Genome-wide Consequences of Deleting Any Single Gene

Teng, Xinchen; Dayhoff-Brannigan, Margaret; Cheng, Wenli; Gilbert, Catherine E; Sing, Cierra N.; Diny, Nicola L.; Wheelan, Sarah J.; Dunham, Maitreya J.; Boeke, Jef D.; Pineda, Fernando; Hardwick, J. Marie

doi:10.1016/j.molcel.2013.09.026

Cited by 175 publications

(208 citation statements)

References 49 publications

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“…Another possible explanation for the viability of the ⌬mhb1 mutant is that the deletion resulted in a genomic imbalance that was compensated for by secondary mutations elsewhere in a genome. In fact, it was shown recently that most gene knockout strains of S. cerevisiae acquired additional mutations selected to counteract the effect(s) of the original genetic defect (86). To explore this possibility, we tried to obtain independent clones lacking the YlMHB1 gene.…”

Section: Discussionmentioning

confidence: 99%

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

et al. 2014

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bMitochondrial DNA (mtDNA) is highly compacted into DNA-protein structures termed mitochondrial nucleoids (mt-nucleoids). The key mt-nucleoid components responsible for mtDNA condensation are HMG box-containing proteins such as mammalian mitochondrial transcription factor A (TFAM) and Abf2p of the yeast Saccharomyces cerevisiae. To gain insight into the function and organization of mt-nucleoids in strictly aerobic organisms, we initiated studies of these DNA-protein structures in Yarrowia lipolytica. We identified a principal component of mt-nucleoids in this yeast and termed it YlMhb1p (Y. lipolytica mitochondrial HMG box-containing protein 1). YlMhb1p contains two putative HMG boxes contributing both to DNA binding and to its ability to compact mtDNA in vitro. Phenotypic analysis of a ⌬mhb1 strain lacking YlMhb1p resulted in three interesting findings. First, although the mutant exhibits clear differences in mt-nucleoids accompanied by a large decrease in the mtDNA copy number and the number of mtDNA-derived transcripts, its respiratory characteristics and growth under most of the conditions tested are indistinguishable from those of the wild-type strain. Second, our results indicate that a potential imbalance between subunits of the respiratory chain encoded separately by nuclear DNA and mtDNA is prevented at a (post)translational level. Third, we found that mtDNA in the ⌬mhb1 strain is more prone to mutations, indicating that mtHMG box-containing proteins protect the mitochondrial genome against mutagenic events. Individual eukaryotic cells contain a population of mitochondrial DNA (mtDNA) molecules, the number of which may reach several thousand copies. For example, aerobically grown diploid cells of the yeast Saccharomyces cerevisiae contain, on average, 100 molecules of 85-kbp mtDNA. With a distance of 0.34 nm between base pairs in DNA, the total length of mtDNA reaches almost 3 mm per cell, while the diameter of the cell does not exceed 3 to 4 m. Analogously to its nuclear counterpart, mtDNA must be packaged into condensed nucleoprotein structures termed mitochondrial nucleoids (mt-nucleoids) (1-6). The size of mt-nucleoids in S. cerevisiae ranges from 0.2 to 0.9 m, meaning that mtDNA in yeasts undergoes compaction of roughly 3 orders of magnitude. Although it is known that the size and shape (oval versus globular) of mt-nucleoids, the number of mt-nucleoids (ranging from 50 to 70) per diploid cell (2), and the number of copies (up to 9) of mtDNA per mt-nucleoid (3) in S. cerevisiae depend on physiological conditions, the molecular mechanisms mediating nucleoid maintenance, dynamics, and roles in mtDNA distribution/segregation during cell division are largely not understood. This is also true for mammalian mt-nucleoids, which, in contrast to their yeast counterparts, contain a relatively small number of mtDNA molecules and are thus more solitary in nature (7,8). Description of these intra-and interspecific differences in the organization of mt-nucleoids would greatly facilitate our understanding of the m...

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Section: Discussionmentioning

confidence: 99%

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

et al. 2014

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“…Indeed, although the Tspo −/− mice were viable and physiologically functional, Banati et al reported a severe bioenergetic deficit in glial cells isolated from these Tspo −/− animals (19). Although adaptive responses in genetically engineered organisms have been reported (31,32), systematic exploration of this question has only just begun to be explored in genetically characterized organisms such as yeast, indicating that genetic adaptation to targeted gene deletion is highly reproducible and widespread (33). Understanding the commonalities and differences between the Tspo-deletion mouse models may provide insight into such processes in higher metazoan genetic models such as mice.…”

Section: Discussionmentioning

confidence: 99%

Conditional steroidogenic cell-targeted deletion of TSPO unveils a crucial role in viability and hormone-dependent steroid formation

Fan

Campioli

Midzak

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

118

112

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Translocator protein (TSPO) is a key member of the mitochondrial cholesterol transport complex in steroidogenic tissues. To assess the function of TSPO, we generated two lines of Cre-mediated Tspo conditional knockout (cKO) mice. First, gonadal somatic celltargeting Amhr2-Cre mice were crossed with Tspo-floxed mice to obtain F1 Tspo Amhr2 cKO mice (Tspo fl/fl ;Amhr2-Cre /+). The unexpected Mendelian ratio of 4.4% cKO mice was confirmed by genotyping of 12.5-day-postcoitum (dpc) embryos. As Amhr2-Cre is expressed in gonads at 12.5 dpc, these findings suggest preimplantation selection of embryos. Analysis of expression databases revealed elevated levels of Amhr2 in two-and eight-cell zygotes, suggesting ectopic Tspo silencing before the morula stage and demonstrating elevated embryonic lethality and involvement of TSPO in embryonic development. To circumvent this issue, steroidogenic cell-targeting Nr5a1-Cre mice were crossed with Tspofloxed mice. The resulting Tspo fl/fl ;Nr5a1-Cre /+ mice were born at a normal Mendelian ratio. Nr5a1-driven Tspo cKO mice exhibited highly reduced Tspo levels in adrenal cortex and gonads. Treatment of mice with human chorionic gonadotropin (hCG) resulted in increased circulating testosterone levels despite extensive lipid droplet depletion. In contrast, Nr5a1-driven Tspo cKO mice lost their ability to form corticosterone in response to adrenocorticotropic hormone (ACTH). Important for ACTH-dependent steroidogenesis, Mc2r, Stard1, and Cypa11a1 levels were unaffected, whereas Scarb1 levels were increased and accumulation of lipid droplets was observed, indicative of a blockade of cholesterol utilization for steroidogenesis. TSPO expression in the adrenal medulla and increased epinephrine production were also observed. In conclusion, TSPO was found necessary for preimplantation embryo development and ACTH-stimulated steroid biosynthesis.translocator protein | anti-Mullerian hormone receptor type II | nuclear receptor subfamily 5 group A member 1 | knockout mice | steroidogenesis T he 18-kDa translocator protein (TSPO) is an outer mitochondrial membrane (OMM) protein, originally named the peripheral benzodiazepine receptor (PBR) due to its discovery as a high-affinity binding site for the benzodiazepine diazepam outside of the central nervous system (1). Pharmacological, biochemical, and structural research has revealed TSPO's ability to bind numerous classes of drugs (1-4). Additional investigations also demonstrated that TSPO is a high-affinity cholesterol-binding protein. Cholesterol binding is localized to the C-terminal portion of its fifth transmembrane helix at a conserved cholesterol recognition/association amino acid consensus motif (1, 5). This finding was further supported by recent structural data (6). A search for additional physiological ligands of TSPO revealed that both the tetrapyrole protoporphyrin IX, a heme precursor, and the polypeptide endozepine, a GABA A modulator, bind TSPO, suggesting a role for TSPO in numerous physiological processes (1, 2, 5). Interestingly...

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“…This may account for the copy number change of Vmn2r111 and Vmn2r112 herein. Engineered copy number change or mutation of single genes has previously been found to induce copy number change of unrelated genes and mutations in secondary effect genes leading to impaired expression [40]. For Vmn2r112 there was moderate and markedly decrease in expression in Glo1(+/+) Gt(..)1Lex and Glo1(+/+) Gt(..)2Lex mutant mice, respectively.…”

Section: Discussionmentioning

confidence: 97%

Reappraisal of putative glyoxalase 1-deficient mouse and dicarbonyl stress on embryonic stem cellsin vitro

Shafie

Xue

Barker

et al. 2016

Biochemical Journal

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Glyoxalase 1 (Glo1) is a cytoplasmic enzyme with a cytoprotective function linked to metabolism of the cytotoxic side product of glycolysis, methylglyoxal (MG). It prevents dicarbonyl stress — the abnormal accumulation of reactive dicarbonyl metabolites, increasing protein and DNA damage. Increased Glo1 expression delays ageing and suppresses carcinogenesis, insulin resistance, cardiovascular disease and vascular complications of diabetes and renal failure. Surprisingly, gene trapping by the International Mouse Knockout Consortium (IMKC) to generate putative Glo1 knockout mice produced a mouse line with the phenotype characterised as normal and healthy. Here, we show that gene trapping mutation was successful, but the presence of Glo1 gene duplication, probably in the embryonic stem cells (ESCs) before gene trapping, maintained wild-type levels of Glo1 expression and activity and sustained the healthy phenotype. In further investigation of the consequences of dicarbonyl stress in ESCs, we found that prolonged exposure of mouse ESCs in culture to high concentrations of MG and/or hypoxia led to low-level increase in Glo1 copy number. In clinical translation, we found a high prevalence of low-level GLO1 copy number increase in renal failure where there is severe dicarbonyl stress. In conclusion, the IMKC Glo1 mutant mouse is not deficient in Glo1 expression through duplication of the Glo1 wild-type allele. Dicarbonyl stress and/or hypoxia induces low-level copy number alternation in ESCs. Similar processes may drive rare GLO1 duplication in health and disease.

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Genome-wide Consequences of Deleting Any Single Gene

Cited by 175 publications

References 49 publications

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

Conditional steroidogenic cell-targeted deletion of TSPO unveils a crucial role in viability and hormone-dependent steroid formation

Reappraisal of putative glyoxalase 1-deficient mouse and dicarbonyl stress on embryonic stem cellsin vitro

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