bioRxiv : the preprint server for biology最新文献

{"title":"Synergistic MAPT mutations as a platform to uncover modifiers of tau pathogenesis.","authors":"Miles R Bryan, Michael Almeida, Kyle Pellegrino, Carli Opland, Ethan Paulakonis, Jake McGillion-Moore, Hanna Trzeciakiewicz, Diamond King, Xu Tian, Jui-Heng Tseng, Jonathan Schisler, Nicholas G Brown, Ben Bahr, Todd J Cohen","doi":"10.1101/2025.02.07.636933","DOIUrl":"10.1101/2025.02.07.636933","url":null,"abstract":"<p><p>The natively unfolded tau (MAPT) protein is extremely soluble, which poses challenges when modeling neurofibrillary tangle (NFT) pathology in Alzheimers disease (AD). To overcome this hurdle, we combined P301L and S320F mutations (PL-SF) to generate a rapid and reliable tau pathology platform to expedite the discovery of factors that modify tau aggregation. Using this model, we evaluated heat-shock proteins (Hsp), which have been linked to tau pathology, but whose role in AD remains enigmatic and controversial. In primary neurons, expression of Hsp70, but not Hsc70 or Hsp90, exacerbated tau aggregation. Conversely, lowering Hsp70 or employing a chaperone-deficient tau mutant (PL-SF-4 delta;) reduced tau phosphorylation and abrogated tau aggregation, highlighting Hsp70 as a key driver of tau aggregation. Hsp70 foci clustered within and surrounding neuritic plaques and NFTs in post-mortem AD brain. Functionally, mature aggregate-bearing neurons showed deficits in neuronal firing and network communication, which were restored by chaperone-binding deficient tau variants that abrogated tau pathology. This study provides a powerful cell-intrinsic model for accelerated tau aggregation, which can be harnessed to identify potent regulators of tau aggregation as promising therapeutic targets.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11839094/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143462039","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"GFAP Degradation in TBI: Linking Novel Modified Products to Astrocyte Pathology and Patient Outcome.","authors":"Ina-Beate Wanner, Julia Halford, Jonathan Lopez, Sean Shen, Yu Chen, Hayden Zhao, Carolina Salas, Rachel R Ogorzalek Loo, Steven Robicsek, Benjamin M Ellingson, Jeffrey Gornbein, Timothy E Van Meter, Gerry Shaw, Joseph A Loo, Paul M Vespa","doi":"10.1101/2025.08.01.668181","DOIUrl":"10.1101/2025.08.01.668181","url":null,"abstract":"<p><strong>Background: </strong>Traumatic brain injury (TBI) is a major public health concern that demands effective patient monitoring to mitigate secondary complications and improve recovery. TBI increases the risk for neurodegeneration by destabilizing proteostasis that increases protein degradation and proteinopathy. Despite the clinical importance of noninvasive TBI biomarkers, such as glial fibrillary acidic protein (GFAP), their mechanistic and cytological underpinnings remain poorly understood, hence are addressed here.</p><p><strong>Methods: </strong>Deep proteomic profiling of GFAP used mass spectrometry (MS) sequencing of immunopurified GFAP breakdown products (BDPs) in TBI patients' cerebrospinal fluid (CSF) and serum. A unique trauma culture model used human neocortical astrocytes for GFAP epitope mapping and GFAP-BDP compartmental localization. Immunofluorescence, protease inhibitors (calpeptin, ZVAD-FMK), and live protease reporters document GFAP proteolysis and citrullination in dye-uptake-identified membrane-wounded astrocytes. Temporal profiles of GFAP and GFAP-BDPs were measured in TBI patients' CSF via calibrated, sub-saturated immunoblot densitometry. Fragment-specific prediction of six-month recovery used the Extended Glasgow Outcome Scale (GOSE).</p><p><strong>Results: </strong>GFAP-BDP sequence sets with mapped posttranslational modifications (PTMs, citrullinations and acetylations) were identified in TBI patients' biofluids. Citrullinated GFAP profiles were distinct from unmodified GFAP in TBI patients' CSF. Novel TBI-specific cleavage fragments differed from those of Alzheimer's and Alexander diseases.Antibody mapping in the culture trauma model confirmed GFAP-BDPs, identified a new trauma cleavage region and revealed GFAP rod-domain harbored coil1-BDPs were selectively released into fluids, while coil2-BDPs remained as intracellular aggregates. Citrullinated coil2-BDPs formed non-filamentous aggregates in dystrophic astrocytes. Calpains and caspases contributed to trauma-generated GFAP-BDPs that were active in distinct astrocyte morphotypes and injury states.TBI patient GFAP proteolysis trajectories show that full-length GFAP and 45-49kDa fragments peaked acutely, then declined, whereas calpain-generated 37-39kDa BDPs remained elevated. Small GFAP-BDP-levels were imbalanced from expected proportions, with 20-26kDa products exceeding 15-19kDa products in CSF, consistent with preferential coil1-BDP release and coil2 retention shown in vitro. GFAP fragment profiles, but not those of uncleaved GFAP, predicted good versus poor outcome of TBI patients.</p><p><strong>Conclusion: </strong>These findings highlight the translational importance of novel proteomic GFAP proteolysis profiles that could serve in neurocritical care monitoring. GFAP use as fluid biomarker, tied to astroglial proteinopathy, is relevant to post-traumatic neurodegeneration.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12324458/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144791386","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Profibrotic monocyte-derived alveolar macrophages as a biomarker and therapeutic target in systemic sclerosis-associated interstitial lung disease.","authors":"Nikolay S Markov, Anthony J Esposito, Karolina J Senkow, Maxwell Schleck, Luisa Cusick, Zhan Yu, Yuliana V Sokolenko, Estefani Diaz, Emmy Jonasson, Suchitra Swaminathan, Ziyan Lu, Radmila Nafikova, Samuel Fenske, Elsie G Bunyan, Xóchitl G Pérez-Leonor, Hiam Abdala-Valencia, Annette S Flozak, Nikita Joshi, A Christine Argento, Elizabeth S Malsin, Paul A Reyfman, Jonathan Puchalski, Mridu Gulati, Mary Carns, Kathleen Aren, Phillip Cooper, Natania S Field, Suror Mohsin, Malek Shawabkeh, Alexandra Soriano, Aaron N Gundersheimer, Isaac A Goldberg, Bailey Damore, Alec Peltekian, Ankit Agrawal, Crystal Cheung, Stephanie Perez, Shannon Teaw, Alyssa Williams, Nicolas Page, Sophia E Kujawski, William Odell, Baran Ilayda Gunes, Michelle Cheng, Morgan Emokpae, R Ian Cumming, Robert M Tighe, Kevin Grudzinski, Hatice Savas, Ami N Rubinowitz, Bashar A Kadhim, Chitaru Kurihara, Ankit Bharat, Vikas Mehta, Jane E Dematte, Bradford C Bemiss, Hadijat M Makinde, Carla M Cuda, Matthew Dapas, Carrie Richardson, Harris Perlman, Anna P Lam, Cara J Gottardi, G R Scott Budinger, Alexander V Misharin, Monique E Hinchcliff","doi":"10.1101/2025.08.07.669006","DOIUrl":"10.1101/2025.08.07.669006","url":null,"abstract":"<p><p>Interstitial lung disease (ILD) is present in over 60% of patients with systemic sclerosis (SSc) and is the leading cause of SSc-related deaths. Profibrotic monocyte-derived alveolar macrophages (MoAM) play a causal role in the pathogenesis of pulmonary fibrosis in animal models where their persistence in the niche requires signaling through Colony Stimulating Factor 1 Receptor (CSF1R). We hypothesized that the presence and proportion of MoAM in bronchoalveolar lavage (BAL) fluid from patients with SSc-ILD may be a biomarker of ILD severity. To test this hypothesis, we analyzed BAL fluid from 9 prospectively enrolled patients with SSc-ILD and 13 healthy controls using flow cytometry and single-cell RNA sequencing. Patients with SSc-ILD had more MoAM and interstitial macrophages in BAL fluid than healthy controls, and their abundance was associated with lung fibrosis severity. We identified changes in the MoAM transcriptome as a function of treatment with mycophenolate, an effective therapy for SSc-ILD. In SSc-ILD lung explants, spatial transcriptomics identified an expanded population of interstitial macrophages spilling into the alveolar space. Our findings suggest that the proportion of profibrotic MoAM and interstitial macrophages in BAL fluid may serve as a biomarker of SSc-ILD and credential them as possible targets for therapy.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12363918/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144884864","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Alternate RNA decoding results in stable and abundant proteins in mammals.","authors":"Shira Tsour, Rainer Machne, Andrew Leduc, Simon Widmer, Eunice Koo, Jeremy Guez, Konrad Karczewski, Nikolai Slavov","doi":"10.1101/2024.08.26.609665","DOIUrl":"10.1101/2024.08.26.609665","url":null,"abstract":"<p><p>Amino acid substitutions may substantially alter protein stability and function, but the contribution of substitutions arising from alternate translation (deviations from the genetic code) is unknown. To explore it, we analyzed deep proteomic and transcriptomic data from over 1,000 human samples, including 6 cancer types and 26 healthy human tissues. This global analysis identified 60,803 fragmentation spectra corresponding to 8,801 unique substitution sites in proteins derived from 1,782 genes, including 2,000 confidently localized sites. Some substitutions are shared across samples, while others exhibit strong tissue-type and cancer specificity. Surprisingly, products of alternate translation are more abundant than their canonical counterparts for hundreds of proteins, suggesting sense codon recoding. Recoded proteins include transcription factors, proteases, signaling proteins, and proteins associated with neurodegeneration. Mechanisms contributing to substitution abundance include protein stability, codon frequency, codon-anticodon mismatches, and RNA modifications. We characterize how alternatively translated proteoform ratios vary across protein domains, tissue types and cancers. The substitution ratios are positively associated with intrinsically disordered regions and genetic polymorphisms in gnomAD, though the polymorphisms cannot account for the substitutions. The sequence, relative abundance, and the tissue-specificity of alternatively translated proteins are conserved between human and mouse. These results demonstrate the contribution of alternate translation to diversifying mammalian proteomes, and its association with protein stability, tissue-specific proteomes, and diseases.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11383030/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142305808","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Overexpression of Ssd1 and calorie restriction extend yeast replicative lifespan by preventing deleterious age-dependent iron uptake.","authors":"J Ignacio Gutierrez, Claudia Edgar, Jessica K Tyler","doi":"10.1101/2025.09.02.673772","DOIUrl":"10.1101/2025.09.02.673772","url":null,"abstract":"<p><p>Overexpression of the mRNA binding protein Ssd1 extends the yeast replicative lifespan. Using microfluidics to trap and image single cells throughout their lifespans, we find that lifespan extension by Ssd1 overexpression is accompanied by formation of cytoplasmic Ssd1 foci. The age-dependent Ssd1 foci are condensates that appear dynamically in a cell cycle-dependent manner and their failure to resolve during mitosis coincided with the end of lifespan. Ssd1 overexpression was epistatic with calorie restriction (CR) for lifespan extension and yeast overexpressing Ssd1 or undergoing CR were resistant to iron supplementation-induced lifespan shortening while their lifespans were reduced by iron chelation. The nuclear translocation of the Aft1 transcriptional regulator of the iron regulon occurred during aging in a manner that predicted remaining lifespan, but was prevented by CR. Accordingly, age-dependent induction of the Fit2 and Arn1 high-affinity iron transporters within the iron regulon was reduced by CR and Ssd1 overexpression. Consistent with age-dependent activation of the iron regulon, intracellular iron accumulated during aging but was prevented by CR and Ssd1 overexpression. Moreover, lifespan extension by Ssd1 overexpression or CR was epistatic to inactivation of the iron regulon. These studies reveal that CR and Ssd1 overexpression extend the yeast replicative lifespan by blocking deleterious age-dependent iron uptake, identifying novel therapeutic targets for lifespan extension and providing insight into how CR may extend the lifespan and healthspan in humans.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12424809/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145067403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Sindbis virus nsP3 opal codon protects viral RNA and fitness by maintaining replication spherule integrity.","authors":"Tamanash Bhattacharya, Tiia S Freeman, Eva M Alleman, Fang Wang, Lyuba Chechik, Michael Emerman, Kevin M Myles, Harmit S Malik","doi":"10.1101/2025.09.27.679005","DOIUrl":"10.1101/2025.09.27.679005","url":null,"abstract":"<p><p>Most alphaviruses encode an in-frame opal stop codon between nsP3 and nsP4 in their non-structural ORF. This opal stop codon mediates a temperature-dependent balance between viral polymerase production and proteolytic processing in vertebrate hosts. Yet, why this opal codon is maintained in insect hosts is unknown. Here, we show that the nsP3 opal stop codon confers a replicative advantage to Sindbis virus (SINV) in RNAi-competent mosquito cells, but not in cells lacking RNAi. Through delays in nsP processing, the lack of opal stop codon disrupts viral replication spherule integrity and increases Dicer 2-dependent cleavage of viral RNA, resulting in higher antiviral siRNA responses to the virus. Moreover, in mammalian cells, the opal codon-mediated spherule integrity also blocks MDA5-dependent viral RNA detection and interferon signaling. Thus, the highly conserved alphavirus opal codon mediates a multipotent viral defensive strategy.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12485792/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145215383","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The $10 proteome: low-cost, deep and quantitative proteome profiling of limited sample amounts using the Orbitrap Astral and timsTOF Ultra 2 mass spectrometers.","authors":"Siqi Huang, Chao Wang, Hsien-Jung L Lin, Ryan T Kelly","doi":"10.1101/2025.07.29.667408","DOIUrl":"10.1101/2025.07.29.667408","url":null,"abstract":"<p><p>Mass spectrometry (MS)-based proteomics remains technically demanding and prohibitively expensive for many large-scale or routine applications, with per-sample costs of hundreds of dollars or more. To democratize access to proteomics and facilitate its integration into more high-throughput multi-omic studies, we present a robust analytical framework for achieving in-depth, quantitative proteome profiling at a cost of approximately $10 per sample, termed the \"$10 proteome.\" Using the Thermo Fisher Orbitrap Astral and Bruker timsTOF Ultra 2 mass spectrometers, we evaluated performance across sample inputs ranging from 200 pg to 100 ng and active gradient lengths from 5 to 60 minutes. Proteome coverage saturated within the low-nanogram input range, with ~8000 protein groups quantified from as little as 10 ng of input and nearly 6000 protein groups from 200 pg. With already demonstrated low-cost one-pot sample preparation workflows that are appropriate for this sample input range, standardized MS acquisition settings, and high-throughput nanoLC operated at ~10 min per sample, the $10 proteome becomes feasible. This study establishes a practical, scalable, and cost-effective foundation for global proteome profiling, paving the way for routine, large-scale applications in systems biology, clinical research and beyond.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12324313/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144791599","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Elucidating lipid nanoparticle properties and structure through biophysical analyses.","authors":"Marshall S Padilla, Sarah J Shepherd, Andrew R Hanna, Martin Kurnik, Xujun Zhang, Michelle Chen, James Byrnes, Ryann A Joseph, Hannah M Yamagata, Adele S Ricciardi, Kaitlin Mrksich, David Issadore, Kushol Gupta, Michael J Mitchell","doi":"10.1101/2024.12.19.629496","DOIUrl":"10.1101/2024.12.19.629496","url":null,"abstract":"<p><p>Designing lipid nanoparticle (LNP) delivery systems with specific targeting, potency and minimal side effects is crucial for their clinical use. However, traditional characterization methods, such as dynamic light scattering, cannot accurately quantify physicochemical properties of LNPs and how they are influenced by the lipid composition and mixing method. Here we structurally characterize polydisperse LNP formulations by applying emerging solution-based biophysical methods that have higher resolution and provide biophysical data beyond size and polydispersity. These techniques include sedimentation velocity analytical ultracentrifugation, field-flow fractionation followed by multi-angle light scattering, and size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering. We show that LNPs have intrinsic polydispersity in size, RNA loading, and shape, which depends on both the formulation technique and lipid composition. Lastly, we predict LNP transfection in vitro and in vivo by examining the relationship between mRNA translation and physicochemical characteristics. Solution-based biophysical methods will be essential for determining LNP structure-function relationships, facilitating the creation of new design rules for LNPs.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11702722/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142974378","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Conduction pathway for potassium through the E. coli pump KdpFABC.","authors":"Adel Hussein, Xihui Zhang, Bjørn Panyella Pedersen, David L Stokes","doi":"10.1101/2025.05.05.652293","DOIUrl":"10.1101/2025.05.05.652293","url":null,"abstract":"<p><p>Under osmotic stress, bacteria express a heterotetrameric protein complex, KdpFABC, which functions as an ATP-dependent K⁺ pump to maintain intracellular potassium levels. The subunit KdpA belongs to the Superfamily of K⁺ Transporters and adopts a pseudo-tetrameric architecture with a membrane embedded selectivity filter as seen in K⁺ channels. KdpB belongs to the superfamily of P-type ATPases with a conserved binding site for ions within the membrane domain and three cytoplasmic domains that orchestrate ATP hydrolysis via an aspartyl phosphate intermediate. Previous work has hypothesized that K⁺ moves parallel to the membrane plane through a 40-Å long tunnel that connects the selectivity filter of KdpA with a canonical binding site in KdpB. In the current work, we have reconstituted KdpFABC into lipid nanodiscs and used cryo-EM to image the wild-type pump under turnover conditions. We present a 2.1 Å structure of the E1~P·ADP conformation, which reveals new features of the conduction pathway. This map shows strong densities within the selectivity filter and at the canonical binding site, consistent with K⁺ bound at each of these sites in this conformation. Many water molecules occupy a vestibule and the proximal end of the tunnel, which becomes markedly hydrophobic and dewetted at the subunit interface. We go on to use ATPase and ion transport assays to assess effects of numerous mutations along this proposed conduction pathway. The results confirm that K⁺ ions pass through the tunnel and support the existence of a low affinity site in KdpB for releasing these ions to the cytoplasm. Taken together, these data shed new light on the unique partnership between a transmembrane channel and an ATP-driven pump in maintaining the large electrochemical K⁺ gradient essential for bacterial survival.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12247778/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628955","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Heterochromatin-based silencing of a foreign tandem repeat in <i>Drosophila melanogaster</i> shows unusual biochemistry and temperature sensitivity.","authors":"Tingting Gu, Elena Gracheva, Michael Lee, Wilson Leung, Sophia Bieser, Alixandria Nielsen, Adam T Smiley, Nhi N T Vuong, Matthias Walther, Gunter Reuter, Sarah C R Elgin, Andrew M Arsham","doi":"10.1101/2025.07.31.667933","DOIUrl":"10.1101/2025.07.31.667933","url":null,"abstract":"<p><p>Eukaryotic genomes are packaged into chromatin, a regulatory nucleoprotein assembly. Establishment, maintenance, and interconversion of chromatin states is required for correct patterns of gene expression, genome integrity, and survival. Transcriptionally repressive heterochromatin minimizes mobilization of transposable elements and limits expansion of other repetitive DNA, but mechanisms for recognition of the latter sequences are not well established. We previously demonstrated in <i>Drosophila melanogaster</i> that transcripts derived from <i>1360</i> and <i>Invader4</i> transposon insertions can trigger local conversion of transcriptionally permissive euchromatin to heterochromatin through the piRNA system, but only in a subset of genomic locations near existing blocks of heterochromatin. Here we show that a ~9 kb tandem array of the 36-nucleotide <i>lac</i> operator (<i>lacO</i>) sequence of <i>Escherichia coli</i> can form ectopic heterochromatin at a similar subset of sites, resulting in variegating expression of an adjacent reporter gene. Heterochromatin Protein 1a (HP1a) and histone deacetylation are required for <i>lacO</i> repeat-induced silencing, but, contrasting with previously described Position Effect Variegation (PEV), we do not observe increased histone H3 lysine 9 methylation. Silencing is effective at 25°C and suppressed at 18°C (in contrast to canonical PEV, which is enhanced at 18°C), indicating involvement of a temperature-sensitive component. Temperature switching experiments show that <i>lacO</i> repeat-induced heterochromatin formation is reversible throughout larval development following an HP1a-dependent initiation step in the early embryo. We conclude that the <i>Drosophila</i> nucleus can recognize a completely foreign tandem repeat as a target for heterochromatin formation, and that the heterochromatin structure established is distinct from that of endogenous tandem arrays.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12324534/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144791438","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}