Progerin impairs 3D genome organization and induces fragile telomeres by limiting the dNTP pools.
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ABSTRACT:
SUBMITTER: Kychygina A
PROVIDER: S-EPMC8222272 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Common fragile sites (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features were suggested to underlie CFS instability, however, these features are prevalent across the genome. Therefore, the molecular mechanisms underlying CFS instability remain unclear. Here, we explore the transcriptional profile and DNA replication timing (RT) under mild replication stress in the context of the 3D genome organization. The results reveal a fragility signature, comprised of a TAD boundary overlapping a highly transcribed large gene with APH-induced RT-delay. This signature enables precise mapping of core fragility regions in known CFSs and identification of novel fragile sites. CFS stability may be compromised by incomplete DNA replication and repair in TAD boundaries core fragility regions leading to genomic instability. The identified fragility signature will allow for a more comprehensive mapping of CFSs and pave the way for investigating mechanisms promoting genomic instability in cancer.
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Project description:Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1, which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.
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Project description:BackgroundWhile the essential role of 3D nuclear architecture on nuclear functions has been demonstrated for various cell types, information available for neutrophils, essential components of the immune system, remains limited. In this study, we analysed the spatial arrangements of telomeres which play a central role in cell fate. Our studies were carried out in swine, which is an excellent model organism for both biomedical research and agronomic applications. We isolated bacterial artificial chromosome (BAC)-containing subtelomeric p and q sequences specific to each porcine chromosome. This allowed us to study the behaviour of p and q telomeres of homologous chromosomes for seven pairs chosen for their difference in length and morphology. This was performed using 3D-FISH on structurally preserved neutrophils, and confocal microscopy. Resting and lipopolysaccharide (LPS)-activated states were investigated to ascertain whether a response to a pathogen aggression modifies this organization.ResultsThe positions of the p and q telomeres relative to the nuclear outer border were determined in the two states. All p telomeres changed their position significantly during the activation process, although the effect was less pronounced for the q telomeres. The patterns of telomeric associations between homologs and their frequencies were analysed for 7 pairs of chromosomes. This analysis revealed that the distribution of pp, qq and pq associations differs significantly among the 7 chromosomes. This distribution does not fit with the theoretical distribution for each chromosome, suggesting that preferential associations occur between subtelomeres.ConclusionsThe percentage of nuclei harbouring at least one telomeric association between homologs varies significantly among the chromosomes, the smallest metacentric chromosome SSC12, which is also the richest in gene-density, harbouring the highest value. The distribution of types of telomeric associations is highly dependent on the chromosomes and is not affected by the activation process. The frequencies of telomeric associations are also highly dependent on the type of association and the type of chromosome. Overall, the LPS-activation process induces only minor changes in these patterns of associations. When telomeric associations occur, the associations of p and q arms from the same chromosome are the most frequent, suggesting that "chromosome bending" occurs in neutrophils as previously observed in gametes.
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Project description:Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here, we show that changes in intracellular dNTP levels affect replication dynamics in budding yeast in different ways. Upregulation of the activity of ribonucleotide reductase (RNR) increases elongation, indicating that dNTP pools are limiting for normal DNA replication. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition to a slow-replication mode within minutes after S-phase entry. Upregulation of RNR activity delays this transition and modulates both fork speed and origin usage under replication stress. Interestingly, we also observed that chromosomal instability (CIN) mutants have increased dNTP pools and show enhanced DNA synthesis in the presence of HU. Since upregulation of RNR promotes fork progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.
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Project description:Ribonucleotide reductase (RNR) plays a critical role in catalyzing the biosynthesis and maintaining the intracellular concentration of 4 deoxyribonucleoside triphosphates (dNTPs). Unbalanced or deficient dNTP pools cause serious genotoxic consequences. Autophagy is the process by which cytoplasmic constituents are degraded in lysosomes to maintain cellular homeostasis and bioenergetics. However, the role of autophagy in regulating dNTP pools is not well understood. Herein, we reported that starvation- or rapamycin-induced autophagy was accompanied by a decrease in RNR activity and dNTP pools in human cancer cells. Furthermore, downregulation of the small subunit of RNR (RRM2) by siRNA or treatment with the RNR inhibitor hydroxyurea substantially induced autophagy. Conversely, cancer cells with abundant endogenous intracellular dNTPs or treated with dNTP precursors were less responsive to autophagy induction by rapamycin, suggesting that autophagy and dNTP pool levels are regulated through a negative feedback loop. Lastly, treatment with si-RRM2 caused an increase in MAP1LC3B, ATG5, BECN1, and ATG12 transcript abundance in xenografted Tu212 tumors in vivo. Together, our results revealed a previously unrecognized reciprocal regulation between dNTP pools and autophagy in cancer cells.
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Project description:Progerin, a truncated unprocessed lamin A protein, causes tissue aging and degeneration. In this study we explored the role of progerin in the pathogenesis of intervertebral disc degeneration (IDD). We also examined the effect of sulforaphane (SFN) on progerin accumulation and mitochondrial dysfunction in IDD. Methods: The role of progerin in IDD was explored using human nucleus pulposus (NP) tissues, rat NP cells, and Lmna G609G knock-in mice. Immunostaining, X-ray imaging, and Western blotting were performed to assess the phenotypes of intervertebral discs. Alterations in senescence and apoptosis were evaluated by SA-?-galactosidase, immunofluorescence, flow cytometry, and TUNEL assays. Mitochondrial function was investigated by JC-1 staining, transmission electron microscopy, and determination of the level of ATP and the activities of mitochondrial enzymes. Results: The progerin level was elevated in degenerated human NP tissues. Lmna G609G/G609G mice displayed IDD, as evidenced by increased matrix metalloproteinase-13 expression and decreased collagen II and aggrecan expression and disc height. Furthermore, progerin overexpression in rat NP cells induced mitochondrial dysfunction (decreased ATP synthesis, mitochondrial membrane potential, and activities of mitochondrial complex enzymes), morphologic abnormalities, and disrupted mitochondrial dynamic (abnormal expression of proteins involved in fission and fusion), resulting in apoptosis and senescence. SFN ameliorated the progerin-induced aging defects and mitochondrial dysfunction in NP cells and IDD in Lmna G609G/G609G mice. Conclusions: Progerin is involved in the pathogenesis of IDD. Also, SFN alleviates progerin?induced IDD, which is associated with amelioration of aging defects and mitochondrial dysfunction. Thus, SFN shows promise for the treatment of IDD.
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Project description:Critically short telomeres activate cellular senescence or apoptosis, as mediated by the tumor suppressor p53, but in the absence of this checkpoint response, telomere dysfunction engenders chromosomal aberrations and cancer. Here, analysis of p53-regulated genes activated in the setting of telomere dysfunction identified Zfp365 (ZNF365 in humans) as a direct p53 target that promotes genome stability. Germline polymorphisms in the ZNF365 locus are associated with increased cancer risk, including those associated with telomere dysfunction. On the mechanistic level, ZNF365 suppresses expression of a subset of common fragile sites (CFS) including telomeres. In the absence of ZNF365, defective telomeres engage in aberrant recombination of telomere ends, leading to increased telomere sister chromatid exchange (T-SCE) and formation of anaphase DNA bridges, including ultra-fine DNA bridges (UFB), and ultimately increased cytokinesis failure and aneuploidy. Thus, the p53-ZNF365 axis contributes to genomic stability in the setting of telomere dysfunction. We expressed an inducible p53 allele encoding a p53-estrogen receptor fusion protein (p53ER) that becomes functional upon addition of 4-hydroxytamoxifen (4-OHT) in TKO cells. We chose a time point of 4 hours post-4-OHT induction to catalog potential direct targets.
2013-08-01 | E-GEOD-44599 | biostudies-arrayexpress
Project description:Critically short telomeres activate cellular senescence or apoptosis, as mediated by the tumor suppressor p53, but in the absence of this checkpoint response, telomere dysfunction engenders chromosomal aberrations and cancer. Here, analysis of p53-regulated genes activated in the setting of telomere dysfunction identified Zfp365 (ZNF365 in humans) as a direct p53 target that promotes genome stability. Germline polymorphisms in the ZNF365 locus are associated with increased cancer risk, including those associated with telomere dysfunction. On the mechanistic level, ZNF365 suppresses expression of a subset of common fragile sites, including telomeres. In the absence of ZNF365, defective telomeres engage in aberrant recombination of telomere ends, leading to increased telomere sister chromatid exchange and formation of anaphase DNA bridges, including ultra-fine DNA bridges, and ultimately increased cytokinesis failure and aneuploidy. Thus, the p53-ZNF365 axis contributes to genomic stability in the setting of telomere dysfunction.
| S-EPMC3710545 | biostudies-literature
Project description:Intracellular levels of deoxyribonucleoside triphosphate (dNTP) must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools have recently been associated with increased mutagenesis, genomic instability and tumorigenesis. However, the mechanisms by which low or imbalanced dNTP pools affect DNA replication remain poorly understood. Here, we have modulated the activity of ribonucleotide reductase (RNR), a key enzyme catalyzing a rate-limiting step of dNTP production, to monitor the effect of altered dNTP levels on replication dynamics in budding yeast. We show that dNTP pools are limiting for normal DNA synthesis as upregulation of RNR activity increases replication fork speed. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition from a regular- to a slow-replication mode within minutes after S-phase entry. Interestingly, we found that upregulation of RNR activity delays this transition and that dNTP levels modulate both fork speed and origin usage under replication stress. Moreover, we report that chromosomal instability (CIN) mutants show increased dNTP pools and enhanced DNA synthesis in the presence of HU. Since upregulation of RNR allows forks to progress faster in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.
2012-01-01 | GSE33686 | GEO
Project description:The integrity of the genome depends on diverse pathways that regulate DNA metabolism. Defects in these pathways result in genome instability, a hallmark of cancer. Deletion of ELG1 in budding yeast, when combined with hypomorphic alleles of PCNA results in spontaneous DNA damage during S phase that elicits upregulation of ribonucleotide reductase (RNR) activity. Increased RNR activity leads to a dramatic expansion of deoxyribonucleotide (dNTP) pools in G1 that allows cells to synthesize significant fractions of the genome in the presence of hydroxyurea in the subsequent S phase. Consistent with the recognized correlation between dNTP levels and spontaneous mutation, compromising ELG1 and PCNA results in a significant increase in mutation rates. Deletion of distinct genome stability genes RAD54, RAD55, and TSA1 also results in increased dNTP levels and mutagenesis, suggesting that this is a general phenomenon. Together, our data point to a vicious circle in which mutations in gatekeeper genes give rise to genomic instability during S phase, inducing expansion of the dNTP pool, which in turn results in high levels of spontaneous mutagenesis.
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