatogenesis. This resulted in the discovery of a special mitochondrial structure formed in spermatocytes of various insect species (LI et al, PNAS 2023), and provided insight into the mechanisms that ensure the maternal inheritance of mtDNA (Wang et al, EMBO, accepted). These findings significantly advance our understanding of mitochondrial biology.
Second, we believe our work showing genetic polymorphisms in one mitochondrial gene can modulate the pathogenic expression of mtDNA mutations in another mitochondrial gene represents the first example of gene interaction inside mitochondria. This work (Chiang et al, Nat Commun 2024) demonstrates the power of our mtDNA recombination system in uncovering multifaceted intra-genome interactions underlying complex mtDNA-linked disorders. Our system opens a new avenue to dissect the molecular basis of how natural mtDNA sequence variations modulate the pathogenicity of a given mtDNA mutation, which could aid disease diagnosis and treatment. Mapping interactions between mitochondrial genes has not been possible until this study.
Finally, the grant also contributed to two other publications that probe mtDNA and mitochondrial dynamics during spermatogenesis. This resulted in the discovery of a special mitochondrial structure formed in spermatocytes of various insect species (LI et al, PNAS 2023), and provided insight into the mechanisms that ensure the maternal inheritance of mtDNA (Wang et al, EMBO, accepted). These findings significantly advance our understanding of mitochondrial biology.