Pub Date : 2025-03-06DOI: 10.1016/j.molcel.2025.02.004
Alexander J. Garvin, Alexander J. Lanz, George E. Ronson, Matthew J.W. Mackintosh, Katarzyna Starowicz, Alexandra K. Walker, Yara Aghabi, Hannah MacKay, Ruth M. Densham, Jai S. Bhachoo, Aneika C. Leney, Joanna R. Morris
The amplitudes of small-modifier protein signaling through ubiquitin and the small ubiquitin-like modifiers, SUMO1–3, are critical to the correct phasing of DNA repair protein accumulation, activity, and clearance and for the completion of mammalian DNA double-strand-break (DSB) repair. However, how SUMO-conjugate signaling in the response is delineated is poorly understood. At the same time, the role of the non-conjugated SUMO protein, SUMO4, has remained enigmatic. Here, we reveal that human SUMO4 is required to prevent excessive DNA-damage-induced SUMOylation and deleterious over-accumulation of RAP80. Mechanistically we show that SUMO4 acts independently of its conjugation and potentiates SENP1 catalytic activity. These data identify SUMO4 as a SUMO deconjugation component and show that SUMO4:SENP1 are critical regulators of DNA-damage-induced SUMO signaling.
{"title":"SUMO4 promotes SUMO deconjugation required for DNA double-strand-break repair","authors":"Alexander J. Garvin, Alexander J. Lanz, George E. Ronson, Matthew J.W. Mackintosh, Katarzyna Starowicz, Alexandra K. Walker, Yara Aghabi, Hannah MacKay, Ruth M. Densham, Jai S. Bhachoo, Aneika C. Leney, Joanna R. Morris","doi":"10.1016/j.molcel.2025.02.004","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.02.004","url":null,"abstract":"The amplitudes of small-modifier protein signaling through ubiquitin and the small ubiquitin-like modifiers, SUMO1–3, are critical to the correct phasing of DNA repair protein accumulation, activity, and clearance and for the completion of mammalian DNA double-strand-break (DSB) repair. However, how SUMO-conjugate signaling in the response is delineated is poorly understood. At the same time, the role of the non-conjugated SUMO protein, SUMO4, has remained enigmatic. Here, we reveal that human SUMO4 is required to prevent excessive DNA-damage-induced SUMOylation and deleterious over-accumulation of RAP80. Mechanistically we show that SUMO4 acts independently of its conjugation and potentiates SENP1 catalytic activity. These data identify SUMO4 as a SUMO deconjugation component and show that SUMO4:SENP1 are critical regulators of DNA-damage-induced SUMO signaling.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"11 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143560750","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
In adaptive immunity, transcription-coupled damage (TCD) is introduced into antibody genes by activation-induced cytidine deaminase (AID) to diversify antibody repertoire. However, the coordination between transcription and DNA damage/repair remains elusive. Here, we find that transcription elongation factor 1 (ELOF1) stabilizes paused RNA polymerase II (RNAPII) at transcription barriers, providing a platform for transcription-coupled DNA damage/repair. Using a genetic screen, we discover that ELOF1 is required for AID targeting and that ELOF1 deficiency results in defective antibody class switch recombination and somatic hypermutation in mice. While downstream transcription-coupled repair factors are dispensable for AID damage, ELOF1 mechanistically facilitates both TCD and repair by stabilizing chromatin-bound RNAPII. In ELOF1-deficient cells, paused RNAPII tends to detach from chromatin and fails to recruit factors to induce or repair DNA damage. Our study places ELOF1 at the center of transcription-coupled DNA metabolism processes and suggests a transition of RNAPII from elongation to a DNA damage/repair scaffold.
{"title":"Transcription-coupled AID deamination damage depends on ELOF1-associated RNA polymerase II","authors":"Pengfei Dai, Yuanqing Tan, Yifeng Luo, Tingting Liu, Yanchao Huang, Yafang Shang, Min Emma Huang, Xiaojing Liu, Senxin Zhang, Yanyan Wang, Qian-Xi Li, Niu Li, Lulu Li, Yining Qin, Junqi Liu, Liu Daisy Liu, Xia Xie, Yanni Cai, Fei Xavier Chen, Xiaoqi Zheng, Fei-Long Meng","doi":"10.1016/j.molcel.2025.02.006","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.02.006","url":null,"abstract":"In adaptive immunity, transcription-coupled damage (TCD) is introduced into antibody genes by activation-induced cytidine deaminase (AID) to diversify antibody repertoire. However, the coordination between transcription and DNA damage/repair remains elusive. Here, we find that transcription elongation factor 1 (ELOF1) stabilizes paused RNA polymerase II (RNAPII) at transcription barriers, providing a platform for transcription-coupled DNA damage/repair. Using a genetic screen, we discover that ELOF1 is required for AID targeting and that ELOF1 deficiency results in defective antibody class switch recombination and somatic hypermutation in mice. While downstream transcription-coupled repair factors are dispensable for AID damage, ELOF1 mechanistically facilitates both TCD and repair by stabilizing chromatin-bound RNAPII. In ELOF1-deficient cells, paused RNAPII tends to detach from chromatin and fails to recruit factors to induce or repair DNA damage. Our study places ELOF1 at the center of transcription-coupled DNA metabolism processes and suggests a transition of RNAPII from elongation to a DNA damage/repair scaffold.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"42 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143546600","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-05DOI: 10.1016/j.molcel.2025.02.007
Lizhen Wu, Anurupa Devi Yadavalli, Filip Senigl, Gabriel Matos-Rodrigues, Dijin Xu, Andreas P. Pintado-Urbanc, Matthew D. Simon, Wei Wu, André Nussenzweig, David G. Schatz
Somatic hypermutation (SHM) and class switch recombination (CSR) diversify immunoglobulin (Ig) genes and are initiated by the activation-induced deaminase (AID), a single-stranded DNA cytidine deaminase thought to engage its substrate during RNA polymerase II (RNAPII) transcription. Through a genetic screen, we identified numerous potential factors involved in SHM, including elongation factor 1 homolog (ELOF1), a component of the RNAPII elongation complex that functions in transcription-coupled nucleotide excision repair (TC-NER) and transcription elongation. Loss of ELOF1 compromises SHM, CSR, and AID action in mammalian B cells and alters RNAPII transcription by reducing RNAPII pausing downstream of transcription start sites and levels of serine 5 but not serine 2 phosphorylated RNAPII throughout transcribed genes. ELOF1 must bind to RNAPII to be a proximity partner for AID and to function in SHM and CSR, and TC-NER is not required for SHM. We propose that ELOF1 helps create the appropriate stalled RNAPII substrate on which AID acts.
{"title":"Transcription elongation factor ELOF1 is required for efficient somatic hypermutation and class switch recombination","authors":"Lizhen Wu, Anurupa Devi Yadavalli, Filip Senigl, Gabriel Matos-Rodrigues, Dijin Xu, Andreas P. Pintado-Urbanc, Matthew D. Simon, Wei Wu, André Nussenzweig, David G. Schatz","doi":"10.1016/j.molcel.2025.02.007","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.02.007","url":null,"abstract":"Somatic hypermutation (SHM) and class switch recombination (CSR) diversify immunoglobulin (Ig) genes and are initiated by the activation-induced deaminase (AID), a single-stranded DNA cytidine deaminase thought to engage its substrate during RNA polymerase II (RNAPII) transcription. Through a genetic screen, we identified numerous potential factors involved in SHM, including elongation factor 1 homolog (ELOF1), a component of the RNAPII elongation complex that functions in transcription-coupled nucleotide excision repair (TC-NER) and transcription elongation. Loss of ELOF1 compromises SHM, CSR, and AID action in mammalian B cells and alters RNAPII transcription by reducing RNAPII pausing downstream of transcription start sites and levels of serine 5 but not serine 2 phosphorylated RNAPII throughout transcribed genes. ELOF1 must bind to RNAPII to be a proximity partner for AID and to function in SHM and CSR, and TC-NER is not required for SHM. We propose that ELOF1 helps create the appropriate stalled RNAPII substrate on which AID acts.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"35 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143546598","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-05DOI: 10.1016/j.molcel.2025.02.028
Emily R. Feierman, Sean Louzon, Nicholas A. Prescott, Tracy Biaco, Qingzeng Gao, Qi Qiu, Kyuhyun Choi, Katherine C. Palozola, Anna J. Voss, Shreya D. Mehta, Camille N. Quaye, Katherine T. Lynch, Marc V. Fuccillo, Hao Wu, Yael David, Erica Korb
(Molecular Cell 84, 2822–2837.e1–e11; August 8, 2024)
{"title":"Histone variant H2BE enhances chromatin accessibility in neurons to promote synaptic gene expression and long-term memory","authors":"Emily R. Feierman, Sean Louzon, Nicholas A. Prescott, Tracy Biaco, Qingzeng Gao, Qi Qiu, Kyuhyun Choi, Katherine C. Palozola, Anna J. Voss, Shreya D. Mehta, Camille N. Quaye, Katherine T. Lynch, Marc V. Fuccillo, Hao Wu, Yael David, Erica Korb","doi":"10.1016/j.molcel.2025.02.028","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.02.028","url":null,"abstract":"(Molecular Cell <em>84</em>, 2822–2837.e1–e11; August 8, 2024)","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"30 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143546531","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-03DOI: 10.1016/j.molcel.2025.02.002
Thomas Hermanns, Susanne Kolek, Matthias Uthoff, Richard A. de Heiden, Monique P.C. Mulder, Ulrich Baumann, Kay Hofmann
Many intracellular bacteria secrete deubiquitinase (DUB) effectors into eukaryotic host cells to keep the bacterial surface or the enclosing vesicle membrane free of ubiquitin marks. This study describes a family of DUBs from several bacterial genera, including Simkania, Parachlamydia, Burkholderia, and Pigmentiphaga, which is structurally related to eukaryotic Josephin-type DUBs but contains members that catalyze a unique destructive substrate deubiquitination. These ubiquitin C-terminal clippases (UCCs) cleave ubiquitin before the C-terminal diGly motif, thereby truncating the modifier and leaving a remnant on the substrate. By comparing the crystal structures of substrate-bound clippases and a closely related conventional DUB, we identified the factors causing this shift and found them to be conserved in other clippases, including one highly specific for M1-linked ubiquitin chains. This enzyme class has great potential to serve as tools for studying the ubiquitin system, particularly aspects involving branched chains.
{"title":"A family of bacterial Josephin-like deubiquitinases with an irreversible cleavage mode","authors":"Thomas Hermanns, Susanne Kolek, Matthias Uthoff, Richard A. de Heiden, Monique P.C. Mulder, Ulrich Baumann, Kay Hofmann","doi":"10.1016/j.molcel.2025.02.002","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.02.002","url":null,"abstract":"Many intracellular bacteria secrete deubiquitinase (DUB) effectors into eukaryotic host cells to keep the bacterial surface or the enclosing vesicle membrane free of ubiquitin marks. This study describes a family of DUBs from several bacterial genera, including <em>Simkania</em>, <em>Parachlamydia</em>, <em>Burkholderia</em>, and <em>Pigmentiphaga</em>, which is structurally related to eukaryotic Josephin-type DUBs but contains members that catalyze a unique destructive substrate deubiquitination. These ubiquitin C-terminal clippases (UCCs) cleave ubiquitin before the C-terminal diGly motif, thereby truncating the modifier and leaving a remnant on the substrate. By comparing the crystal structures of substrate-bound clippases and a closely related conventional DUB, we identified the factors causing this shift and found them to be conserved in other clippases, including one highly specific for M1-linked ubiquitin chains. This enzyme class has great potential to serve as tools for studying the ubiquitin system, particularly aspects involving branched chains.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"66 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143532452","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-03DOI: 10.1016/j.molcel.2025.02.003
Ning Tsao, Patrick M. Lombardi, Ajin Park, Jennifer Olabode, Rebecca Rodell, Hua Sun, Shilpa Padmanaban, Joshua R. Brickner, Miaw-Sheue Tsai, Elizabeth A. Pollina, Chun-Kan Chen, Nima Mosammaparast
Certain environmental toxins and chemotherapeutics are nucleic acid-damaging agents, causing adducts in DNA and RNA. While most of these adducts occur in RNA, the consequences of RNA damage are largely unexplored. Here, we demonstrate that nuclear RNA damage can result in loss of genome integrity in human cells. Specifically, we show that YTHDC1 regulates alkylation damage responses with the THO complex (THOC). In addition to its established binding to N6-methyladenosine (m6A), YTHDC1 binds to chemically induced N1-methyladenosine (m1A). Without YTHDC1, cells have greater alkylation damage sensitivity and increased DNA breaks, which are rescued by an RNA-specific dealkylase. These RNA-damage-induced DNA breaks (RDIBs) depend on R-loop formation, which is converted to DNA breaks by the XPG nuclease. Strikingly, in the absence of YTHDC1 or THOC, a nuclear RNA m1A methyltransferase is sufficient to induce DNA breaks. Our results provide mechanistic insight into how damaged RNAs can impact genomic integrity.
{"title":"YTHDC1 cooperates with the THO complex to prevent RNA-damage-induced DNA breaks","authors":"Ning Tsao, Patrick M. Lombardi, Ajin Park, Jennifer Olabode, Rebecca Rodell, Hua Sun, Shilpa Padmanaban, Joshua R. Brickner, Miaw-Sheue Tsai, Elizabeth A. Pollina, Chun-Kan Chen, Nima Mosammaparast","doi":"10.1016/j.molcel.2025.02.003","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.02.003","url":null,"abstract":"Certain environmental toxins and chemotherapeutics are nucleic acid-damaging agents, causing adducts in DNA and RNA. While most of these adducts occur in RNA, the consequences of RNA damage are largely unexplored. Here, we demonstrate that nuclear RNA damage can result in loss of genome integrity in human cells. Specifically, we show that YTHDC1 regulates alkylation damage responses with the THO complex (THOC). In addition to its established binding to <em>N</em>6-methyladenosine (m<sup>6</sup>A), YTHDC1 binds to chemically induced <em>N</em>1-methyladenosine (m<sup>1</sup>A). Without YTHDC1, cells have greater alkylation damage sensitivity and increased DNA breaks, which are rescued by an RNA-specific dealkylase. These RNA-damage-induced DNA breaks (RDIBs) depend on R-loop formation, which is converted to DNA breaks by the XPG nuclease. Strikingly, in the absence of YTHDC1 or THOC, a nuclear RNA m<sup>1</sup>A methyltransferase is sufficient to induce DNA breaks. Our results provide mechanistic insight into how damaged RNAs can impact genomic integrity.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"84 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143532351","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-26DOI: 10.1016/j.molcel.2025.01.023
Weiwei Liu, Lijun Deng, Ming Wang, Xiaojun Liu, Xuan Ouyang, Yuan Wang, Na Miao, Xiu Luo, Xueming Wu, Xiaohua Lu, Xiangjin Xv, Tianyu Zhang, Yu Li, Jinyao Ji, Zhenghao Qiao, Sheng Wang, Li Guan, Dong Li, Yunkun Dang, Chao Liu, Yang Yu
The PIWI-interacting RNA (piRNA) pathway plays a crucial role in protecting animal germ cells by repressing transposons. However, the mechanism of piRNA-guided heterochromatin formation and its relationship to transcriptional termination remains elusive. Through RNA interference screening, we discovered Pcf11 and PNUTS as essential for piRNA-guided silencing in Drosophila germ line. Enforced tethering of Pcf11 leads to co-transcriptional repression and RNA polymerase II (RNA Pol II) stalling, and both are dependent on an α-helical region of Pcf11 capable of forming condensates. An intrinsically disordered region can substitute for the α-helical region of Pcf11 in its silencing capacity and support animal development, arguing for a causal relationship between phase separation and Pcf11’s function. Pcf11 stalls RNA Pol II by preferentially forming condensates with the unphosphorylated Spt5, promoted by the PP1/PNUTS phosphatase during termination. We propose that Pcf11/Spt5 condensates control termination by decelerating polymerase elongation, a property exploited by piRNAs to silence transposons and initiate RNA-mediated heterochromatin formation.
{"title":"Pcf11/Spt5 condensates stall RNA polymerase II to facilitate termination and piRNA-guided heterochromatin formation","authors":"Weiwei Liu, Lijun Deng, Ming Wang, Xiaojun Liu, Xuan Ouyang, Yuan Wang, Na Miao, Xiu Luo, Xueming Wu, Xiaohua Lu, Xiangjin Xv, Tianyu Zhang, Yu Li, Jinyao Ji, Zhenghao Qiao, Sheng Wang, Li Guan, Dong Li, Yunkun Dang, Chao Liu, Yang Yu","doi":"10.1016/j.molcel.2025.01.023","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.01.023","url":null,"abstract":"The PIWI-interacting RNA (piRNA) pathway plays a crucial role in protecting animal germ cells by repressing transposons. However, the mechanism of piRNA-guided heterochromatin formation and its relationship to transcriptional termination remains elusive. Through RNA interference screening, we discovered Pcf11 and PNUTS as essential for piRNA-guided silencing in <em>Drosophila</em> germ line. Enforced tethering of Pcf11 leads to co-transcriptional repression and RNA polymerase II (RNA Pol II) stalling, and both are dependent on an α-helical region of Pcf11 capable of forming condensates. An intrinsically disordered region can substitute for the α-helical region of Pcf11 in its silencing capacity and support animal development, arguing for a causal relationship between phase separation and Pcf11’s function. Pcf11 stalls RNA Pol II by preferentially forming condensates with the unphosphorylated Spt5, promoted by the PP1/PNUTS phosphatase during termination. We propose that Pcf11/Spt5 condensates control termination by decelerating polymerase elongation, a property exploited by piRNAs to silence transposons and initiate RNA-mediated heterochromatin formation.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"25 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143495770","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-26DOI: 10.1016/j.molcel.2025.01.032
Merle Skribbe, Charlotte Soneson, Michael B. Stadler, Michaela Schwaiger, Vishnu N. Suma Sreechakram, Vytautas Iesmantavicius, Daniel Hess, Eliza Pandini Figueiredo Moreno, Sigurd Braun, Jan Seebacher, Sebastien A. Smallwood, Marc Bühler
Transcription factors (TFs) are key regulators of gene expression, yet many of their targets and modes of action remain unknown. In Schizosaccharomyces pombe, one-third of TFs are solely homology predicted, with few experimentally validated. We created a comprehensive library of 89 endogenously tagged S. pombe TFs, mapping their protein and chromatin interactions using immunoprecipitation-mass spectrometry and chromatin immunoprecipitation sequencing. Our study identified protein interactors for half the TFs, with over a quarter potentially forming stable complexes. We discovered DNA-binding sites for most TFs across 2,027 unique genomic regions, revealing motifs for 38 TFs and uncovering a complex network of extensive TF cross- and autoregulation. Characterization of the largest TF family revealed conserved DNA sequence preferences but diverse binding patterns and identified a repressive heterodimer, Ntu1/Ntu2, linked to perinuclear gene localization. Our TFexplorer webtool makes all data interactively accessible, offering insights into TF interactions and regulatory mechanisms with broad biological relevance.
{"title":"A comprehensive Schizosaccharomyces pombe atlas of physical transcription factor interactions with proteins and chromatin","authors":"Merle Skribbe, Charlotte Soneson, Michael B. Stadler, Michaela Schwaiger, Vishnu N. Suma Sreechakram, Vytautas Iesmantavicius, Daniel Hess, Eliza Pandini Figueiredo Moreno, Sigurd Braun, Jan Seebacher, Sebastien A. Smallwood, Marc Bühler","doi":"10.1016/j.molcel.2025.01.032","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.01.032","url":null,"abstract":"Transcription factors (TFs) are key regulators of gene expression, yet many of their targets and modes of action remain unknown. In <em>Schizosaccharomyces pombe</em>, one-third of TFs are solely homology predicted, with few experimentally validated. We created a comprehensive library of 89 endogenously tagged <em>S. pombe</em> TFs, mapping their protein and chromatin interactions using immunoprecipitation-mass spectrometry and chromatin immunoprecipitation sequencing. Our study identified protein interactors for half the TFs, with over a quarter potentially forming stable complexes. We discovered DNA-binding sites for most TFs across 2,027 unique genomic regions, revealing motifs for 38 TFs and uncovering a complex network of extensive TF cross- and autoregulation. Characterization of the largest TF family revealed conserved DNA sequence preferences but diverse binding patterns and identified a repressive heterodimer, Ntu1/Ntu2, linked to perinuclear gene localization. Our TFexplorer webtool makes all data interactively accessible, offering insights into TF interactions and regulatory mechanisms with broad biological relevance.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"36 1 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143495772","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-26DOI: 10.1016/j.molcel.2025.01.034
Ernst W. Schmid, Johannes C. Walter
Protein-protein interactions (PPIs) are ubiquitous in biology, yet a comprehensive structural characterization of the PPIs underlying cellular processes is lacking. AlphaFold-Multimer (AF-M) has the potential to fill this knowledge gap, but standard AF-M confidence metrics do not reliably separate relevant PPIs from an abundance of false positive predictions. To address this limitation, we used machine learning on curated datasets to train a structure prediction and omics-informed classifier (SPOC) that effectively separates true and false AF-M predictions of PPIs, including in proteome-wide screens. We applied SPOC to an all-by-all matrix of nearly 300 human genome maintenance proteins, generating ∼40,000 predictions that can be viewed at predictomes.org, where users can also score their own predictions with SPOC. High-confidence PPIs discovered using our approach enable hypothesis generation in genome maintenance. Our results provide a framework for interpreting large-scale AF-M screens and help lay the foundation for a proteome-wide structural interactome.
{"title":"Predictomes, a classifier-curated database of AlphaFold-modeled protein-protein interactions","authors":"Ernst W. Schmid, Johannes C. Walter","doi":"10.1016/j.molcel.2025.01.034","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.01.034","url":null,"abstract":"Protein-protein interactions (PPIs) are ubiquitous in biology, yet a comprehensive structural characterization of the PPIs underlying cellular processes is lacking. AlphaFold-Multimer (AF-M) has the potential to fill this knowledge gap, but standard AF-M confidence metrics do not reliably separate relevant PPIs from an abundance of false positive predictions. To address this limitation, we used machine learning on curated datasets to train a structure prediction and omics-informed classifier (SPOC) that effectively separates true and false AF-M predictions of PPIs, including in proteome-wide screens. We applied SPOC to an all-by-all matrix of nearly 300 human genome maintenance proteins, generating ∼40,000 predictions that can be viewed at <span><span>predictomes.org</span><svg aria-label=\"Opens in new window\" focusable=\"false\" height=\"20\" viewbox=\"0 0 8 8\"><path d=\"M1.12949 2.1072V1H7V6.85795H5.89111V2.90281L0.784057 8L0 7.21635L5.11902 2.1072H1.12949Z\"></path></svg></span>, where users can also score their own predictions with SPOC. High-confidence PPIs discovered using our approach enable hypothesis generation in genome maintenance. Our results provide a framework for interpreting large-scale AF-M screens and help lay the foundation for a proteome-wide structural interactome.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"68 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143495768","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-25DOI: 10.1016/j.molcel.2025.02.001
Anthony D. Rish, Elizabeth Fosuah, Zhangfei Shen, Ila A. Marathe, Vicki H. Wysocki, Tian-Min Fu
Leveraging AlphaFold models and integrated experiments, we characterized the HerA-DUF4297 (DUF) anti-phage defense system, focusing on DUF’s undefined biochemical functions. Guided by structure-based genomic analyses, we found DUF homologs to be universally distributed across diverse bacterial immune systems. Notably, one such homolog, Cap4, is a nuclease. Inspired by this evolutionary clue, we tested DUF’s nuclease activity and observed that DUF cleaves DNA substrates only when bound to its partner protein HerA. To dissect the mechanism of DUF activation, we determined the structures of DUF and HerA-DUF. Although DUF forms large oligomeric assemblies both alone and with HerA, oligomerization alone was insufficient to elicit nuclease activity. Instead, HerA binding induces a profound architecture remodeling that propagates throughout the complex. This remodeling reconfigures DUF into an active nuclease capable of robust DNA cleavage. Together, we highlight an architecture remodeling-driven mechanism that may inform the activation of other immune systems.
{"title":"Architecture remodeling activates the HerA-DUF anti-phage defense system","authors":"Anthony D. Rish, Elizabeth Fosuah, Zhangfei Shen, Ila A. Marathe, Vicki H. Wysocki, Tian-Min Fu","doi":"10.1016/j.molcel.2025.02.001","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.02.001","url":null,"abstract":"Leveraging AlphaFold models and integrated experiments, we characterized the HerA-DUF4297 (DUF) anti-phage defense system, focusing on DUF’s undefined biochemical functions. Guided by structure-based genomic analyses, we found DUF homologs to be universally distributed across diverse bacterial immune systems. Notably, one such homolog, Cap4, is a nuclease. Inspired by this evolutionary clue, we tested DUF’s nuclease activity and observed that DUF cleaves DNA substrates only when bound to its partner protein HerA. To dissect the mechanism of DUF activation, we determined the structures of DUF and HerA-DUF. Although DUF forms large oligomeric assemblies both alone and with HerA, oligomerization alone was insufficient to elicit nuclease activity. Instead, HerA binding induces a profound architecture remodeling that propagates throughout the complex. This remodeling reconfigures DUF into an active nuclease capable of robust DNA cleavage. Together, we highlight an architecture remodeling-driven mechanism that may inform the activation of other immune systems.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"19 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143486523","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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