Protein function depends on interactions with other protein partners, ultimately leading to the formation of intricate protein-protein interaction (PPI) networks. These molecular networks (or interactomes) are formed progressively, each interaction influencing the next one. Accordingly, a same protein can lead to the formation of different interactomes depending on its first associated cofactor. Therefore, capturing PPIs of specific dimeric protein complexes is key for understanding the molecular rules underlying the diverse cell- and/or subcellular- functions of a bait protein of interest. Here, we introduce an innovative method called Bi-nano-ID that is based on bicolor bimolecular fluorescence complementation and the specific binding of a nanobody fused to a proximity-dependent biotinylating enzyme to tackle this issue. Bi-nano-ID was used to capture endogenous interactomes of the cytoplasmic TAZ/14-3-3e and nuclear TAZ/TEAD2 complexes, which are major downstream effectors of the Hippo signaling pathway. Among the different specific interactions, we revealed the role of a particular family of protease inhibitors for stabilizing and promoting the proliferative activity of TAZ/14-3-3e complexes in mesenchymal stem cells. Overall, our work establishes a novel sensitive method for capturing and visualizing specific interactions of binary bait protein complexes in human living cells.
{"title":"A nanobody-based approach to capture and visualize interactions of binary protein complexes in living cells","authors":"Nawal Hajj Sleiman, Julie Carnesecchi, Yunlong Jia, Frederic Delolme, Laurent Gilquin, Patrice Gouet, Samir Merabet","doi":"10.1101/2024.09.12.612471","DOIUrl":"https://doi.org/10.1101/2024.09.12.612471","url":null,"abstract":"Protein function depends on interactions with other protein partners, ultimately leading to the formation of intricate protein-protein interaction (PPI) networks. These molecular networks (or interactomes) are formed progressively, each interaction influencing the next one. Accordingly, a same protein can lead to the formation of different interactomes depending on its first associated cofactor. Therefore, capturing PPIs of specific dimeric protein complexes is key for understanding the molecular rules underlying the diverse cell- and/or subcellular- functions of a bait protein of interest. Here, we introduce an innovative method called Bi-nano-ID that is based on bicolor bimolecular fluorescence complementation and the specific binding of a nanobody fused to a proximity-dependent biotinylating enzyme to tackle this issue. Bi-nano-ID was used to capture endogenous interactomes of the cytoplasmic TAZ/14-3-3e and nuclear TAZ/TEAD2 complexes, which are major downstream effectors of the Hippo signaling pathway. Among the different specific interactions, we revealed the role of a particular family of protease inhibitors for stabilizing and promoting the proliferative activity of TAZ/14-3-3e complexes in mesenchymal stem cells. Overall, our work establishes a novel sensitive method for capturing and visualizing specific interactions of binary bait protein complexes in human living cells.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"187 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253416","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.11.612397
Feba Shaji, Jamshaid Ali, Rakesh S. Laishram
Endonucleolytic cleavage step of the pre-mRNA 3'-end processing is imprecise and results in heterogeneity of cleavage site (CS). On the contrary, we show that cleavage imprecision is tightly regulated leading to CS heterogeneity (CSH) and controls antioxidant response gene expression. CSH centres at a primary CS followed by subsidiary cleavages determined by the position of the CS. Globally and using targeted antioxidant mRNAs, we discovered an inverse relationship between the number of CS and the gene expression with highest cleavage efficiency from the primary CS. Strikingly, reducing CSH and increasing primary CS usage induces gene expression. Under oxidative stress (tBHQ, H2O2 or NaAsO2), CSH is decreased and the primary CS usage is stimulated that induces antioxidant response gene expression. Concomitantly, ectopic anti-oxidant protein expression from the primary CS or reduction in CSH imparts cellular oxidative stress tolerance. Genome-wide CS analysis of stress response genes also shows a concomitant result. We show that oxidative stress induces affinity/strength of cleavage complex assembly increasing the fidelity of cleavage at the primary CS thereby reducing CSH inducing antioxidant response.
{"title":"Cleavage site heterogeneity at the pre-mRNA 3'-untranslated region regulates gene expression","authors":"Feba Shaji, Jamshaid Ali, Rakesh S. Laishram","doi":"10.1101/2024.09.11.612397","DOIUrl":"https://doi.org/10.1101/2024.09.11.612397","url":null,"abstract":"Endonucleolytic cleavage step of the pre-mRNA 3'-end processing is imprecise and results in heterogeneity of cleavage site (CS). On the contrary, we show that cleavage imprecision is tightly regulated leading to CS heterogeneity (CSH) and controls antioxidant response gene expression. CSH centres at a primary CS followed by subsidiary cleavages determined by the position of the CS. Globally and using targeted antioxidant mRNAs, we discovered an inverse relationship between the number of CS and the gene expression with highest cleavage efficiency from the primary CS. Strikingly, reducing CSH and increasing primary CS usage induces gene expression. Under oxidative stress (tBHQ, H2O2 or NaAsO2), CSH is decreased and the primary CS usage is stimulated that induces antioxidant response gene expression. Concomitantly, ectopic anti-oxidant protein expression from the primary CS or reduction in CSH imparts cellular oxidative stress tolerance. Genome-wide CS analysis of stress response genes also shows a concomitant result. We show that oxidative stress induces affinity/strength of cleavage complex assembly increasing the fidelity of cleavage at the primary CS thereby reducing CSH inducing antioxidant response.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212724","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
To date, RNA-targeted chemical matter is under explored due to a lack of robust screening assays. In this study, we present a novel RNA-targeted small molecule screening approach using a specialized DNA-encoded library (DEL). Our findings reveal that the specialized DEL library, called DEL Zipper, can significantly reduce single-stranded DNA-RNA region interaction signals during various kinds of RNA selection. By performing the selection against both G-quadruplex, we have identified novel hits that interact with RNA targets and the results are validated through binding. This study demonstrates that the DEL Zipper method is a robust screening assay that has potential for discovering small molecule ligands for diverse RNA targets.
迄今为止，由于缺乏稳健的筛选检测方法，RNA 靶向化学物质的研究仍处于探索阶段。在本研究中，我们利用专门的 DNA 编码文库（DEL）提出了一种新的 RNA 靶向小分子筛选方法。我们的研究结果表明，被称为 DEL Zipper 的专用 DEL 文库能在各种 RNA 筛选过程中显著减少单链 DNA-RNA 区域相互作用信号。通过对 G-四链体进行选择，我们发现了与 RNA 靶标相互作用的新命中物，并通过结合验证了结果。这项研究表明，DEL Zipper 方法是一种稳健的筛选检测方法，具有发现多种 RNA 靶标小分子配体的潜力。
{"title":"Development of a DNA-encoded library screening method DEL Zipper to empower the study of RNA-targeted chemical matter","authors":"Zhongyao Ma, Bin Zou, Jiannan Zhao, Rui Zhang, Qiaoqiao Zhu, Xiaofeng Wang, Linan Xu, Xiang Gao, Xinyue Hu, Wei Feng, Wen Luo, Min Wang, Yunyun He, Zhifeng Yu, Weiren Cui, Qi Zhang, Letian Kuai, Wenji Su","doi":"10.1101/2024.09.13.612806","DOIUrl":"https://doi.org/10.1101/2024.09.13.612806","url":null,"abstract":"To date, RNA-targeted chemical matter is under explored due to a lack of robust screening assays. In this study, we present a novel RNA-targeted small molecule screening approach using a specialized DNA-encoded library (DEL). Our findings reveal that the specialized DEL library, called DEL Zipper, can significantly reduce single-stranded DNA-RNA region interaction signals during various kinds of RNA selection. By performing the selection against both G-quadruplex, we have identified novel hits that interact with RNA targets and the results are validated through binding. This study demonstrates that the DEL Zipper method is a robust screening assay that has potential for discovering small molecule ligands for diverse RNA targets.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"66 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253424","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.12.612644
Susana Rodriguez-Navarro, Ana Tejada-Colon, Joan Serrano-Quilez, Carme Nuno-Cabanes
Cells not only adapt to environmental changes, but they also "remember" specific signals, allowing them to respond more rapidly to future stressors. This phenomenon, known as transcriptional memory, is orchestrated by a complex interplay of epigenetics, transcription regulators and RNA metabolism factors. Although transcriptional memory has been well-studied in various contexts, its role in the heat shock (HS) response of yeast remains largely unexplored. In our study, we delve into the dynamics of HS memory in wild-type yeast and a mip6Δ mutant, which lacks the RNA-binding protein Mip6. Notably, Mip6 has been shown to influence the expression of key stress-related genes and maintain low Msn2/4-dependent transcript levels under standard conditions. Our transcriptomic analysis offers novel insights into how yeast cells remember HS exposure. We uncover striking differences in gene expression patterns depending on whether genes are induced or repressed during HS memory. Furthermore, we find that an initial 15-minute heat shock triggers a response that becomes attenuated with just 5 additional minutes of stress. While the response kinetics between memory and non-memory conditions are similar, we report a subtle but important role for Mip6 in fine-tuning transcriptional memory and adaptation to heat stress. Biochemical and genetic evidence also suggests that Mip6 cooperates with alternative survival pathways independent of MSN2/4, and functionally interacts with the Rpd3 histone deacetylase complex, a key player in transcriptional memory and the HS response. These findings open up new avenues for understanding the molecular mechanisms behind heat stress adaptation in eukaryotes.
{"title":"Decoding Transcriptional Memory in Yeast Heat Shock and the Functional Implication of the RNA Binding Protein Mip6","authors":"Susana Rodriguez-Navarro, Ana Tejada-Colon, Joan Serrano-Quilez, Carme Nuno-Cabanes","doi":"10.1101/2024.09.12.612644","DOIUrl":"https://doi.org/10.1101/2024.09.12.612644","url":null,"abstract":"Cells not only adapt to environmental changes, but they also \"remember\" specific signals, allowing them to respond more rapidly to future stressors. This phenomenon, known as transcriptional memory, is orchestrated by a complex interplay of epigenetics, transcription regulators and RNA metabolism factors. Although transcriptional memory has been well-studied in various contexts, its role in the heat shock (HS) response of yeast remains largely unexplored. In our study, we delve into the dynamics of HS memory in wild-type yeast and a mip6Δ mutant, which lacks the RNA-binding protein Mip6. Notably, Mip6 has been shown to influence the expression of key stress-related genes and maintain low Msn2/4-dependent transcript levels under standard conditions. Our transcriptomic analysis offers novel insights into how yeast cells remember HS exposure. We uncover striking differences in gene expression patterns depending on whether genes are induced or repressed during HS memory. Furthermore, we find that an initial 15-minute heat shock triggers a response that becomes attenuated with just 5 additional minutes of stress. While the response kinetics between memory and non-memory conditions are similar, we report a subtle but important role for Mip6 in fine-tuning transcriptional memory and adaptation to heat stress. Biochemical and genetic evidence also suggests that Mip6 cooperates with alternative survival pathways independent of MSN2/4, and functionally interacts with the Rpd3 histone deacetylase complex, a key player in transcriptional memory and the HS response. These findings open up new avenues for understanding the molecular mechanisms behind heat stress adaptation in eukaryotes.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"24 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253419","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.13.612259
Joanna Krwawicz, Caroline J Sheeba, Katie Hains, Thomas McMahon, Yimo Zhang, Skirmantas Kriaucionis, Peter Sarkies
DNA methylation at the 5 position of cytosine (5mC) is an ancient epigenetic mark in eukaryotes. The levels of total 5mC vary enormously between different species, and the DNA methyltransferases that introduce 5mC have been repeatedly lost in several independent lineages. DNA methyltransferases are a threat to genomic stability due to the increased mutagenicity of 5mC bases and the propensity of DNA methyltransferases themselves to introduce DNA alkylation damage as an off-target effect. However, whether alkylation damage explains why 5mC is frequently lost in evolution is unclear. Here we tested the fitness consequences of DNA methyltransferase-induced alkylation damage by introducing a eukaryotic-like 5mC system into E. coli. We showed that introducing 5mC genome-wide leads to increased sensitivity to alkylating agents, which is strongly enhanced by removal of the 3mC repair enzyme AlkB. Unexpectedly, we discovered that 5mC introduction led to increased sensitivity to oxidative stress. We showed that this is due to increased formation of reactive oxygen in the presence of 5mC. We determined that reactive oxygen species led to non-enzymatic oxidation of 5mC, producing modified cytosines such as 5fC that are recognised as DNA base damage in E. coli. Overall, our work identifies increased sensitivity to oxidative stress, as well as alkylating agents, as a negative consequence of genome-wide 5mC. Oxidative stress is frequently encountered by organisms in their environment, thus offering a plausible reason for total loss of 5mC in some species.
胞嘧啶 5 位上的 DNA 甲基化（5mC）是真核生物中一种古老的表观遗传标记。5mC 总含量在不同物种之间存在巨大差异，而引入 5mC 的 DNA 甲基转移酶在多个独立种系中一再消失。DNA 甲基转移酶对基因组的稳定性构成威胁，这是因为 5mC 碱基的突变性增加，而且 DNA 甲基转移酶本身也倾向于引入 DNA 烷基化损伤作为脱靶效应。然而，烷基化损伤是否能解释 5mC 在进化过程中频繁丢失的原因尚不清楚。在这里，我们通过在大肠杆菌中引入类似真核生物的 5mC 系统，测试了 DNA 甲基转移酶诱导的烷基化损伤对健康的影响。我们发现，在全基因组范围内引入 5mC 会导致对烷基化药剂的敏感性增加，而去除 3mC 修复酶 AlkB 则会大大提高这种敏感性。意想不到的是，我们发现引入 5mC 会增加对氧化应激的敏感性。我们证明，这是由于在 5mC 存在下活性氧的形成增加所致。我们确定，活性氧导致 5mC 非酶性氧化，产生修饰的胞嘧啶，如 5fC，在大肠杆菌中被识别为 DNA 碱基损伤。总之，我们的研究发现，对氧化应激和烷化剂的敏感性增加是全基因组 5mC 的负面影响。生物在其生存环境中经常会遇到氧化应激，这就为某些物种 5mC 的完全丧失提供了一个合理的原因。
{"title":"Introduction of cytosine-5 DNA methylation sensitizes cells to oxidative damage","authors":"Joanna Krwawicz, Caroline J Sheeba, Katie Hains, Thomas McMahon, Yimo Zhang, Skirmantas Kriaucionis, Peter Sarkies","doi":"10.1101/2024.09.13.612259","DOIUrl":"https://doi.org/10.1101/2024.09.13.612259","url":null,"abstract":"DNA methylation at the 5 position of cytosine (5mC) is an ancient epigenetic mark in eukaryotes. The levels of total 5mC vary enormously between different species, and the DNA methyltransferases that introduce 5mC have been repeatedly lost in several independent lineages. DNA methyltransferases are a threat to genomic stability due to the increased mutagenicity of 5mC bases and the propensity of DNA methyltransferases themselves to introduce DNA alkylation damage as an off-target effect. However, whether alkylation damage explains why 5mC is frequently lost in evolution is unclear. Here we tested the fitness consequences of DNA methyltransferase-induced alkylation damage by introducing a eukaryotic-like 5mC system into E. coli. We showed that introducing 5mC genome-wide leads to increased sensitivity to alkylating agents, which is strongly enhanced by removal of the 3mC repair enzyme AlkB. Unexpectedly, we discovered that 5mC introduction led to increased sensitivity to oxidative stress. We showed that this is due to increased formation of reactive oxygen in the presence of 5mC. We determined that reactive oxygen species led to non-enzymatic oxidation of 5mC, producing modified cytosines such as 5fC that are recognised as DNA base damage in E. coli. Overall, our work identifies increased sensitivity to oxidative stress, as well as alkylating agents, as a negative consequence of genome-wide 5mC. Oxidative stress is frequently encountered by organisms in their environment, thus offering a plausible reason for total loss of 5mC in some species.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253420","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.11.612530
Karly A Nisson, Rishi S Patel, Yennifer Delgado, Mehdi Bouhaddou, Lucie Etienne, Oliver I Fregoso
The lentiviral accessory protein Vpr engages an extensive network of cellular pathways to drive diverse host consequences. Of its many phenotypes, CRL4A-E3 ubiquitin ligase complex co-option, DNA damage response (DDR) engagement, and G2/M arrest are conserved and thus proposed to be functionally important. How Vpr effects these functions and whether they explain how Vpr dysregulates additional cellular pathways remain unclear. Here we leverage the ability of Vpr to deplete the nucleolar protein CCDC137 to understand how Vpr-induced DDR activation impacts nucleolar processes. We characterize CCDC137 as an indirect Vpr target whose degradation does not correlate with Vpr-induced G2/M arrest. Yet, degradation is conserved among Vpr from the pandemic HIV-1 and related SIVcpz/SIVgor, and it is triggered by genomic insults that activate a nucleolar ATR pathway in a manner similar to camptothecin. We determine that Vpr causes ATR-dependent features of nucleolar stress that correlate with CCDC137 degradation, including redistribution of nucleolar proteins, altered nucleolar morphology, and repressed ribosome biogenesis. Together, this data distinguishes CCDC137 as a non-canonical Vpr target that may serve as a sensor of nucleolar disruption, and in doing so, identifies a novel role for Vpr in nucleolar stress.
{"title":"HIV Vpr activates a nucleolar-specific ATR pathway to degrade the nucleolar stress sensor CCDC137","authors":"Karly A Nisson, Rishi S Patel, Yennifer Delgado, Mehdi Bouhaddou, Lucie Etienne, Oliver I Fregoso","doi":"10.1101/2024.09.11.612530","DOIUrl":"https://doi.org/10.1101/2024.09.11.612530","url":null,"abstract":"The lentiviral accessory protein Vpr engages an extensive network of cellular pathways to drive diverse host consequences. Of its many phenotypes, CRL4A-E3 ubiquitin ligase complex co-option, DNA damage response (DDR) engagement, and G2/M arrest are conserved and thus proposed to be functionally important. How Vpr effects these functions and whether they explain how Vpr dysregulates additional cellular pathways remain unclear. Here we leverage the ability of Vpr to deplete the nucleolar protein CCDC137 to understand how Vpr-induced DDR activation impacts nucleolar processes. We characterize CCDC137 as an indirect Vpr target whose degradation does not correlate with Vpr-induced G2/M arrest. Yet, degradation is conserved among Vpr from the pandemic HIV-1 and related SIVcpz/SIVgor, and it is triggered by genomic insults that activate a nucleolar ATR pathway in a manner similar to camptothecin. We determine that Vpr causes ATR-dependent features of nucleolar stress that correlate with CCDC137 degradation, including redistribution of nucleolar proteins, altered nucleolar morphology, and repressed ribosome biogenesis. Together, this data distinguishes CCDC137 as a non-canonical Vpr target that may serve as a sensor of nucleolar disruption, and in doing so, identifies a novel role for Vpr in nucleolar stress.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"22 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212718","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The in vivo mechanism, regulations by cis-acting roadblocks, and biological functions of loop extrusion by eukaryotic SMC complexes are incompletely defined. Here, using Hi-C, we identified two condensin-dependent contact stripes at the Recombination Enhancer (RE) and the rDNA in S. cerevisiae. We show that oriented, unidirectional loop extrusion proceeds from these sites with an estimated processivity ~170 kb and a density ~0.04-0.18 that varies across the cell cycle. Centromeres and highly-transcribed RNA PolII-dependent genes are permeable condensin roadblocks. Other positionally labile elements such as replication forks and Smc5/6 complexes bound to substrates generated in the absence of Top2 also hinder loop extrusion by condensin. Cohesin is not an obstacle for condensin. Finally, a DNA double-strand break at MAT blocks condensin, which results in the rapid establishment of a long-range RE-MAT loop that juxtaposes the recombination machinery with its HMLα donor target. Hence, all budding yeast SMCs are involved in recombinational DNA repair. We propose a revised model for donor selection during MAT switching that exploits specific properties of loop extrusion by condensin. It can serve as a paradigm for the establishment of other types of selective interactions along chromosomes.
真核 SMC 复合物挤出环路的体内机制、顺式作用路障的调控和生物学功能尚未完全明确。在这里，我们利用 Hi-C 技术在 S. cerevisiae 的重组增强子（RE）和 rDNA 上发现了两条依赖于凝集素的接触带。我们的研究表明，定向、单向的环状挤压从这些位点进行，其过程活性约为 170 kb，密度约为 0.04-0.18，在整个细胞周期中各不相同。中心粒和高度转录的 RNA PolII 依赖性基因是可渗透的凝集素路障。其他位置易变的元素，如复制叉和与 Top2 缺失时产生的底物结合的 Smc5/6 复合物，也会阻碍凝集素的环挤出。粘合素并不是冷凝蛋白的障碍。最后，MAT 处的 DNA 双链断裂会阻碍凝缩素，从而导致长程 RE-MAT 环的快速建立，该环将重组机制与其 HMLα 供体目标并列。因此，所有芽殖酵母 SMC 都参与了 DNA 重组修复。我们提出了一个在 MAT 转换过程中供体选择的修正模型，该模型利用了冷凝蛋白挤出环的特殊性质。它可以作为沿染色体建立其他类型选择性相互作用的范例。
{"title":"Condensin loop extrusion properties, roadblocks, and role in homology search in S. cerevisiae","authors":"Vinciane Piveteau, Hossein Salari, Agnes Dumont, Jerome Savocco, Chloe Dupont, Daniel Jost, Aurele Piazza","doi":"10.1101/2024.09.12.612585","DOIUrl":"https://doi.org/10.1101/2024.09.12.612585","url":null,"abstract":"The in vivo mechanism, regulations by cis-acting roadblocks, and biological functions of loop extrusion by eukaryotic SMC complexes are incompletely defined. Here, using Hi-C, we identified two condensin-dependent contact stripes at the Recombination Enhancer (RE) and the rDNA in S. cerevisiae. We show that oriented, unidirectional loop extrusion proceeds from these sites with an estimated processivity ~170 kb and a density ~0.04-0.18 that varies across the cell cycle. Centromeres and highly-transcribed RNA PolII-dependent genes are permeable condensin roadblocks. Other positionally labile elements such as replication forks and Smc5/6 complexes bound to substrates generated in the absence of Top2 also hinder loop extrusion by condensin. Cohesin is not an obstacle for condensin. Finally, a DNA double-strand break at MAT blocks condensin, which results in the rapid establishment of a long-range RE-MAT loop that juxtaposes the recombination machinery with its HMLα donor target. Hence, all budding yeast SMCs are involved in recombinational DNA repair. We propose a revised model for donor selection during MAT switching that exploits specific properties of loop extrusion by condensin. It can serve as a paradigm for the establishment of other types of selective interactions along chromosomes.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253423","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Break-induced replication (BIR) is mutagenic, and thus its use requires tight regulation, yet the underlying mechanisms remain elusive. Here we uncover an important role of 53BP1 in suppressing BIR after end resection at double strand breaks (DSBs), distinct from its end protection activity, providing insight into the mechanisms governing BIR regulation and DSB repair pathway selection. We demonstrate that loss of 53BP1 induces BIR-like hyperrecombination, in a manner dependent on Polα-primase-mediated end fill-in DNA synthesis on single-stranded DNA (ssDNA) overhangs at DSBs, leading to PCNA ubiquitination and PIF1 recruitment to activate BIR. On broken replication forks, where BIR is required for repairing single-ended DSBs (seDSBs), SMARCAD1 displaces 53BP1 to facilitate the localization of ubiquitinated PCNA and PIF1 to DSBs for BIR activation. Hyper BIR associated with 53BP1 deficiency manifests template switching and large deletions, underscoring another aspect of 53BP1 in suppressing genome instability. The synthetic lethal interaction between the 53BP1 and BIR pathways provides opportunities for targeted cancer treatment.
断裂诱导复制（BIR）具有致突变性，因此其使用需要严格的调控，但其潜在机制仍然难以捉摸。在这里，我们发现了 53BP1 在双链断裂（DSB）末端切除后抑制 BIR 的重要作用，这与其末端保护活性不同，从而为 BIR 调节和 DSB 修复途径选择的机制提供了见解。我们证明，53BP1 的缺失会诱导类似 BIR 的过度重组，其方式依赖于 Polα-primase 介导的在 DSB 处单链 DNA（ssDNA）悬垂上的末端填充 DNA 合成，从而导致 PCNA 泛素化和 PIF1 招募以激活 BIR。在断裂的复制叉上，BIR 需要修复单端 DSB（seDSB），SMARCAD1 会取代 53BP1 以促进泛素化的 PCNA 和 PIF1 定位于 DSB，从而激活 BIR。与 53BP1 缺乏相关的超 BIR 表现为模板切换和大量缺失，这从另一个方面强调了 53BP1 在抑制基因组不稳定性方面的作用。53BP1 和 BIR 通路之间的合成致死相互作用为癌症的靶向治疗提供了机会。
{"title":"53BP1 deficiency leads to hyperrecombination using break-induced replication (BIR)","authors":"Sameer Bikram Shah, Youhang Li, Shibo Li, Qing Hu, Tong Wu, Yanmeng Shi, Tran Nguyen, Isaac Ive, Linda Shi, Hailong Wang, Xiaohua Wu","doi":"10.1101/2024.09.11.612483","DOIUrl":"https://doi.org/10.1101/2024.09.11.612483","url":null,"abstract":"Break-induced replication (BIR) is mutagenic, and thus its use requires tight regulation, yet the underlying mechanisms remain elusive. Here we uncover an important role of 53BP1 in suppressing BIR after end resection at double strand breaks (DSBs), distinct from its end protection activity, providing insight into the mechanisms governing BIR regulation and DSB repair pathway selection. We demonstrate that loss of 53BP1 induces BIR-like hyperrecombination, in a manner dependent on Polα-primase-mediated end fill-in DNA synthesis on single-stranded DNA (ssDNA) overhangs at DSBs, leading to PCNA ubiquitination and PIF1 recruitment to activate BIR. On broken replication forks, where BIR is required for repairing single-ended DSBs (seDSBs), SMARCAD1 displaces 53BP1 to facilitate the localization of ubiquitinated PCNA and PIF1 to DSBs for BIR activation. Hyper BIR associated with 53BP1 deficiency manifests template switching and large deletions, underscoring another aspect of 53BP1 in suppressing genome instability. The synthetic lethal interaction between the 53BP1 and BIR pathways provides opportunities for targeted cancer treatment.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253422","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The dual interaction of many transcription factors (TFs) with both DNA and RNA is an underexplored issue that could fundamentally reshape our understanding of gene regulation. We address this central issue by investigating the RNA binding activity of the Drosophila Hox TF Ultrabithorax (Ubx) in alternative splicing and morphogenesis. Relying on molecular and genetic interactions, we uncover a homodimerization-dependent mechanism by which Ubx regulates splicing. Notably, this mechanism enables the decoupling of Ubx-DNA and -RNA binding activity in splicing. We identify a critical residue for Ubx-RNA binding and demonstrate the essential role of Ubx-RNA binding ability for its homeotic functions. Overall, we uncover a unique mechanism for Ubx-mediated splicing and underscore the critical contribution of synergistic DNA/RNA binding for its morphogenetic functions. These findings advance our understanding of co-transcriptional regulation and highlight the significance of TF-DNA/RNA synergistic function in shaping gene regulatory networks in living organisms.
{"title":"Synergistic DNA and RNA binding of the Hox transcription factor Ultrabithorax coordinates splicing and shapes in vivo homeotic functions","authors":"Constanza Blanco, Wan Xiang, Panagiotis Boumpas, Maily Scorcelletti, Ashley Suraj Hermon, Jiemin Wong, Samir Merabet, Julie Carnesecchi","doi":"10.1101/2024.09.10.612310","DOIUrl":"https://doi.org/10.1101/2024.09.10.612310","url":null,"abstract":"The dual interaction of many transcription factors (TFs) with both DNA and RNA is an underexplored issue that could fundamentally reshape our understanding of gene regulation. We address this central issue by investigating the RNA binding activity of the Drosophila Hox TF Ultrabithorax (Ubx) in alternative splicing and morphogenesis. Relying on molecular and genetic interactions, we uncover a homodimerization-dependent mechanism by which Ubx regulates splicing. Notably, this mechanism enables the decoupling of Ubx-DNA and -RNA binding activity in splicing. We identify a critical residue for Ubx-RNA binding and demonstrate the essential role of Ubx-RNA binding ability for its homeotic functions. Overall, we uncover a unique mechanism for Ubx-mediated splicing and underscore the critical contribution of synergistic DNA/RNA binding for its morphogenetic functions. These findings advance our understanding of co-transcriptional regulation and highlight the significance of TF-DNA/RNA synergistic function in shaping gene regulatory networks in living organisms.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"33 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212719","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-12DOI: 10.1101/2024.09.10.612373
Ranu Singh, Raghuvir Singh Tomar
Yeast flocculation relies on cell surface flocculin proteins encoded by the FLO1 gene. The expression of FLO1 is antagonistically regulated by the Tup1-Cyc8 and the Swi-Snf complexes. The Post translational modifications of core histones regulate the transcription of Tup1-Cyc8-regulated genes. However, the mechanisms by which the physical presence of tail residues regulate FLO1 transcription process and flocculation is yet to be completely understood. Through screening we have identified a new region within the N-terminal tail of histone H3 regulating the transcription of FLO1 and FLO5. One of the histone H3 N-terminal truncation mutants H3del(17 to 24) showed higher FLO1 expression compared to wild type H3. Results revealed that in absence of 17 to 24 stretch the occupancy of Cyc8 decreases from the upstream regions of FLO1. Additionally, analysis suggests that Hda1 is required for the Cyc8-mediated repression of FLO1. Altogether we demonstrate that 17 to 24 stretch is essential for the Tup1 independent binding of Cyc8 at the promoters assisted by Hda1, leading to the strong repression of FLO1 transcription. In the absence of the 17 to 24 stretch, Cyc8 cannot bind, resulting in uncontrolled transcription of FLO1.
{"title":"Screening of histone mutants reveals a domain within the N-terminal tail of histone H3 that regulates the Tup1-independent repressive role of Cyc8 at the active FLO1","authors":"Ranu Singh, Raghuvir Singh Tomar","doi":"10.1101/2024.09.10.612373","DOIUrl":"https://doi.org/10.1101/2024.09.10.612373","url":null,"abstract":"Yeast flocculation relies on cell surface flocculin proteins encoded by the FLO1 gene. The expression of FLO1 is antagonistically regulated by the Tup1-Cyc8 and the Swi-Snf complexes. The Post translational modifications of core histones regulate the transcription of Tup1-Cyc8-regulated genes. However, the mechanisms by which the physical presence of tail residues regulate FLO1 transcription process and flocculation is yet to be completely understood. Through screening we have identified a new region within the N-terminal tail of histone H3 regulating the transcription of FLO1 and FLO5. One of the histone H3 N-terminal truncation mutants H3del(17 to 24) showed higher FLO1 expression compared to wild type H3. Results revealed that in absence of 17 to 24 stretch the occupancy of Cyc8 decreases from the upstream regions of FLO1. Additionally, analysis suggests that Hda1 is required for the Cyc8-mediated repression of FLO1. Altogether we demonstrate that 17 to 24 stretch is essential for the Tup1 independent binding of Cyc8 at the promoters assisted by Hda1, leading to the strong repression of FLO1 transcription. In the absence of the 17 to 24 stretch, Cyc8 cannot bind, resulting in uncontrolled transcription of FLO1.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"25 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212720","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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