Enhancers modulate gene expression by interacting with promoters.Models of enhancer-promoter interactions(EPIs)in the literature involve the activity of many components,including transcription factors and nucleic acid...Enhancers modulate gene expression by interacting with promoters.Models of enhancer-promoter interactions(EPIs)in the literature involve the activity of many components,including transcription factors and nucleic acid.However,the role that sequence similarity plays in EPIs remains largely unexplored.Herein,we report that Alu-derived sequences dominate sequence similarity between enhancers and promoters.After rejecting alternative DNA:DNA and DNA:RNA triplex models,we propose that enhancer-associated RNAs(eRNAs)may directly contact their targeted promoters by forming trans-acting R-loops at those Alu sequences.We show how the characteristic distribution of functional genomic data,such as RNA-DNA proximate ligation reads,binding of transcription factors,and RNA-binding proteins,all align with the Alu sequences of EPIs.We also show that these aligned Alu sequences may be subject to the constraint of coevolution,further implying the functional significance of these R-loop hybrids.Finally,our results imply that eRNA and Alu elements associate in a manner previously unrecognized in EPIs and the evolution of gene regulation networks in mammals.展开更多
R-loops play various roles in many physiological processes,however,their role in meiotic division remains largely unknown.Here we show that R-loops and their regulator RNase H1 are present at centromeres during oocyte...R-loops play various roles in many physiological processes,however,their role in meiotic division remains largely unknown.Here we show that R-loops and their regulator RNase H1 are present at centromeres during oocyte meiotic divisions.Proper centromeric R-loops are essential to ensure chromosome alignment in oocytes during metaphase I(MI).Remarkably,both Rnaseh1 knockout and overexpression in oocytes lead to severe spindle assembly defects and chromosome misalignment due to dysregulation of R-loops at centromeres.Furthermore,we find that replication protein A(RPA)is recruited to centromeric R-loops,facilitating the deposition of ataxia telangiectasia-mutated and Rad3-related(ATR)kinase at centromeres by interacting with the ATR-interaction protein(ATRIP).The ATR kinase deposition triggers the activity of CHK1,stimulating the phosphorylation of Aurora B to finally promote proper spindle assembly and chromosome alignment at the equatorial plate.Most importantly,the application of ATR,CHK1,and Aurora B inhibitors could efficiently rescue the defects in spindle assembly and chromosome alignment due to RNase H1 deficiency in oocytes.Overall,our findings uncover a critical role of R-loops during mouse oocyte meiotic divisions,suggesting that dysregulation of R-loops may be associated with female infertility.Additionally,ATR,CHK1,and Aurora B inhibitors may potentially be used to treat some infertile patients.展开更多
R-loops,three-strand nucleic acid structures,have emerged as crucial players in various physiological processes,including the regulation of gene expression,DNA replication,and class switch recombination.However,their ...R-loops,three-strand nucleic acid structures,have emerged as crucial players in various physiological processes,including the regulation of gene expression,DNA replication,and class switch recombination.However,their presence also poses a significant threat to genome stability.A particularly challenging aspect is understanding the dynamic balance between R-loops’“light”and“dark”sites,especially concerning maintaining genome integrity.The complex and multifaceted roles of R-loops in genome stability necessitate a deeper understanding.This review offers a comprehensive exploration of the formation,resolution,and implications of R-loops,particularly in the context of DNA damage and human disease.We delve into the dualistic nature of R-loops,highlighting their role in DNA damage response and repair,and discuss the therapeutic potential arising from our evolving understanding of these enigmatic entities.Emphasizing recent advancements and unresolved questions,this review aims to provide a cohesive overview of R-loops,inviting further inquiry and investigation into their complex biological significance.展开更多
Centromeres play a vital role in cellular division by facilitating kinetochore assembly and spindle attachments.Despite their conserved functionality,centromeric DNA sequences exhibit rapid evolution,presenting divers...Centromeres play a vital role in cellular division by facilitating kinetochore assembly and spindle attachments.Despite their conserved functionality,centromeric DNA sequences exhibit rapid evolution,presenting diverse sizes and compositions across species.The functional significance of rye centromeric DNA sequences,particularly in centromere identity,remains unclear.In this study,we comprehensively characterized the sequence composition and organization of rye centromeres.Our findings revealed that these centromeres are primarily composed of long terminal repeat retrotransposons(LTR-RTs)and interspersed minisatellites.We systematically classified LTR-RTs into five categories,highlighting the prevalence of younger CRS1,CRS2,and CRS3 of CRSs(centromeric retrotransposons of Secale cereale)were primarily located in the core centromeres and exhibited a higher association with CENH3 nucleosomes.The minisatellites,mainly derived from retrotransposons,along with CRSs,played a pivotal role in establishing functional centromeres in rye.Additionally,we observed the formation of R-loops at specific regions of CRS1,CRS2,and CRS3,with both rye pericentromeres and centromeres exhibiting enrichment in R-loops.Notably,these R-loops selectively formed at binding regions of the CENH3 nucleosome in rye centromeres,suggesting a potential role in mediating the precise loading of CENH3 to centromeres and contributing to centromere specification.Our work provides insights into the DNA sequence composition,distribution,and potential function of R-loops in rye centromeres.This knowledge contributes valuable information to understanding the genetics and epigenetics of rye centromeres,offering implications for the development of synthetic centromeres in future plant modifications and beyond.展开更多
Telomeres are specialized structures at the ends of linear chromosomes that protect genome stability.The telomeric repeat-containing RNA(TERRA)that is transcribed from subtelomeric regions can invade into double-stran...Telomeres are specialized structures at the ends of linear chromosomes that protect genome stability.The telomeric repeat-containing RNA(TERRA)that is transcribed from subtelomeric regions can invade into double-stranded DNA regions and form RNA:DNA hybrid-containing structure called R-loop.In tumor cells,R-loop formation is closely linked to gene expression and the alternative lengthening of telomeres(ALT)pathway.Dysregulated R-loops can cause stalled replication forks and telomere instability.However,how R-loops are recognized and regulated,particularly at telomeres,is not well understood.We discovered that ILF3 selectively associates with telomeric R-loops and safeguards telomeres from abnormal homologous recombination.Knocking out ILF3 results in excessive R-loops at telomeres and triggers telomeric DNA damage responses.In addition,ILF3 deficiency disrupts telomere homeostasis and causes abnormalities in the ALT pathway.Using the proximity-dependent biotin identification(BioID)technology,we mapped the ILF3 interactome and discovered that ILF3 could interact with several DNA/RNA helicases,including DHX9.Importantly,ILF3 may aid in the resolution of telomeric R-loops through its interaction with DHX9.Our findings suggest that ILF3 may function as a reader of telomeric R-loops,helping to prevent abnormal homologous recombination and maintain telomere homeostasis.展开更多
R-loop structures （RNA：DNA hybrids） have important functions in many biological processes, including transcriptional regulation and genome instability among diverse organisms. DNA topoisomerase 1 （TOP1）, an essen...R-loop structures （RNA：DNA hybrids） have important functions in many biological processes, including transcriptional regulation and genome instability among diverse organisms. DNA topoisomerase 1 （TOP1）, an essential manipulator of DNA topology during RNA transcription and DNA replication processes, can prevent R-loop accumulation by removing the positive and negative DNA supercoiling that is made by RNA polymerases during transcription. TOP1 is required for plant development, but little is known about its function in preventing co-transcriptional R-loop accumulation in various biological processes in plants. Here we show that knockdown of OsTOP1 strongly affects rice development, causing defects in root archi- tecture and gravitropism, which are the consequences of misregulation of auxin signaling and transporter genes. We found that R-loops are naturally formed at rice auxin-related gene loci, and overaccumulate when OsTOP1 is knocked down or OsTOP1 protein activity is inhibited. OsTOP1 therefore sets the accurate expression levels of auxin-related genes by preventing the overaccumulation of inherent R-loops. Our data reveal R-loops as important factors in polar auxin transport and plant root development, and highlight that OsTOP1 functions as a key to link transcriptional R-loops with plant hormone signaling, provide new in- sights into transcriptional regulation of hormone signaling in plants.展开更多
An R-loop is a three-stranded chromatin structure that consists of a displaced single strand of DNA and an RNA:DNA hybrid duplex,which was thought to be a rare by-product of transcription.However,recent genome-wide da...An R-loop is a three-stranded chromatin structure that consists of a displaced single strand of DNA and an RNA:DNA hybrid duplex,which was thought to be a rare by-product of transcription.However,recent genome-wide data have shown that R-loops are widespread and pervasive in a variety of genomes,and a growing body of experimental evidence indicates that R-loops have both beneficial and harmful effects on an organism.To maximize benefit and avoid harm,organisms have evolved several means by which they tightly regulate R-loop levels.Here,we summarize our current understanding of the biogenesis and effects of R-loops,the mechanisms that regulate them,and methods of R-loop profiling,reviewing recent research advances on R-loops in plants.Furthermore,we provide perspectives on future research directions for R-loop biology in plants,which might lead to a more comprehensive understanding of R-loop functions in plant genome regulation and contribute to future agricultural improvements.展开更多
基金the National Natural Science Foundation of China of China(91940304,31871331,31671342)Beijing Natural Science Foundation(Z200021)+2 种基金Special Investigation on Science and Technology Basic Resources of MOST,China(2019FY100102)the National Key R&D Program of China(2018YFC2000400)the Beijing Advanced Discipline Fund(115200S001)。
文摘Enhancers modulate gene expression by interacting with promoters.Models of enhancer-promoter interactions(EPIs)in the literature involve the activity of many components,including transcription factors and nucleic acid.However,the role that sequence similarity plays in EPIs remains largely unexplored.Herein,we report that Alu-derived sequences dominate sequence similarity between enhancers and promoters.After rejecting alternative DNA:DNA and DNA:RNA triplex models,we propose that enhancer-associated RNAs(eRNAs)may directly contact their targeted promoters by forming trans-acting R-loops at those Alu sequences.We show how the characteristic distribution of functional genomic data,such as RNA-DNA proximate ligation reads,binding of transcription factors,and RNA-binding proteins,all align with the Alu sequences of EPIs.We also show that these aligned Alu sequences may be subject to the constraint of coevolution,further implying the functional significance of these R-loop hybrids.Finally,our results imply that eRNA and Alu elements associate in a manner previously unrecognized in EPIs and the evolution of gene regulation networks in mammals.
基金supported by the National Natural Science Foundation of China(32230029,81925015,32270898,32400709,and 32400714)the National Key Research and Development Program of China(2022YFC2702600)+2 种基金the China Postdoctoral Science Foundation(2024M760638 and 2024M760640)the Science and Technology Project of Guangzhou(2023A03J0886)the plan on enhancing scientific research in GMU.
文摘R-loops play various roles in many physiological processes,however,their role in meiotic division remains largely unknown.Here we show that R-loops and their regulator RNase H1 are present at centromeres during oocyte meiotic divisions.Proper centromeric R-loops are essential to ensure chromosome alignment in oocytes during metaphase I(MI).Remarkably,both Rnaseh1 knockout and overexpression in oocytes lead to severe spindle assembly defects and chromosome misalignment due to dysregulation of R-loops at centromeres.Furthermore,we find that replication protein A(RPA)is recruited to centromeric R-loops,facilitating the deposition of ataxia telangiectasia-mutated and Rad3-related(ATR)kinase at centromeres by interacting with the ATR-interaction protein(ATRIP).The ATR kinase deposition triggers the activity of CHK1,stimulating the phosphorylation of Aurora B to finally promote proper spindle assembly and chromosome alignment at the equatorial plate.Most importantly,the application of ATR,CHK1,and Aurora B inhibitors could efficiently rescue the defects in spindle assembly and chromosome alignment due to RNase H1 deficiency in oocytes.Overall,our findings uncover a critical role of R-loops during mouse oocyte meiotic divisions,suggesting that dysregulation of R-loops may be associated with female infertility.Additionally,ATR,CHK1,and Aurora B inhibitors may potentially be used to treat some infertile patients.
基金supported by research funding from the National Natural Science Foundation of China(No.32201061)the Natural Science Foundation of Shandong,China(No.ZR2021QC083).
文摘R-loops,three-strand nucleic acid structures,have emerged as crucial players in various physiological processes,including the regulation of gene expression,DNA replication,and class switch recombination.However,their presence also poses a significant threat to genome stability.A particularly challenging aspect is understanding the dynamic balance between R-loops’“light”and“dark”sites,especially concerning maintaining genome integrity.The complex and multifaceted roles of R-loops in genome stability necessitate a deeper understanding.This review offers a comprehensive exploration of the formation,resolution,and implications of R-loops,particularly in the context of DNA damage and human disease.We delve into the dualistic nature of R-loops,highlighting their role in DNA damage response and repair,and discuss the therapeutic potential arising from our evolving understanding of these enigmatic entities.Emphasizing recent advancements and unresolved questions,this review aims to provide a cohesive overview of R-loops,inviting further inquiry and investigation into their complex biological significance.
基金supported by the National Natural Science Foundation of China(31991212,31920103006)。
文摘Centromeres play a vital role in cellular division by facilitating kinetochore assembly and spindle attachments.Despite their conserved functionality,centromeric DNA sequences exhibit rapid evolution,presenting diverse sizes and compositions across species.The functional significance of rye centromeric DNA sequences,particularly in centromere identity,remains unclear.In this study,we comprehensively characterized the sequence composition and organization of rye centromeres.Our findings revealed that these centromeres are primarily composed of long terminal repeat retrotransposons(LTR-RTs)and interspersed minisatellites.We systematically classified LTR-RTs into five categories,highlighting the prevalence of younger CRS1,CRS2,and CRS3 of CRSs(centromeric retrotransposons of Secale cereale)were primarily located in the core centromeres and exhibited a higher association with CENH3 nucleosomes.The minisatellites,mainly derived from retrotransposons,along with CRSs,played a pivotal role in establishing functional centromeres in rye.Additionally,we observed the formation of R-loops at specific regions of CRS1,CRS2,and CRS3,with both rye pericentromeres and centromeres exhibiting enrichment in R-loops.Notably,these R-loops selectively formed at binding regions of the CENH3 nucleosome in rye centromeres,suggesting a potential role in mediating the precise loading of CENH3 to centromeres and contributing to centromere specification.Our work provides insights into the DNA sequence composition,distribution,and potential function of R-loops in rye centromeres.This knowledge contributes valuable information to understanding the genetics and epigenetics of rye centromeres,offering implications for the development of synthetic centromeres in future plant modifications and beyond.
基金National Natural Science Foundation(Grant Nos.82271598,81871109,82071587,31930058,32330023 and 32170757)National Key Research and Development Program of China(2018YFA0107003)Guang Dong Basic and Applied Basic Research Foundation(2020A1515010462).
文摘Telomeres are specialized structures at the ends of linear chromosomes that protect genome stability.The telomeric repeat-containing RNA(TERRA)that is transcribed from subtelomeric regions can invade into double-stranded DNA regions and form RNA:DNA hybrid-containing structure called R-loop.In tumor cells,R-loop formation is closely linked to gene expression and the alternative lengthening of telomeres(ALT)pathway.Dysregulated R-loops can cause stalled replication forks and telomere instability.However,how R-loops are recognized and regulated,particularly at telomeres,is not well understood.We discovered that ILF3 selectively associates with telomeric R-loops and safeguards telomeres from abnormal homologous recombination.Knocking out ILF3 results in excessive R-loops at telomeres and triggers telomeric DNA damage responses.In addition,ILF3 deficiency disrupts telomere homeostasis and causes abnormalities in the ALT pathway.Using the proximity-dependent biotin identification(BioID)technology,we mapped the ILF3 interactome and discovered that ILF3 could interact with several DNA/RNA helicases,including DHX9.Importantly,ILF3 may aid in the resolution of telomeric R-loops through its interaction with DHX9.Our findings suggest that ILF3 may function as a reader of telomeric R-loops,helping to prevent abnormal homologous recombination and maintain telomere homeostasis.
基金This work was supported by the National Natural Science Foundation of China （30900070, to C.C.）, Tsinghua University initiative Scientific Research Program, Tsinghua-Peking Joint Center for Life Sciences, and 1000 Young Talent Program of China （to The Sun Lab）. S.S. and E.W. were supported by postdoctoral fellowships from Tsinghua-Peking Joint Center for Life Sciences.
文摘R-loop structures （RNA：DNA hybrids） have important functions in many biological processes, including transcriptional regulation and genome instability among diverse organisms. DNA topoisomerase 1 （TOP1）, an essential manipulator of DNA topology during RNA transcription and DNA replication processes, can prevent R-loop accumulation by removing the positive and negative DNA supercoiling that is made by RNA polymerases during transcription. TOP1 is required for plant development, but little is known about its function in preventing co-transcriptional R-loop accumulation in various biological processes in plants. Here we show that knockdown of OsTOP1 strongly affects rice development, causing defects in root archi- tecture and gravitropism, which are the consequences of misregulation of auxin signaling and transporter genes. We found that R-loops are naturally formed at rice auxin-related gene loci, and overaccumulate when OsTOP1 is knocked down or OsTOP1 protein activity is inhibited. OsTOP1 therefore sets the accurate expression levels of auxin-related genes by preventing the overaccumulation of inherent R-loops. Our data reveal R-loops as important factors in polar auxin transport and plant root development, and highlight that OsTOP1 functions as a key to link transcriptional R-loops with plant hormone signaling, provide new in- sights into transcriptional regulation of hormone signaling in plants.
基金the National Natural Science Foundation of China(Grant Nos.91740105 and 31822028 to Q.Sun32100428 to J.Zhou+2 种基金and 32070651 to W.Zhang)supported by the Tsinghua-Peking Center for Life Sciencessupported by postdoc fellowships from the Tsinghua-Peking Center for Life Sciences。
文摘An R-loop is a three-stranded chromatin structure that consists of a displaced single strand of DNA and an RNA:DNA hybrid duplex,which was thought to be a rare by-product of transcription.However,recent genome-wide data have shown that R-loops are widespread and pervasive in a variety of genomes,and a growing body of experimental evidence indicates that R-loops have both beneficial and harmful effects on an organism.To maximize benefit and avoid harm,organisms have evolved several means by which they tightly regulate R-loop levels.Here,we summarize our current understanding of the biogenesis and effects of R-loops,the mechanisms that regulate them,and methods of R-loop profiling,reviewing recent research advances on R-loops in plants.Furthermore,we provide perspectives on future research directions for R-loop biology in plants,which might lead to a more comprehensive understanding of R-loop functions in plant genome regulation and contribute to future agricultural improvements.
