Pirh2-dependent DNA damage in neurons induced by the G-quadruplex ligand pyridostatin
2023; Elsevier BV; Volume: 299; Issue: 10 Linguagem: Inglês
10.1016/j.jbc.2023.105157
ISSN1083-351X
AutoresRocio Diaz Escarcega, Abhijeet A. Patil, Jose F. Moruno-Manchon, Akihiko Urayama, Sean P. Marrelli, Nayun Kim, David Monchaud, Louise D. McCullough, Andrey S. Tsvetkov,
Tópico(s)RNA Interference and Gene Delivery
ResumoNoncanonical base pairing between four guanines (G) within single-stranded G-rich sequences leads to formation of а G-quartet. Self-stacking of G-quartets results in a columnar four-stranded DNA structure known as the G-quadruplex (G4 or G4-DNA). In cancer cells, G4-DNA regulates multiple DNA-dependent processes, including transcription, replication, and telomere function. How G4s function in neurons is poorly understood. Here, we performed a genome-wide gene expression analysis (RNA-Seq) to identify genes modulated by a G4-DNA ligand, pyridostatin (PDS), in primary cultured neurons. PDS promotes stabilization of G4 structures, thus allowing us to define genes directly or indirectly responsive to G4 regulation. We found that 901 genes were differentially expressed in neurons treated with PDS out of a total of 18,745 genes with measured expression. Of these, 505 genes were downregulated and 396 genes were upregulated and included gene networks regulating p53 signaling, the immune response, learning and memory, and cellular senescence. Within the p53 network, the E3 ubiquitin ligase Pirh2 (Rchy1), a modulator of DNA damage responses, was upregulated by PDS. Ectopically overexpressing Pirh2 promoted the formation of DNA double-strand breaks, suggesting a new DNA damage mechanism in neurons that is regulated by G4 stabilization. Pirh2 downregulated DDX21, an RNA helicase that unfolds G4-RNA and R-loops. Finally, we demonstrated that Pirh2 increased G4-DNA levels in the neuronal nucleolus. Our data reveal the genes that are responsive to PDS treatment and suggest similar transcriptional regulation by endogenous G4-DNA ligands. They also connect G4-dependent regulation of transcription and DNA damage mechanisms in neuronal cells. Noncanonical base pairing between four guanines (G) within single-stranded G-rich sequences leads to formation of а G-quartet. Self-stacking of G-quartets results in a columnar four-stranded DNA structure known as the G-quadruplex (G4 or G4-DNA). In cancer cells, G4-DNA regulates multiple DNA-dependent processes, including transcription, replication, and telomere function. How G4s function in neurons is poorly understood. Here, we performed a genome-wide gene expression analysis (RNA-Seq) to identify genes modulated by a G4-DNA ligand, pyridostatin (PDS), in primary cultured neurons. PDS promotes stabilization of G4 structures, thus allowing us to define genes directly or indirectly responsive to G4 regulation. We found that 901 genes were differentially expressed in neurons treated with PDS out of a total of 18,745 genes with measured expression. Of these, 505 genes were downregulated and 396 genes were upregulated and included gene networks regulating p53 signaling, the immune response, learning and memory, and cellular senescence. Within the p53 network, the E3 ubiquitin ligase Pirh2 (Rchy1), a modulator of DNA damage responses, was upregulated by PDS. Ectopically overexpressing Pirh2 promoted the formation of DNA double-strand breaks, suggesting a new DNA damage mechanism in neurons that is regulated by G4 stabilization. Pirh2 downregulated DDX21, an RNA helicase that unfolds G4-RNA and R-loops. Finally, we demonstrated that Pirh2 increased G4-DNA levels in the neuronal nucleolus. Our data reveal the genes that are responsive to PDS treatment and suggest similar transcriptional regulation by endogenous G4-DNA ligands. They also connect G4-dependent regulation of transcription and DNA damage mechanisms in neuronal cells. The G-quadruplex (G4) is a four-stranded higher-order nucleic acid structure that folds from single-stranded guanine (G)-rich DNA or RNA sequences. Intramolecular folding of G-enriched sequences is promoted by the self-association of four Gs to form G-quartets, which are then stacked on top of each other. Physiological cations, such as K+ or Na+, promote G4-DNA and G4-RNA formation and stabilization by decreasing the electron density within the central stem of the G4 structure that results from the presence of inwardly pointing carbonyl groups of Gs (four carbonyls per а G-quartet). G4-DNA and G4-RNA are thermodynamically highly stable structures with fast and reversible formation kinetics (1Lane A.N. Chaires J.B. Gray R.D. Trent J.O. Stability and kinetics of G-quadruplex structures.Nucleic Acids Res. 2008; 36: 5482-5515Crossref PubMed Scopus (575) Google Scholar) and are functionally modulated in cells by chaperones and helicases (2Meier-Stephenson V. G4-quadruplex-binding proteins: review and insights into selectivity.Biophys. Rev. 2022; 14: 635-654Crossref PubMed Scopus (30) Google Scholar, 3Sauer M. Paeschke K. G-quadruplex unwinding helicases and their function in vivo.Biochem. Soc. Trans. 2017; 45: 1173-1182Crossref PubMed Scopus (110) Google Scholar, 4Lejault P. Mitteaux J. Sperti F.R. Monchaud D. How to untie G-quadruplex knots and why?.Cell Chem. Biol. 2021; 28: 436-455Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). All these features make G4s ideal genetic levers to control DNA- and RNA-dependent processes, including gene expression at the transcriptional and translational levels (5Rhodes D. Lipps H.J. G-quadruplexes and their regulatory roles in biology.Nucleic Acids Res. 2015; 43: 8627-8637Crossref PubMed Scopus (1026) Google Scholar, 6Maizels N. Gray L.T. The G4 genome.PLoS Genet. 2013; 9e1003468Crossref PubMed Scopus (400) Google Scholar). Putative G4-forming sequences (QFSes) were identified in the human genome by both in silico (7Puig Lombardi E. Londoño-Vallejo A. A guide to computational methods for G-quadruplex prediction.Nucleic Acids Res. 2019; 48: 1-15Crossref Scopus (106) Google Scholar) and genome-wide (e.g., G4-Seq, G4 chromatin immunoprecipitation-Seq) or transcriptome-wide methods (e.g., rG4-Seq, G4RP-Seq) (8Chambers V.S. Marsico G. Boutell J.M. Di Antonio M. Smith G.P. Balasubramanian S. High-throughput sequencing of DNA G-quadruplex structures in the human genome.Nat. Biotechnol. 2015; 33: 877-881Crossref PubMed Scopus (795) Google Scholar). QFSes are often located in gene promoters (9Marsico G. Chambers V.S. Sahakyan A.B. McCauley P. Boutell J.M. Antonio M.D. et al.Whole genome experimental maps of DNA G-quadruplexes in multiple species.Nucleic Acids Res. 2019; 47: 3862-3874Crossref PubMed Scopus (212) Google Scholar), near the replication start sites (10Besnard E. Babled A. Lapasset L. Milhavet O. Parrinello H. Dantec C. et al.Unraveling cell type-specific and reprogrammable human replication origin signatures associated with G-quadruplex consensus motifs.Nat. Struct. Mol. Biol. 2012; 19: 837-844Crossref PubMed Scopus (305) Google Scholar), and in mitochondrial DNA (11Damas J. Carneiro J. Goncalves J. Stewart J.B. Samuels D.C. Amorim A. et al.Mitochondrial DNA deletions are associated with non-B DNA conformations.Nucleic Acids Res. 2012; 40: 7606-7621Crossref PubMed Scopus (52) Google Scholar). G4 chromatin immunoprecipitation-Seq demonstrated that G4-DNA forms at nucleosome-depleted and transcriptionally active sites (12Spiegel J. Cuesta S.M. Adhikari S. Hänsel-Hertsch R. Tannahill D. Balasubramanian S. G-quadruplexes are transcription factor binding hubs in human chromatin.Genome Biol. 2021; 22: 117Crossref PubMed Scopus (89) Google Scholar), thus functionally linking G4s to active DNA-dependent processes (8Chambers V.S. Marsico G. Boutell J.M. Di Antonio M. Smith G.P. Balasubramanian S. High-throughput sequencing of DNA G-quadruplex structures in the human genome.Nat. Biotechnol. 2015; 33: 877-881Crossref PubMed Scopus (795) Google Scholar). Analyses of "active" G4-DNA structures revealed that G4 landscapes differ between cell types (13Hansel-Hertsch R. Beraldi D. Lensing S.V. Marsico G. Zyner K. Parry A. et al.G-quadruplex structures mark human regulatory chromatin.Nat. Genet. 2016; 48: 1267-1272Crossref PubMed Scopus (548) Google Scholar, 14Biffi G. Tannahill D. Miller J. Howat W.J. Balasubramanian S. Elevated levels of G-quadruplex formation in human stomach and liver cancer tissues.PLoS One. 2014; 9e102711Crossref Scopus (144) Google Scholar), and these G4 landscapes are regulated by G4-DNA-binding proteins and G4-DNA helicases (3Sauer M. Paeschke K. G-quadruplex unwinding helicases and their function in vivo.Biochem. Soc. Trans. 2017; 45: 1173-1182Crossref PubMed Scopus (110) Google Scholar, 4Lejault P. Mitteaux J. Sperti F.R. Monchaud D. How to untie G-quadruplex knots and why?.Cell Chem. Biol. 2021; 28: 436-455Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). We previously showed that the predominant cells in the brain—neurons, astrocytes, and microglia—basally exhibit different G4 landscapes (15Tabor N. Ngwa C. Mitteaux J. Meyer M.D. Moruno-Manchon J.F. Zhu L. et al.Differential responses of neurons, astrocytes, and microglia to G-quadruplex stabilization.Aging (Albany NY). 2021; 13: 15917-15941Crossref PubMed Scopus (9) Google Scholar). Many small-molecule G4-DNA ligands that bind to G4s have been discovered over the last decade. Pyridostatin (PDS), the most studied G4 ligand, is an efficient G4-DNA stabilizer and a potential chemotherapy agent as it promotes DNA double-strand breaks (DSBs) at G4 sites (16Rodriguez R. Miller K.M. Forment J.V. Bradshaw C.R. Nikan M. Britton S. et al.Small-molecule–induced DNA damage identifies alternative DNA structures in human genes.Nat. Chem. Biol. 2012; 8: 301-310Crossref PubMed Scopus (520) Google Scholar, 17Olivieri M. Cho T. Álvarez-Quilón A. Li K. Schellenberg M.J. Zimmermann M. et al.A genetic map of the response to DNA damage in human cells.Cell. 2020; 182: 481-496.e421Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 18Bossaert M. Pipier A. Riou J.-F. Noirot C. Nguyên L.-T. Serre R.-F. et al.Transcription-associated topoisomerase 2α (TOP2A) activity is a major effector of cytotoxicity induced by G-quadruplex ligands.Elife. 2021; 10e65184Crossref PubMed Scopus (32) Google Scholar, 19Zell J. Rota Sperti F. Britton S. Monchaud D. DNA folds threaten genetic stability and can be leveraged for chemotherapy.RSC Chem. Biol. 2021; 2: 47-76Crossref PubMed Google Scholar). Intriguingly, in cancer cells, PDS activates a number of innate immune genes, suggesting that G4 ligands may act as immunomodulators (20Miglietta G. Russo M. Duardo R.C. Capranico G. G-quadruplex binders as cytostatic modulators of innate immune genes in cancer cells.Nucleic Acids Res. 2021; 49: 6673-6686Crossref PubMed Scopus (20) Google Scholar). G4 ligands have been extensively studied in yeast and cancer cells, providing insights into G4 functions in these cell types. However, little is known about how G4 ligands modulate DNA-based mechanisms in many other cell types, such as postmitotic neurons. Genome integrity is critical for neurons, and thus, these cells devote substantial resources to minimize genomic instability (21Maynard S. Fang E.F. Scheibye-Knudsen M. Croteau D.L. Bohr V.A. DNA damage, DNA repair, aging, and neurodegeneration.Cold Spring Harb. Perspect. Med. 2015; 5a025130Crossref PubMed Scopus (256) Google Scholar). DNA damage and repair occur in neurons during normal physiologic activity (22Suberbielle E. Sanchez P.E. Kravitz A.V. Wang X. Ho K. Eilertson K. et al.Physiologic brain activity causes DNA double-strand breaks in neurons, with exacerbation by amyloid-beta.Nat. Neurosci. 2013; 16: 613-621Crossref PubMed Scopus (340) Google Scholar). However, as neuronal cells age, the DNA repair machinery (DNA damage response [DDR]) is less efficient, leading to genomic instability and cellular dysfunction (23Madabhushi R. Pan L. Tsai L.H. DNA damage and its links to neurodegeneration.Neuron. 2014; 83: 266-282Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). DNA damage is associated with many age-associated neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease (24Canugovi C. Misiak M. Ferrarelli L.K. Croteau D.L. Bohr V.A. The role of DNA repair in brain related disease pathology.DNA Repair. 2013; 12: 578-587Crossref PubMed Scopus (105) Google Scholar, 25Penndorf D. Witte O. Kretz A. DNA plasticity and damage in amyotrophic lateral sclerosis.Neural Regen. Res. 2018; 13: 173-180Crossref PubMed Scopus (19) Google Scholar). G4-DNA is also involved in DNA recombination, deletion, and gross chromosomal rearrangements (26Katapadi V.K. Nambiar M. Raghavan S.C. Potential G-quadruplex formation at breakpoint regions of chromosomal translocations in cancer may explain their fragility.Genomics. 2012; 100: 72-80Crossref PubMed Scopus (61) Google Scholar, 27Wiedemann E.-M. Peycheva M. Pavri R. DNA replication origins in immunoglobulin switch regions regulate class switch recombination in an r-loop-dependent manner.Cell Rep. 2016; 17: 2927-2942Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 28Ribeyre C. Lopes J. Boule J.B. Piazza A. Guedin A. Zakian V.A. et al.The yeast Pif1 helicase prevents genomic instability caused by G-quadruplex-forming CEB1 sequences in vivo.PLoS Genet. 2009; 5e1000475Crossref PubMed Scopus (285) Google Scholar, 29Piazza A. Largy E. Boulé J.-B. Lopes J. Mingo K. Teulade-Fichou M.-P. et al.Genetic instability triggered by G-quadruplex interacting Phen-DC compounds in Saccharomyces cerevisiae.Nucleic Acids Res. 2010; 38: 4337-4348Crossref PubMed Scopus (136) Google Scholar, 30Massey T.H. Jones L. The central role of DNA damage and repair in CAG repeat diseases.Dis. Model. Mech. 2018; 11dmm031930Crossref PubMed Scopus (69) Google Scholar). Overly stabilized G4-DNA structures act as physical barriers for polymerases, leading to DNA damage via transcription-coupled repair poisoning (31Puget N. Miller K.M. Legube G. Non-canonical DNA/RNA structures during transcription-coupled double-strand break repair: roadblocks or bona fide repair intermediates?.DNA Repair (Amst). 2019; 81102661Crossref PubMed Scopus (62) Google Scholar) and/or Top2 trapping at G4 sites (17Olivieri M. Cho T. Álvarez-Quilón A. Li K. Schellenberg M.J. Zimmermann M. et al.A genetic map of the response to DNA damage in human cells.Cell. 2020; 182: 481-496.e421Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 18Bossaert M. Pipier A. Riou J.-F. Noirot C. Nguyên L.-T. Serre R.-F. et al.Transcription-associated topoisomerase 2α (TOP2A) activity is a major effector of cytotoxicity induced by G-quadruplex ligands.Elife. 2021; 10e65184Crossref PubMed Scopus (32) Google Scholar, 32Bruno P.M. Lu M. Dennis K.A. Inam H. Moore C.J. Sheehe J. et al.The primary mechanism of cytotoxicity of the chemotherapeutic agent CX-5461 is topoisomerase II poisoning.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 4053-4060Crossref PubMed Scopus (87) Google Scholar). Various G4-regulating proteins suppress G4-associated genomic instability in yeast (28Ribeyre C. Lopes J. Boule J.B. Piazza A. Guedin A. Zakian V.A. et al.The yeast Pif1 helicase prevents genomic instability caused by G-quadruplex-forming CEB1 sequences in vivo.PLoS Genet. 2009; 5e1000475Crossref PubMed Scopus (285) Google Scholar, 33Paeschke K. Bochman M.L. Garcia P.D. Cejka P. Friedman K.L. Kowalczykowski S.C. et al.Pif1 family helicases suppress genome instability at G-quadruplex motifs.Nature. 2013; 497: 458-462Crossref PubMed Scopus (353) Google Scholar, 34Yadav P. Owiti N. Kim N. The role of topoisomerase I in suppressing genome instability associated with a highly transcribed guanine-rich sequence is not restricted to preventing RNA:DNA hybrid accumulation.Nucleic Acids Res. 2016; 44: 718-729Crossref PubMed Scopus (32) Google Scholar, 35Lopez C.R. Singh S. Hambarde S. Griffin W.C. Gao J. Chib S. et al.Yeast Sub1 and human PC4 are G-quadruplex binding proteins that suppress genome instability at co-transcriptionally formed G4 DNA.Nucleic Acids Res. 2017; 45: 5850-5862Crossref PubMed Scopus (35) Google Scholar) and cancer cells (36Clynes D. Jelinska C. Xella B. Ayyub H. Scott C. Mitson M. et al.Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX.Nat. Commun. 2015; 6: 7538Crossref PubMed Scopus (191) Google Scholar, 37Wang Y. Yang J. Wild A.T. Wu W.H. Shah R. Danussi C. et al.G-quadruplex DNA drives genomic instability and represents a targetable molecular abnormality in ATRX-deficient malignant glioma.Nat. Commun. 2019; 10: 943Crossref PubMed Scopus (115) Google Scholar). We reported that aged mouse brains contain higher levels of G4-DNA than young brains (38Moruno-Manchon J.F. Lejault P. Wang Y. McCauley B. Honarpisheh P. Morales Scheihing D.A. et al.Small-molecule G-quadruplex stabilizers reveal a novel pathway of autophagy regulation in neurons.Elife. 2020; 9e52283Crossref PubMed Scopus (48) Google Scholar), and that stabilizing G4-DNA by PDS in neuronal cells contributes to genomic instability (39Moruno-Manchon J.F. Koellhoffer E.C. Gopakumar J. Hambarde S. Kim N. McCullough L.D. et al.The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons.Aging (Albany NY). 2017; 9: 1957-1970Crossref PubMed Scopus (48) Google Scholar). However, the molecular mechanisms of G4-mediated genomic instability in neurons are still poorly understood (40Vijay Kumar M.J. Morales R. Tsvetkov A.S. G-quadruplexes and associated proteins in aging and Alzheimer's disease.Front. Aging. 2023; 41164057Crossref PubMed Scopus (3) Google Scholar). In this study, we performed genome-wide gene expression analysis (RNA-Seq) to identify genes modulated by PDS in primary cultured neurons. We found that 901 genes were differentially expressed in neurons treated with PDS out of a total of 18,745 genes with measured expression. Our findings indicate that G4s represent an important mechanism of transcriptional regulation in neuronal cells, involving a network of genes regulating p53 signaling, immune response, learning and memory, cellular senescence, and others. We also found that the E3 ubiquitin ligase Pirh2 (Rchy1), which promotes the degradation of DDR proteins, was upregulated by PDS. Interestingly, ectopically overexpressing Pirh2 promotes the formation of DNA DSBs in neurons. We discovered that Pirh2 downregulates DDX21, an RNA helicase that unfolds G4-RNA and R-loops that form cotranscriptionally (41Duquette M.L. Handa P. Vincent J.A. Taylor A.F. Maizels N. Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA.Genes Dev. 2004; 18: 1618-1629Crossref PubMed Scopus (417) Google Scholar, 42Miglietta G. Russo M. Capranico G. G-quadruplex–R-loop interactions and the mechanism of anticancer G-quadruplex binders.Nucleic Acids Res. 2020; 48: 11942-11957Crossref PubMed Scopus (65) Google Scholar). We also found that Pirh2 promotes G4-DNA formation in the neuronal nucleolus. Our findings indicate that G4s are an important mechanism for modulating gene expression and DDRs in neurons. Previously, we reported that in neurons, stabilizing G4s with PDS promotes changes in chromatin structure (15Tabor N. Ngwa C. Mitteaux J. Meyer M.D. Moruno-Manchon J.F. Zhu L. et al.Differential responses of neurons, astrocytes, and microglia to G-quadruplex stabilization.Aging (Albany NY). 2021; 13: 15917-15941Crossref PubMed Scopus (9) Google Scholar) and reduces transcription of several genes that contain G4-DNA motifs (15Tabor N. Ngwa C. Mitteaux J. Meyer M.D. Moruno-Manchon J.F. Zhu L. et al.Differential responses of neurons, astrocytes, and microglia to G-quadruplex stabilization.Aging (Albany NY). 2021; 13: 15917-15941Crossref PubMed Scopus (9) Google Scholar, 38Moruno-Manchon J.F. Lejault P. Wang Y. McCauley B. Honarpisheh P. Morales Scheihing D.A. et al.Small-molecule G-quadruplex stabilizers reveal a novel pathway of autophagy regulation in neurons.Elife. 2020; 9e52283Crossref PubMed Scopus (48) Google Scholar, 39Moruno-Manchon J.F. Koellhoffer E.C. Gopakumar J. Hambarde S. Kim N. McCullough L.D. et al.The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons.Aging (Albany NY). 2017; 9: 1957-1970Crossref PubMed Scopus (48) Google Scholar). Therefore, we decided to investigate whether and how stabilizing G4-DNA with PDS would affect the whole transcriptome expression in primary cultured neurons. We hypothesized that comparing transcription in neurons treated with PDS or vehicle would reveal physiologically relevant G4-dependent molecular mechanisms. Primary mouse cortical neurons were cultured for 14 days (14 days in vitro [DIV]) until they were synaptically established. Neurons were then treated with PDS or vehicle (overnight) and analyzed by RNA-Seq. With QIAGEN RNA-Seq, we found that high-quality data were obtained for all samples (Supplemental Data 1 and 2). Principal component analysis (PCA) of RNA-Seq results revealed that the differences between control and PDS-treated samples were well pronounced (Fig. 1A). PDS-treated samples showed greater variability in clustering compared with control, which is commonly observed in RNA-Seq analyses. By RNA-Seq, 901 genes were differentially expressed in neurons treated with PDS out of a total of 18,745 genes with measured expression. Of those, 505 genes were downregulated and 396 genes were upregulated (Fig. 1, B–E). The upregulated genes tended to have a higher fold change compared with downregulated genes (Fig. 1, C–E); however, the number of genes downregulated was higher (Fig. 1, B and C). Since PCA of RNA-Seq data demonstrated separation of one individual sample in the PDS group, we removed the separated dataset and reran our analyses, which did not considerably affect the results (Fig. S1, A and B). With that analysis, 517 genes were downregulated and 394 genes were upregulated (Fig. S1, A and B), indicating that variability in the PDS cohort did not significantly alter the outcome data. Overall, we discovered that, similar to a previous study in cancer cells (20Miglietta G. Russo M. Duardo R.C. Capranico G. G-quadruplex binders as cytostatic modulators of innate immune genes in cancer cells.Nucleic Acids Res. 2021; 49: 6673-6686Crossref PubMed Scopus (20) Google Scholar), PDS treatment results in transcriptional changes, both positive and negative. RNA-Seq data were analyzed in the context of pathways obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (43Kanehisa M. Goto S. KEGG: kyoto encyclopedia of genes and genomes.Nucleic Acids Res. 2000; 28: 27-30Crossref PubMed Scopus (20816) Google Scholar, 44Kanehisa M. Goto S. Kawashima S. Nakaya A. The KEGG databases at GenomeNet.Nucleic Acids Res. 2002; 30: 42-46Crossref PubMed Google Scholar) and gene ontologies from the Gene Ontology (GO) Consortium database (45Ashburner M. Ball C.A. Blake J.A. Botstein D. Butler H. Cherry J.M. et al.Gene ontology: tool for the unification of biology. The gene ontology consortium.Nat. Genet. 2000; 25: 25-29Crossref PubMed Scopus (29031) Google Scholar). The underlying pathway topologies, including genes and their directional interactions, were obtained from the KEGG database using iPathwayGuide (Advaita Bioinformatics). In our analyses, 1583 GO terms were found to be significantly enriched in PDS-treated samples. Among them, neuron-specific and neuron-nonspecific networks were enriched (Fig. 2, A–C). For example, nervous system development, neuronal differentiation, neurogenesis, and learning and memory networks were enriched. Cellular senescence, cell death, re-entry into mitotic cell cycle, DDR, and negative and positive regulation of autophagosome networks were affected (Fig. 2, A and B). Immune response genes were also affected by PDS, which was also seen in a study with cancer cells (20Miglietta G. Russo M. Duardo R.C. Capranico G. G-quadruplex binders as cytostatic modulators of innate immune genes in cancer cells.Nucleic Acids Res. 2021; 49: 6673-6686Crossref PubMed Scopus (20) Google Scholar). Intriguingly, the positive regulation of amyloid precursor protein network was affected by PDS (Fig. 2A). Our data indicate that PDS induces neuron-specific and neuron-nonspecific transcriptional changes in primary cultured neurons. One of the most affected pathways was the p53 pathway (46Kastenhuber E.R. Lowe S.W. Putting p53 in context.Cell. 2017; 170: 1062-1078Abstract Full Text Full Text PDF PubMed Scopus (1134) Google Scholar) (KEGG: 04115) with 17 genes dysregulated (16 genes upregulated, one downregulated, Fig. 3, A–C). Among upregulated genes was the Pirh2 (Rchy1) gene (Fig. 3, C and D). Its corresponding protein interacts with G4 helicases in cancer cells (47Daks A. Petukhov A. Fedorova O. Shuvalov O. Kizenko A. Tananykina E. et al.The RNA-binding protein HuR is a novel target of Pirh2 E3 ubiquitin ligase.Cell Death Dis. 2021; 12: 581Crossref PubMed Scopus (14) Google Scholar), suggesting an interesting connection between PDS, Pirh2, and G4 helicases. With quantitative PCR (qPCR), we confirmed that PDS indeed upregulates the Pirh2 gene in cultured primary neurons (Fig. 3E). Since PCA of RNA-Seq data showed separation of one individual sample in the PDS group, we again removed the separated dataset and performed our iPathwayGuide analyses. Removing the dataset did not have a considerable effect on our findings (Fig. S2). In particular, the same 16 genes, including Pirh2, were upregulated and one gene was downregulated with small changes in the p and fold-change values (Fig. S2). Overall, the p53 signaling pathway may be affected by PDS-induced DNA damage and/or altered transcription of specific genes, triggering positive and negative feedback loops, which enhance and/or attenuate the p53 protein and integrate its functions with other signaling pathways. We then used the QFS mapper (QGRS) to computationally analyze the Pirh2 gene and its promoter in several species for the presence of putative G4-DNA motifs (Fig. S3). In Homo sapiens and Mus musculus, Pirh2 has two putative G4-DNA sequences, and its promoter has none. In Rattus norvegicus, Pirh2 and its promoter form have no QFSes (Fig. S3). A recent study generated whole-genome experimental maps of G4-DNA in multiple species (9Marsico G. Chambers V.S. Sahakyan A.B. McCauley P. Boutell J.M. Antonio M.D. et al.Whole genome experimental maps of DNA G-quadruplexes in multiple species.Nucleic Acids Res. 2019; 47: 3862-3874Crossref PubMed Scopus (212) Google Scholar). In agreement with our computational data, the Pirh2 promoter was found to contain no G4-DNA (9Marsico G. Chambers V.S. Sahakyan A.B. McCauley P. Boutell J.M. Antonio M.D. et al.Whole genome experimental maps of DNA G-quadruplexes in multiple species.Nucleic Acids Res. 2019; 47: 3862-3874Crossref PubMed Scopus (212) Google Scholar), indicating that the effect of PDS on Pirh2 might be indirect rather than direct. While analyzing our data from 14 DIV neurons treated with PDS, we noticed differences compared with our earlier data generated with four DIV neurons (39Moruno-Manchon J.F. Koellhoffer E.C. Gopakumar J. Hambarde S. Kim N. McCullough L.D. et al.The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons.Aging (Albany NY). 2017; 9: 1957-1970Crossref PubMed Scopus (48) Google Scholar). In synaptically developed 14 DIV neurons, the most affected pathway was the neuroactive ligand–receptor interaction pathway (KEGG: 04080) that included synaptic receptor genes (Fig. 3A). Many of these genes were not expressed in cultured neurons soon after plating, and thus, the data generated with freshly plated neurons would be expected to be different. In our earlier four DIV data generated with RT–qPCR, we showed that transcription of the Atg7 gene was diminished by PDS; however, the current 14 DIV data show that Atg7 was not affected. Transcription of the Brca1 gene was reduced by PDS in four DIV neurons (shown by RT–qPCR), but the current 14 DIV data show that Brca1 was not affected. We, therefore, tested if there was a difference in the expression of Atg7 and Brca1 in 14 DIV and four DIV neurons measured by PCR using Tbp as the internal control that we discovered earlier (39Moruno-Manchon J.F. Koellhoffer E.C. Gopakumar J. Hambarde S. Kim N. McCullough L.D. et al.The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons.Aging (Albany NY). 2017; 9: 1957-1970Crossref PubMed Scopus (48) Google Scholar). The Tbp gene was not affected by PDS in the current RNA-Seq dataset either (Supplemental Data 1). About 14 DIV and four DIV neurons were treated with PDS, and then mRNA was extracted and analyzed (Fig. S4). Levels of both Atg7 and Brca1 went down in four DIV neurons treated with PDS, whereas 14 DIV neuronal cells only exhibited a nonsignificant trend (Fig. S4). Interestingly, PDS robustly upregulated Pirh2 in both 14 DIV and four DIV neurons, indicating that the p53 pathway is altered by PDS in developed and freshly plated cells. We conclude that freshly plated developing neurons and synaptically established cells respond to PDS differently, at least in part, likely reflecting developmental differences in transcriptional activity. These results implicate PDS-mediated changes in neuronal transcriptional activity rather than a DNA damage–mediated pathway. Pirh2 localizes to the cytoplasm, as well as to intranuclear foci in cancer cells, although the nature of those foci was not clear (48Logan I.R. Sapountzi V. Gaughan L. Neal D.E. Robson C.N. Control of human PIRH2 protein stability: involvement of TIP60 and the proteosome.J. Biol. Chem. 2004; 279: 11696-11704Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Pirh2 interacts with and ubiquitinates a number of proteins in the nucleus and in the cytoplasm (49Wang Z. Yang B. Dong L. Peng B. He X. Liu W. A novel oncoprotein Pirh2: rising from the shadow of MDM2.Cancer Sci. 2011; 102: 909-917Crossref P
Referência(s)