Meiotic recombination ensures accurate chromosome segregation and genetic diversity during gametogenesis, and its disruption leads to infertility. The dual histone methylation writer-reader system, in which PRDM9 deposits H3K4me3 and H3K36me3 marks at nucleosomes to define recombination hotspots and ZCWPW1 acts as a reader recognizing these marks, is essential for meiotic recombination. However, the regulatory mechanisms of this system remain unclear. Here, we showed that deficiency of ZCWPW2 causes recombination defects in humans and mice, including impaired homologous chromosome synapsis and defective DNA double-strand break repair. CUT&Tag analysis revealed that ZCWPW2 exhibits increased enrichment at dual H3K4me3 and H3K36me3 sites in the presence of PRDM9, while binding to promoter regions independently of PRDM9 to regulate meiotic transcription. Mass spectrometry further showed that ZCWPW2 forms a complex with ZCWPW1 and interacts with recombination-associated proteins in a ZCWPW1-dependent manner. Mechanistically, we demonstrate that the ZCWPW1-ZCWPW2 complex enhances the functions of key lactylation regulators LDHA and EP300, thereby promoting lactylation of recombination-associated proteins and stabilizing their abundance. Collectively, we identify ZCWPW2 as a previously unrecognized but essential factor in meiotic recombination, elucidate the molecular mechanism of the PRDM9/ZCWPW1/ZCWPW2 system in regulating recombination, and uncover a critical role for lactylation in meiosis.
{"title":"A novel dual histone mark reader ZCWPW2 regulates meiotic recombination through lactylation and transcriptional regulation in humans and mice.","authors":"Tiechao Ruan,Jun Ma,Gan Shen,Xiang Wang,Yihong Yang,Liangchai Zhuo,Chuan Jiang,Guicheng Zhao,Yunchuan Tian,Shikun Zhao,Ruixi Zhou,Mohan Liu,Xinyao Tang,Yingteng Zhang,Chanjuan Zhao,Jincheng Zhang,Dingming Li,Xiaohui Jiang,Dezhi Mu,Lingbo Wang,Ying Shen","doi":"10.1093/nar/gkag049","DOIUrl":"https://doi.org/10.1093/nar/gkag049","url":null,"abstract":"Meiotic recombination ensures accurate chromosome segregation and genetic diversity during gametogenesis, and its disruption leads to infertility. The dual histone methylation writer-reader system, in which PRDM9 deposits H3K4me3 and H3K36me3 marks at nucleosomes to define recombination hotspots and ZCWPW1 acts as a reader recognizing these marks, is essential for meiotic recombination. However, the regulatory mechanisms of this system remain unclear. Here, we showed that deficiency of ZCWPW2 causes recombination defects in humans and mice, including impaired homologous chromosome synapsis and defective DNA double-strand break repair. CUT&Tag analysis revealed that ZCWPW2 exhibits increased enrichment at dual H3K4me3 and H3K36me3 sites in the presence of PRDM9, while binding to promoter regions independently of PRDM9 to regulate meiotic transcription. Mass spectrometry further showed that ZCWPW2 forms a complex with ZCWPW1 and interacts with recombination-associated proteins in a ZCWPW1-dependent manner. Mechanistically, we demonstrate that the ZCWPW1-ZCWPW2 complex enhances the functions of key lactylation regulators LDHA and EP300, thereby promoting lactylation of recombination-associated proteins and stabilizing their abundance. Collectively, we identify ZCWPW2 as a previously unrecognized but essential factor in meiotic recombination, elucidate the molecular mechanism of the PRDM9/ZCWPW1/ZCWPW2 system in regulating recombination, and uncover a critical role for lactylation in meiosis.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"30 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146021584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Precise control of gene expression is essential for establishing and maintaining cell identities in response to endogenous signals and environmental cues in multicellular organisms. An example is the regulation of the class B and C floral organ identity genes-APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG)-which are repressed in leaves and activated in floral meristems. Their repression in leaves is associated with CURLY LEAF (CLF), a core component of the polycomb repressive complex 2 (PRC2). However, since CLF is expressed in both leaf and floral primordia, its target specificity is likely mediated by additional factors. In this study, we found that ZINC FINGER PROTEIN 1 (ZP1) and ZFP8 physically interact with PRC2 components CLF, SWINGER (SWN), and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) to repress AP3, PI, and AG. In plants ectopically expressing ZP1 and ZFP8, loss of CLF partially restored floral organ identity and reactivated the expression of AP3, PI, and AG, indicating that this repression is PRC2-dependent. Functional studies revealed that the zinc finger domain is necessary and sufficient for ZFP8 activity, whereas both the zinc finger and EAR domains are required for ZP1 function.
{"title":"ZINC FINGER PROTEIN 1 and 8 interact with polycomb repressive complex 2 to repress class B and C floral organ identity genes.","authors":"Tieqiang Hu,Liren Du,Mingli Xu","doi":"10.1093/nar/gkag045","DOIUrl":"https://doi.org/10.1093/nar/gkag045","url":null,"abstract":"Precise control of gene expression is essential for establishing and maintaining cell identities in response to endogenous signals and environmental cues in multicellular organisms. An example is the regulation of the class B and C floral organ identity genes-APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG)-which are repressed in leaves and activated in floral meristems. Their repression in leaves is associated with CURLY LEAF (CLF), a core component of the polycomb repressive complex 2 (PRC2). However, since CLF is expressed in both leaf and floral primordia, its target specificity is likely mediated by additional factors. In this study, we found that ZINC FINGER PROTEIN 1 (ZP1) and ZFP8 physically interact with PRC2 components CLF, SWINGER (SWN), and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) to repress AP3, PI, and AG. In plants ectopically expressing ZP1 and ZFP8, loss of CLF partially restored floral organ identity and reactivated the expression of AP3, PI, and AG, indicating that this repression is PRC2-dependent. Functional studies revealed that the zinc finger domain is necessary and sufficient for ZFP8 activity, whereas both the zinc finger and EAR domains are required for ZP1 function.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"6 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146021643","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Nicolas G Bologna,Alexis Sarazin,Gregory Schott,Antoine Bouet,Natalia P Achkar,Belen Moro,Florence Jay,Uciel Chorostecki,Emanuel A Devers,Olivier Voinnet
In plants, DICER-LIKE1 (DCL1) orchestrates microRNA (miRNA) biogenesis by cleaving imperfect stem-loop precursors within primary transcripts (pri-miRNAs). However, the full spectrum of DCL1 RNA substrates remains unexplored. Here, we report transcriptome-wide RNA immunoprecipitation and deep-sequencing (RIP-Seq) analyses of the Arabidopsis catalytically inactive DCL1 (DCL1ci), designed to bind but not cleave its targets. In inflorescences, DCL1ci-RIP retrieved nearly all evolutionarily conserved MIRNA loci and uncovered many hitherto unknown young MIRNA loci. Extensive interactions with both pre-miRNA stem-loops and flanking single-stranded regions were detected, suggesting that DCL1 scans pri-miRNAs prior to stem-loop cleavage. Quantitative binding profiles resolved the specific contribution of paralogous MIRNA family members in inflorescences, enabling tissue-level discrimination of pri-miRNA engagement. The analysis also identified hundreds of DCL1ci-interacting non-MIRNA loci, including protein-coding genes, transposons, and intergenic regions, with many lacking canonical stem-loop structures. We show that DCL1 promotes 24-nt small RNA biogenesis mostly from helitron-derived transcripts via a pathway genetically distinct from RNA-directed DNA methylation. Moreover, we identify a conserved stem-loop in the DCL1 5'-UTR suggesting autoregulatory feedback control. Collectively, our study establishes DCLci-RIP as a robust noninvasive approach for profiling DCL substrates, broadens DCL1's functional landscape, and provides a foundation for dissecting dynamic DCL-RNA interactions across developmental and stress contexts.
{"title":"Transcriptome-wide analysis of Arabidopsis DICER-LIKE1 RNA substrates.","authors":"Nicolas G Bologna,Alexis Sarazin,Gregory Schott,Antoine Bouet,Natalia P Achkar,Belen Moro,Florence Jay,Uciel Chorostecki,Emanuel A Devers,Olivier Voinnet","doi":"10.1093/nar/gkaf1434","DOIUrl":"https://doi.org/10.1093/nar/gkaf1434","url":null,"abstract":"In plants, DICER-LIKE1 (DCL1) orchestrates microRNA (miRNA) biogenesis by cleaving imperfect stem-loop precursors within primary transcripts (pri-miRNAs). However, the full spectrum of DCL1 RNA substrates remains unexplored. Here, we report transcriptome-wide RNA immunoprecipitation and deep-sequencing (RIP-Seq) analyses of the Arabidopsis catalytically inactive DCL1 (DCL1ci), designed to bind but not cleave its targets. In inflorescences, DCL1ci-RIP retrieved nearly all evolutionarily conserved MIRNA loci and uncovered many hitherto unknown young MIRNA loci. Extensive interactions with both pre-miRNA stem-loops and flanking single-stranded regions were detected, suggesting that DCL1 scans pri-miRNAs prior to stem-loop cleavage. Quantitative binding profiles resolved the specific contribution of paralogous MIRNA family members in inflorescences, enabling tissue-level discrimination of pri-miRNA engagement. The analysis also identified hundreds of DCL1ci-interacting non-MIRNA loci, including protein-coding genes, transposons, and intergenic regions, with many lacking canonical stem-loop structures. We show that DCL1 promotes 24-nt small RNA biogenesis mostly from helitron-derived transcripts via a pathway genetically distinct from RNA-directed DNA methylation. Moreover, we identify a conserved stem-loop in the DCL1 5'-UTR suggesting autoregulatory feedback control. Collectively, our study establishes DCLci-RIP as a robust noninvasive approach for profiling DCL substrates, broadens DCL1's functional landscape, and provides a foundation for dissecting dynamic DCL-RNA interactions across developmental and stress contexts.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"64 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146015323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yujie Ma,Tianyi Zhang,Xiao Albert Zhou,Zhanzhan Xu,Jiadong Zhou,Abudureyimujiang Aili,Pei Li,Chen Nie,Yundong Xiong,Xiaoman Li,Baoshan Cao,Shiwei Li,Jiadong Wang
The spatial distribution and dynamics of double-strand break (DSBs) repair controlled by microtubules are essential for preserving genomic stability. However, the processes through which extranuclear microtubules govern intranuclear DSB repair across the nuclear envelope (NE) remain poorly understood. This study uncovers a mechanism by which the microtubule-depolymerizing kinesin KIF2A regulates nonhomologous end joining (NHEJ) repair by mediating NE invagination. Our investigation reveals that damage-induced α-tubulin tyrosination triggers KIF2A binding to microtubules, subsequently inducing NE invagination through the microtubule-the linker of nucleoskeleton and cytoskeleton (LINC) complex and lamin B1. This invagination, in turn, provides a larger region of a stable NHEJ repair environment close to the NE, facilitating efficient NHEJ repair. Loss of KIF2A disrupts the formation of invaginations after DNA damage, impacting the formation of 53BP1 foci. Our study establishes KIF2A-mediated NE invagination as a critical regulator of the intricate relationships among microtubules, NE dynamics, and NHEJ repair, shedding light on a previously obscure pathway crucial for genome stability.
{"title":"KIF2A-mediated microtubule-dependent nuclear envelope invagination drives nonhomologous end joining.","authors":"Yujie Ma,Tianyi Zhang,Xiao Albert Zhou,Zhanzhan Xu,Jiadong Zhou,Abudureyimujiang Aili,Pei Li,Chen Nie,Yundong Xiong,Xiaoman Li,Baoshan Cao,Shiwei Li,Jiadong Wang","doi":"10.1093/nar/gkag004","DOIUrl":"https://doi.org/10.1093/nar/gkag004","url":null,"abstract":"The spatial distribution and dynamics of double-strand break (DSBs) repair controlled by microtubules are essential for preserving genomic stability. However, the processes through which extranuclear microtubules govern intranuclear DSB repair across the nuclear envelope (NE) remain poorly understood. This study uncovers a mechanism by which the microtubule-depolymerizing kinesin KIF2A regulates nonhomologous end joining (NHEJ) repair by mediating NE invagination. Our investigation reveals that damage-induced α-tubulin tyrosination triggers KIF2A binding to microtubules, subsequently inducing NE invagination through the microtubule-the linker of nucleoskeleton and cytoskeleton (LINC) complex and lamin B1. This invagination, in turn, provides a larger region of a stable NHEJ repair environment close to the NE, facilitating efficient NHEJ repair. Loss of KIF2A disrupts the formation of invaginations after DNA damage, impacting the formation of 53BP1 foci. Our study establishes KIF2A-mediated NE invagination as a critical regulator of the intricate relationships among microtubules, NE dynamics, and NHEJ repair, shedding light on a previously obscure pathway crucial for genome stability.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"2 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146056754","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Marie B A Peters,Richard Lindqvist,Priyanka Madhu,Richard Lundmark,Ylva Ivarsson,Anna K Överby
The nuclear pore complex (NPC) is composed of multiple nucleoporins (NUPs) and enables the exchange of RNA and proteins between the nucleus and cytoplasm. NUP98 is one of the major components of the NPC, being involved in the RNA export pathway by interacting with several transport factors. Previous studies have suggested both proviral and antiviral functions of NUP98 in viral infection, yet little is known about its function in orthoflavivirus infection. In this study we show that NUP98 is a proviral cellular protein that is recruited to the cytoplasm during orthoflavivirus infection. We observe that NUP98 is found specifically in the vicinity of the replication vesicles during infections with tick-borne encephalitis virus, Japanese encephalitis virus, and yellow fever virus. Furthermore, using surface plasmon resonance, cross-link immunoprecipitation, and cross-link immunoprecipitation-sequencing we observe that the C-terminal domain of NUP98 directly interacts with a conserved site of the viral RNA (vRNA) in the E coding region promoting viral replication. We identified a peptide that binds to NUP98 that is antivirally active against several orthoflaviviruses by outcompeting the binding between NUP98 and vRNA, making NUP98 an attractive target for antiviral development.
{"title":"NUP98 regulates orthoflavivirus replication through interaction with vRNA and can be targeted for antiviral purposes.","authors":"Marie B A Peters,Richard Lindqvist,Priyanka Madhu,Richard Lundmark,Ylva Ivarsson,Anna K Överby","doi":"10.1093/nar/gkag027","DOIUrl":"https://doi.org/10.1093/nar/gkag027","url":null,"abstract":"The nuclear pore complex (NPC) is composed of multiple nucleoporins (NUPs) and enables the exchange of RNA and proteins between the nucleus and cytoplasm. NUP98 is one of the major components of the NPC, being involved in the RNA export pathway by interacting with several transport factors. Previous studies have suggested both proviral and antiviral functions of NUP98 in viral infection, yet little is known about its function in orthoflavivirus infection. In this study we show that NUP98 is a proviral cellular protein that is recruited to the cytoplasm during orthoflavivirus infection. We observe that NUP98 is found specifically in the vicinity of the replication vesicles during infections with tick-borne encephalitis virus, Japanese encephalitis virus, and yellow fever virus. Furthermore, using surface plasmon resonance, cross-link immunoprecipitation, and cross-link immunoprecipitation-sequencing we observe that the C-terminal domain of NUP98 directly interacts with a conserved site of the viral RNA (vRNA) in the E coding region promoting viral replication. We identified a peptide that binds to NUP98 that is antivirally active against several orthoflaviviruses by outcompeting the binding between NUP98 and vRNA, making NUP98 an attractive target for antiviral development.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"74 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146056759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anders Fuglsang,Sweta Suman Rout,Eliska Bartl Koutna,Nicholas Sofos,Alejandro Redondo Gallego,Guillermo Montoya
The type I-FHNH CRISPR-Cas system is a non-canonical Class 1 effector complex distinguished by the replacement of the Cas3 recruitment domain with a catalytic HNH domain in Cas8, enabling autonomous DNA cleavage without accessory nucleases. Using cryo-EM, we determined high-resolution structures of the effector complex in three catalytic states-precatalytic, NTS-cleaved, and post-catalytic-revealing a dynamic trajectory of the HNH domain through inward, middle, and outward conformations. Biochemical assays demonstrated that the complex cleaves the nontarget strand (NTS) prior to the target strand (TS), consistent with a sequential cleavage mechanism similar to Cas12 effectors but notably lacking trans-cleavage activity on single-stranded DNA. Structural comparisons confirmed a minimal PAM requirement (5'-CN) and a constrained HNH catalytic site poised for precise strand scission. We engineered a ΔLinker variant of Cas8 that repositions the HNH domain, selectively abolishing TS cleavage and converting the system into a programmable NTS-specific nickase. Importantly, we validated the functionality of both wild-type and mutant complexes in human cells. While the wild-type system induced indels and base substitutions, the ΔLinker variant triggered targeted single-strand nicks without double-stranded breaks. Together, our work establishes type I-FHNH as a compact and precise genome editing platform with in vivo efficacy.
{"title":"Conformational dynamics of CRISPR-Cas type I-F-HNH inform nickase engineering in a cascade scaffold.","authors":"Anders Fuglsang,Sweta Suman Rout,Eliska Bartl Koutna,Nicholas Sofos,Alejandro Redondo Gallego,Guillermo Montoya","doi":"10.1093/nar/gkag053","DOIUrl":"https://doi.org/10.1093/nar/gkag053","url":null,"abstract":"The type I-FHNH CRISPR-Cas system is a non-canonical Class 1 effector complex distinguished by the replacement of the Cas3 recruitment domain with a catalytic HNH domain in Cas8, enabling autonomous DNA cleavage without accessory nucleases. Using cryo-EM, we determined high-resolution structures of the effector complex in three catalytic states-precatalytic, NTS-cleaved, and post-catalytic-revealing a dynamic trajectory of the HNH domain through inward, middle, and outward conformations. Biochemical assays demonstrated that the complex cleaves the nontarget strand (NTS) prior to the target strand (TS), consistent with a sequential cleavage mechanism similar to Cas12 effectors but notably lacking trans-cleavage activity on single-stranded DNA. Structural comparisons confirmed a minimal PAM requirement (5'-CN) and a constrained HNH catalytic site poised for precise strand scission. We engineered a ΔLinker variant of Cas8 that repositions the HNH domain, selectively abolishing TS cleavage and converting the system into a programmable NTS-specific nickase. Importantly, we validated the functionality of both wild-type and mutant complexes in human cells. While the wild-type system induced indels and base substitutions, the ΔLinker variant triggered targeted single-strand nicks without double-stranded breaks. Together, our work establishes type I-FHNH as a compact and precise genome editing platform with in vivo efficacy.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"102 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146056901","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Saccharomyces cerevisiae Fzf1 is a transcriptional regulator with five Cys2His2 zinc fingers, controlling the expression of SSU1, YHB1, DDI2/3, and YNR064C genes through a shared promoter sequence CS2. After exposure to chemicals such as cyanamide (CY) or methyl methanesulfonate (MMS), Fzf1-regulated gene expression increases in yeast cells without concomitant changes in Fzf1 levels, suggesting that chemical modification of Fzf1 leads to increased transcription of target genes. Here, we showed that Fzf1 binds to the four known CS2 promoter sequences with comparable nanomolar affinity, while treatment of Fzf1 with inducing chemicals CY or MMS modestly increased its binding affinity for CS2 sequences. Crystallographic analysis of the N-terminal three zinc fingers of Fzf1 bound to a 26-bp YHB1 CS2 DNA containing the consensus sequence 3'-C3G4T5C6T7G8A9T10A11G12T13-5' reveals non-canonical recognition of duplex DNA for a zinc finger transcription factor. The first zinc finger interacts with the 5'-end dG3' of the standard non-recognition strand, while the second and third fingers read two (3'-T7G8-5') and four (3'-T10A11G12T13-5') adjacent base pairs on the recognition strand, respectively. Recognition of the DNA phosphodiester backbone by Fzf1 mostly resembles other zinc finger proteins. Future work will aim to elucidate how chemical modification of Fzf1 increases transcriptional activation in vivo.
{"title":"Zinc-finger transcription factor Fzf1 binds to its target DNA in a non-canonical manner.","authors":"Chaoqun Ma,Ying Du,Wei Xiao,Stanley A Moore","doi":"10.1093/nar/gkag054","DOIUrl":"https://doi.org/10.1093/nar/gkag054","url":null,"abstract":"Saccharomyces cerevisiae Fzf1 is a transcriptional regulator with five Cys2His2 zinc fingers, controlling the expression of SSU1, YHB1, DDI2/3, and YNR064C genes through a shared promoter sequence CS2. After exposure to chemicals such as cyanamide (CY) or methyl methanesulfonate (MMS), Fzf1-regulated gene expression increases in yeast cells without concomitant changes in Fzf1 levels, suggesting that chemical modification of Fzf1 leads to increased transcription of target genes. Here, we showed that Fzf1 binds to the four known CS2 promoter sequences with comparable nanomolar affinity, while treatment of Fzf1 with inducing chemicals CY or MMS modestly increased its binding affinity for CS2 sequences. Crystallographic analysis of the N-terminal three zinc fingers of Fzf1 bound to a 26-bp YHB1 CS2 DNA containing the consensus sequence 3'-C3G4T5C6T7G8A9T10A11G12T13-5' reveals non-canonical recognition of duplex DNA for a zinc finger transcription factor. The first zinc finger interacts with the 5'-end dG3' of the standard non-recognition strand, while the second and third fingers read two (3'-T7G8-5') and four (3'-T10A11G12T13-5') adjacent base pairs on the recognition strand, respectively. Recognition of the DNA phosphodiester backbone by Fzf1 mostly resembles other zinc finger proteins. Future work will aim to elucidate how chemical modification of Fzf1 increases transcriptional activation in vivo.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"53 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146069955","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Megan Biller, Sara Kabir, Sarah Nipper, Sydney Allen, Yara Kayali, Skyler Kuncik, Hiroyuki Sasanuma, Pei Zhou, Cyrus Vaziri, Junya Tomida
Ataxia-telangiectasia mutated and RAD3-related (ATR) and its partner ATR-interacting protein (ATRIP) function as a critical proximal sensor and transducer of the DNA damage response (DDR). Several ATR substrates, including p53 and CHK1, are crucial for the coordination of cell cycle phase transitions, transcription, and DNA repair when cells sustain DNA damage. While much is known about ATR activation mechanisms, it is less clear how ATR signaling is negatively regulated in cells. Here, we identify the DNA repair protein REV7 as a novel direct binding partner of ATRIP. We define a REV7-interaction motif in ATRIP, which, when mutated, abrogates the REV7–ATRIP interaction in vitro and in intact cells. Using in vitro kinase assays, we show that REV7 inhibits ATR-mediated phosphorylation of its substrates, including p53. Disruption of the REV7–ATRIP interaction also enhances phosphorylation of CHK1 at Ser317 in intact cells. Taken together, our results establish REV7 as a critical negative regulator of ATR signaling. REV7 has pleiotropic roles in multiple DDR pathways, including Translesion Synthesis, DNA double-strand break resection, and p53 stability and may play a central role in the integration of multiple genome maintenance pathways.
{"title":"REV7 associates with ATRIP and inhibits ATR kinase activity","authors":"Megan Biller, Sara Kabir, Sarah Nipper, Sydney Allen, Yara Kayali, Skyler Kuncik, Hiroyuki Sasanuma, Pei Zhou, Cyrus Vaziri, Junya Tomida","doi":"10.1093/nar/gkaf1527","DOIUrl":"https://doi.org/10.1093/nar/gkaf1527","url":null,"abstract":"Ataxia-telangiectasia mutated and RAD3-related (ATR) and its partner ATR-interacting protein (ATRIP) function as a critical proximal sensor and transducer of the DNA damage response (DDR). Several ATR substrates, including p53 and CHK1, are crucial for the coordination of cell cycle phase transitions, transcription, and DNA repair when cells sustain DNA damage. While much is known about ATR activation mechanisms, it is less clear how ATR signaling is negatively regulated in cells. Here, we identify the DNA repair protein REV7 as a novel direct binding partner of ATRIP. We define a REV7-interaction motif in ATRIP, which, when mutated, abrogates the REV7–ATRIP interaction in vitro and in intact cells. Using in vitro kinase assays, we show that REV7 inhibits ATR-mediated phosphorylation of its substrates, including p53. Disruption of the REV7–ATRIP interaction also enhances phosphorylation of CHK1 at Ser317 in intact cells. Taken together, our results establish REV7 as a critical negative regulator of ATR signaling. REV7 has pleiotropic roles in multiple DDR pathways, including Translesion Synthesis, DNA double-strand break resection, and p53 stability and may play a central role in the integration of multiple genome maintenance pathways.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"31 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146005987","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Joanna Stefano, Lara E Elcavage, Sue-Jean Hong, David P Bartel, Benjamin Kleaveland
The miR-200a and miR-200b families control mouse ovulation and are essential for female fertility. The ZEB1 transcription factor is a conserved target of both families and has been implicated as a key player in female fertility at multiple levels. Using gene-edited mice that express a miR-200a/b-resistant form of Zeb1, we found that derepression of Zeb1 in the female pituitary caused decreased production of luteinizing hormone and anovulatory infertility. These phenotypes were accompanied by widespread changes in pituitary gene expression characterized by decreased levels of ZEB1 targets, which include the miR-200a/b microRNAs (miRNAs), as expected from the miR-200a/b-ZEB1 double-negative feedback loop. Also observed were increased levels of mesenchymal genes, neuronal genes, and miR-200a/b targets. These results show that a double-negative feedback loop centered on the miRNA regulation of a single transcription factor can significantly influence the expression of thousands of genes and have dramatic phenotypic consequences.
{"title":"Derepression of a single microRNA target causes female infertility in mice.","authors":"Joanna Stefano, Lara E Elcavage, Sue-Jean Hong, David P Bartel, Benjamin Kleaveland","doi":"10.1093/nar/gkaf1357","DOIUrl":"10.1093/nar/gkaf1357","url":null,"abstract":"<p><p>The miR-200a and miR-200b families control mouse ovulation and are essential for female fertility. The ZEB1 transcription factor is a conserved target of both families and has been implicated as a key player in female fertility at multiple levels. Using gene-edited mice that express a miR-200a/b-resistant form of Zeb1, we found that derepression of Zeb1 in the female pituitary caused decreased production of luteinizing hormone and anovulatory infertility. These phenotypes were accompanied by widespread changes in pituitary gene expression characterized by decreased levels of ZEB1 targets, which include the miR-200a/b microRNAs (miRNAs), as expected from the miR-200a/b-ZEB1 double-negative feedback loop. Also observed were increased levels of mesenchymal genes, neuronal genes, and miR-200a/b targets. These results show that a double-negative feedback loop centered on the miRNA regulation of a single transcription factor can significantly influence the expression of thousands of genes and have dramatic phenotypic consequences.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"54 2","pages":""},"PeriodicalIF":13.1,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12802903/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145984922","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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