Miria Ricchetti

Continued Colonization of the Human Genome by Mitochondrial DNA

Integration of mitochondrial DNA fragments into nuclear chromosomes (giving rise to nuclear DNA sequences of mitochondrial origin, or NUMTs) is an ongoing process that shapes nuclear genomes. In yeast this process depends on double-strand-break repair. Since NUMTs lack amplification and specific integration mechanisms, they represent the prototype of exogenous insertions in the nucleus. From sequence analysis of the genome of Homo sapiens, followed by sampling humans from different ethnic backgrounds, and chimpanzees, we have identified 27 NUMTs that are specific to humans and must have colonized human chromosomes in the last 4–6 million years. Thus, we measured the fixation rate of NUMTs in the human genome. Six such NUMTs show insertion polymorphism and provide a useful set of DNA markers for human population genetics. We also found that during recent human evolution, Chromosomes 18 and Y have been more susceptible to colonization by NUMTs. Surprisingly, 23 out of 27 human-specific NUMTs are inserted in known or predicted genes, mainly in introns. Some individuals carry a NUMT insertion in a tumor-suppressor gene and in a putative angiogenesis inhibitor. Therefore in humans, but not in yeast, NUMT integrations preferentially target coding or regulatory sequences. This is indeed the case for novel insertions associated with human diseases and those driven by environmental insults. We thus propose a mutagenic phenomenon that may be responsible for a variety of genetic diseases in humans and suggest that genetic or environmental factors that increase the frequency of chromosome breaks provide the impetus for the continued colonization of the human genome by mitochondrial DNA.

Skeletal muscle stem cells adopt a dormant cell state post mortem and retain regenerative capacity

The accessibility to stem cells from healthy or diseased individuals, and the maintenance of their potency are challenging issues for stem cell biology. Here we report the isolation of viable and functional skeletal myogenic cells from humans up to 17 days, and mice up to 14 days post mortem, much longer beyond previous reports. Muscle stem cells are enriched in post mortem tissue, suggesting a selective survival advantage compared with other cell types. Transplantation of mouse muscle and haematopoietic stem cells regenerates tissues robustly. Cellular quiescence contributes to this cell viability where cells adopt a reversible dormant state characterized by reduced metabolic activity, a prolonged lag phase before the first cell division, elevated levels of reactive oxygen species and a transcriptional status less primed for commitment. Finally, severe hypoxia, or anoxia is critical for maintaining stem cell viability and regenerative capacity. Thus, these cells provide a useful resource for studying stem cell biology.

Distance from the chromosome end determines the efficiency of double strand break repair in subtelomeres of haploid yeast.

Double strand break (DSB) repair plays an important role in chromosome evolution. We have investigated the fate of DSBs as a function of their location along the yeast chromosome XI, in a system where no conventional homologous recombination can occur. We report that the relative frequency of non-homologous endjoining (NHEJ), which is the exclusive mode of DSB repair in the internal chromosomal portion, decreases gradually towards the telomere, keeping the absolute frequency nearly constant, and that other repair mechanisms, which generally involve the loss of the distal chromosomal fragment, appear in subtelomeric regions. Distance of the DSB from chromosome ends plays a critical role in the global frequency of these repair mechanisms. Direct telomere additions are rare, and other events such as break-induced replication, plasmid incorporation, and gene conversion, involve acquisition of heterologous sequences. Therefore, in subtelomeric regions, cell survival to DSBs is higher and alternative modes of repair allow new genomic combinations to be generated. Furthermore, subtelomeric rearrangements depend on the recombination process, which, unexpectedly, also promotes the joining of heterologous sequences. Finally, we report that the Rad52 protein increases the efficiency of NHEJ.

An end-joining repair mechanism in Escherichia coli

Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and stability of eukaryote genomes. Although some bacteria possess a simplified NHEJ mechanism, the human commensal Escherichia coli is thought to rely exclusively on homology-directed mechanisms to repair DNA double-strand breaks (DSBs). We show here that laboratory and pathogenic E. coli strains possess a distinct end-joining activity that repairs DSBs and generates genome rearrangements. This mechanism, named alternative end-joining (A-EJ), does not rely on the key NHEJ proteins Ku and Ligase-D which are absent in E. coli. Differently from classical NHEJ, A-EJ is characterized by extensive end-resection largely due to RecBCD, by overwhelming usage of microhomology and extremely rare DNA synthesis. We also show that A-EJ is dependent on the essential Ligase-A and independent on Ligase-B. Importantly, mutagenic repair requires a functional Ligase-A. Although generally mutagenic, accurate A-EJ also occurs and is frequent in some pathogenic bacteria. Furthermore, we show the acquisition of an antibiotic-resistance gene via A-EJ, refuting the notion that bacteria gain exogenous sequences only by recombination-dependent mechanisms. This finding demonstrates that E. coli can integrate unrelated, nonhomologous exogenous sequences by end-joining and it provides an alternative strategy for horizontal gene transfer in the bacterial genome. Thus, A-EJ contributes to bacterial genome evolution and adaptation to environmental challenges. Interestingly, the key features of A-EJ also appear in A-NHEJ, an alternative end-joining mechanism implicated in chromosomal translocations associated with human malignancies, and we propose that this mutagenic repair might have originated in bacteria.

Sepsis induces long-term metabolic and mitochondrial muscle stem cell dysfunction amenable by mesenchymal stem cell therapy

Sepsis, or systemic inflammatory response syndrome, is the major cause of critical illness resulting in admission to intensive care units. Sepsis is caused by severe infection and is associated with mortality in 60% of cases. Morbidity due to sepsis is complicated by neuromyopathy, and patients face long-term disability due to muscle weakness, energetic dysfunction, proteolysis and muscle wasting. These processes are triggered by pro-inflammatory cytokines and metabolic imbalances and are aggravated by malnutrition and drugs. Skeletal muscle regeneration depends on stem (satellite) cells. Herein we show that mitochondrial and metabolic alterations underlie the sepsis-induced long-term impairment of satellite cells and lead to inefficient muscle regeneration. Engrafting mesenchymal stem cells improves the septic status by decreasing cytokine levels, restoring mitochondrial and metabolic function in satellite cells, and improving muscle strength. These findings indicate that sepsis affects quiescent muscle stem cells and that mesenchymal stem cells might act as a preventive therapeutic approach for sepsis-related morbidity.

Large-scale exploration of growth inhibition caused by overexpression of genomic fragments in Saccharomyces cerevisiae.

We have screened the genome of Saccharomyces cerevisiae for fragments that confer a growth-retardation phenotype when overexpressed in a multicopy plasmid with a tetracycline-regulatable (Tet-off) promoter. We selected 714 such fragments with a mean size of 700 base-pairs out of around 84,000 clones tested. These include 493 in-frame open reading frame fragments corresponding to 454 distinct genes (of which 91 are of unknown function), and 162 out-of-frame, antisense and intergenic genomic fragments, representing the largest collection of toxic inserts published so far in yeast.

Nitroso-redox balance and mitochondrial homeostasis are regulated by STOX1, a pre-eclampsia-associated gene.

AIMS Storkhead box 1 (STOX1) is a winged-helix transcription factor that is implicated in the genetic forms of a high-prevalence human gestational disease, pre-eclampsia. STOX1 overexpression confers pre-eclampsia-like transcriptomic features to trophoblastic cell lines and pre-eclampsia symptoms to pregnant mice. The aim of this work was to evaluate the impact of STOX1 on free radical equilibrium and mitochondrial function, both in vitro and in vivo. RESULTS Transcriptome analysis of STOX1-transgenic versus nontransgenic placentas at 16.5 days of gestation revealed alterations of mitochondria-related pathways. Placentas overexpressing STOX1 displayed altered mitochondrial mass and were severely biased toward protein nitration, indicating nitroso-redox imbalance in vivo. Trophoblast cells overexpressing STOX1 displayed an increased mitochondrial activity at 20% O2 and in hypoxia, despite reduction of the mitochondrial mass in the former. STOX1 overexpression is, therefore, associated with hyperactive mitochondria, resulting in increased free radical production. Moreover, nitric oxide (NO) production pathways were activated, resulting in peroxynitrite formation. At low oxygen pressure, STOX1 overexpression switched the free radical balance from reactive oxygen species (ROS) to reactive nitrogen species (RNS) in the placenta as well as in a trophoblast cell line. INNOVATION In pre-eclamptic placentas, NO interacts with ROS and generates peroxynitrite and nitrated proteins as end products. This process will deprive the maternal organism of NO, a crucial vasodilator molecule. CONCLUSION Our data posit STOX1 as a genetic switch in the ROS/RNS balance and suggest an explanation for elevated blood pressure in pre-eclampsia.

More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny.

The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.

Reversal of mitochondrial defects with CSB-dependent serine protease inhibitors in patient cells of the progeroid Cockayne syndrome

Significance Ageing is dramatically accelerated in Cockayne syndrome (CS), but the impairments that lead to this phenotype have not been elucidated. The DNA repair proteins CSA or CSB are mutated in CS, but premature ageing is not caused by the DNA repair defect. CSB also affects mitochondrial turnover. Our data reveal a novel pathway that is affected in CS cells. We show that CSB deregulates the expression of a serine protease, which degrades mitochondrial DNA polymerase gamma and impairs mitochondrial function. We rescue this defect, by two independent strategies, in primary cells from patients. Our findings open novel possibilities for developing treatments, which are presently missing for CS patients. Abnormalities revealed here might occur at a slower rate during normal physiological ageing. UV-sensitive syndrome (UVSS) and Cockayne syndrome (CS) are human disorders caused by CSA or CSB gene mutations; both conditions cause defective transcription-coupled repair and photosensitivity. Patients with CS also display neurological and developmental abnormalities and dramatic premature aging, and their cells are hypersensitive to oxidative stress. We report CSA/CSB-dependent depletion of the mitochondrial DNA polymerase-γ catalytic subunit (POLG1), due to HTRA3 serine protease accumulation in CS, but not in UVsS or control fibroblasts. Inhibition of serine proteases restored physiological POLG1 levels in either CS fibroblasts and in CSB-silenced cells. Moreover, patient-derived CS cells displayed greater nitroso-redox imbalance than UVSS cells. Scavengers of reactive oxygen species and peroxynitrite normalized HTRA3 and POLG1 levels in CS cells, and notably, increased mitochondrial oxidative phosphorylation, which was altered in CS cells. These data reveal critical deregulation of proteases potentially linked to progeroid phenotypes in CS, and our results suggest rescue strategies as a therapeutic option.

Prevalent coordination of mitochondrial DNA transcription and initiation of replication with the cell cycle

Nuclear (nDNA) and mitochondrial DNA (mtDNA) communication is essential for cell function, but it remains unclear whether the replication of these genomes is linked. We inspected human cells with a novel fluorescence in situ hybridization protocol (mitochondrial Transcription and Replication Imaging Protocol) that identifies mitochondrial structures engaged in initiation of mtDNA replication and unique transcript profiles, and reconstruct the temporal series of mitochondrial and nuclear events in single cells during the cell cycle. We show that mtDNA transcription and initiation of replication are prevalently coordinated with the cell cycle, preceding nuclear DNA synthesis, and being reactivated towards the end of S-phase. This coordination is achieved by modulating the fraction of mitochondrial structures that intiate mtDNA synthesis and/or contain transcript at a given time. Thus, although replication of the mitochondrial genome is active through the entire cell cycle, but in a limited fraction of mitochondrial structures, peaks of these activities are synchronized with nDNA synthesis. After release from blockage of mtDNA replication with either nocodazole or double thymidine treatment, prevalent mtDNA and nDNA synthesis occurred simultaneously, indicating that mitochondrial coordination with the nuclear phase can be adjusted in response to physiological alterations. These findings will help redefine other nuclear–mitochondrial links in cell function.