Chong Li, Marc Jan Bonder, Sabriya Syed, Matthew Jensen, Human Genome Structural Variation Consortium (HGSVC), HGSVC Functional Analysis Working Group, Mark B. Gerstein, Michael C. Zody, Mark J.P. Chaisson, Michael E. Talkowski, Tobias Marschall, Jan O. Korbel, Evan E. Eichler, Charles Lee, Xinghua Shi
The human genome is packaged within a three-dimensional (3D) nucleus and organized into structural units known as compartments, topologically associating domains (TADs), and loops. TAD boundaries, separating adjacent TADs, have been found to be well conserved across mammalian species and more evolutionarily constrained than TADs themselves. Recent studies show that structural variants (SVs) can modify 3D genomes through the disruption of TADs, which play an essential role in insulating genes from outside regulatory elements’ aberrant regulation. However, how SV affects the 3D genome structure and their association among different aspects of gene regulation and candidate cis-regulatory elements (cCREs) have rarely been studied systematically. Here, we assess the impact of SVs intersecting with TAD boundaries by developing an integrative Hi-C analysis pipeline, which enables the generation of an in-depth catalog of TADs and TAD boundaries in human lymphoblastoid cell lines (LCLs) to fill the gap of limited resources. Our catalog contains 18,865 TADs, including 4596 sub-TADs, with 185 SVs (TAD–SVs) that alter chromatin architecture. By leveraging the ENCODE registry of cCREs in humans, we determine that 34 of 185 TAD–SVs intersect with cCREs and observe significant enrichment of TAD–SVs within cCREs. This study provides a database of TADs and TAD–SVs in the human genome that will facilitate future investigations of the impact of SVs on chromatin structure and gene regulation in health and disease.
{"title":"An integrative TAD catalog in lymphoblastoid cell lines discloses the functional impact of deletions and insertions in human genomes","authors":"Chong Li, Marc Jan Bonder, Sabriya Syed, Matthew Jensen, Human Genome Structural Variation Consortium (HGSVC), HGSVC Functional Analysis Working Group, Mark B. Gerstein, Michael C. Zody, Mark J.P. Chaisson, Michael E. Talkowski, Tobias Marschall, Jan O. Korbel, Evan E. Eichler, Charles Lee, Xinghua Shi","doi":"10.1101/gr.279419.124","DOIUrl":"https://doi.org/10.1101/gr.279419.124","url":null,"abstract":"The human genome is packaged within a three-dimensional (3D) nucleus and organized into structural units known as compartments, topologically associating domains (TADs), and loops. TAD boundaries, separating adjacent TADs, have been found to be well conserved across mammalian species and more evolutionarily constrained than TADs themselves. Recent studies show that structural variants (SVs) can modify 3D genomes through the disruption of TADs, which play an essential role in insulating genes from outside regulatory elements’ aberrant regulation. However, how SV affects the 3D genome structure and their association among different aspects of gene regulation and candidate <em>cis</em>-regulatory elements (cCREs) have rarely been studied systematically. Here, we assess the impact of SVs intersecting with TAD boundaries by developing an integrative Hi-C analysis pipeline, which enables the generation of an in-depth catalog of TADs and TAD boundaries in human lymphoblastoid cell lines (LCLs) to fill the gap of limited resources. Our catalog contains 18,865 TADs, including 4596 sub-TADs, with 185 SVs (TAD–SVs) that alter chromatin architecture. By leveraging the ENCODE registry of cCREs in humans, we determine that 34 of 185 TAD–SVs intersect with cCREs and observe significant enrichment of TAD–SVs within cCREs. This study provides a database of TADs and TAD–SVs in the human genome that will facilitate future investigations of the impact of SVs on chromatin structure and gene regulation in health and disease.","PeriodicalId":12678,"journal":{"name":"Genome research","volume":"199 1","pages":""},"PeriodicalIF":7.0,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142783298","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Arne Sahm, Konstantin Riege, Marco Groth, Martin Bens, Johann Kraus, Martin Fischer, Hans Kestler, Christoph Englert, Ralf Schaible, Matthias Platzer, Steve Hoffmann
Growing evidence suggests that somatic mutations may be a major cause of the aging process. However, it remains to be tested whether the predictions of the theory also apply to species with longer life spans than humans. Hydra is a genus of freshwater polyps with remarkable regeneration abilities and a potentially unlimited life span under laboratory conditions. By genome sequencing of single cells and whole animals, we found that the mutation rates in Hydra’s stem cells are even slightly higher than in humans or mice. A potential explanation for this deviation from the prediction of the theory may lie in the adaptability offered by a higher mutation rate, as we were able to show that the genome of the widely studied Hydra magnipapillata strain 105 has undergone a process of strong positive selection since the strain's cultivation 50 years ago. This most likely represents a rapid adaptation to the drastically altered environmental conditions associated with the transition from the wild to laboratory conditions. Processes under positive selection in captive animals include pathways associated with Hydra’s simple nervous system, its nucleic acid metabolic process, cell migration, and hydrolase activity.
{"title":"Hydra has mammal-like mutation rates facilitating fast adaptation despite its nonaging phenotype","authors":"Arne Sahm, Konstantin Riege, Marco Groth, Martin Bens, Johann Kraus, Martin Fischer, Hans Kestler, Christoph Englert, Ralf Schaible, Matthias Platzer, Steve Hoffmann","doi":"10.1101/gr.279025.124","DOIUrl":"https://doi.org/10.1101/gr.279025.124","url":null,"abstract":"Growing evidence suggests that somatic mutations may be a major cause of the aging process. However, it remains to be tested whether the predictions of the theory also apply to species with longer life spans than humans. <em>Hydra</em> is a genus of freshwater polyps with remarkable regeneration abilities and a potentially unlimited life span under laboratory conditions. By genome sequencing of single cells and whole animals, we found that the mutation rates in <em>Hydra</em>’s stem cells are even slightly higher than in humans or mice. A potential explanation for this deviation from the prediction of the theory may lie in the adaptability offered by a higher mutation rate, as we were able to show that the genome of the widely studied <em>Hydra magnipapillata s</em>train 105 has undergone a process of strong positive selection since the strain's cultivation 50 years ago. This most likely represents a rapid adaptation to the drastically altered environmental conditions associated with the transition from the wild to laboratory conditions. Processes under positive selection in captive animals include pathways associated with <em>Hydra</em>’s simple nervous system, its nucleic acid metabolic process, cell migration, and hydrolase activity.","PeriodicalId":12678,"journal":{"name":"Genome research","volume":"27 1","pages":""},"PeriodicalIF":7.0,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142777005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jonathan Cahn, James P.B. Lloyd, Ino D. Karemaker, Pascal W.T.C. Jansen, Jahnvi Pflueger, Owen Duncan, Jakob Petereit, Ozren Bogdanovic, A. Harvey Millar, Michiel Vermeulen, Ryan Lister
In plants, cytosine DNA methylation (mC) is largely associated with transcriptional repression of transposable elements, but it can also be found in the body of expressed genes, referred to as gene body methylation (gbM). gbM is correlated with ubiquitously expressed genes; however, its function, or absence thereof, is highly debated. The different outputs that mC can have raise questions as to how it is interpreted—or read—differently in these sequence and genomic contexts. To screen for potential mC-binding proteins, we performed an unbiased DNA affinity pull-down assay combined with quantitative mass spectrometry using methylated DNA probes for each DNA sequence context. All mC readers known to date preferentially bind to the methylated probes, along with a range of new mC-binding protein candidates. Functional characterization of these mC readers, focused on the MBD and SUVH families, was undertaken by ChIP-seq mapping of genome-wide binding sites, their protein interactors, and the impact of high-order mutations on transcriptomic and epigenomic profiles. Together, these results highlight specific context preferences for these proteins, and in particular the ability of MBD2 to bind predominantly to gbM. This comprehensive analysis of Arabidopsis mC readers emphasizes the complexity and interconnectivity between DNA methylation and chromatin remodeling processes in plants.
{"title":"Characterization of DNA methylation reader proteins of Arabidopsis thaliana","authors":"Jonathan Cahn, James P.B. Lloyd, Ino D. Karemaker, Pascal W.T.C. Jansen, Jahnvi Pflueger, Owen Duncan, Jakob Petereit, Ozren Bogdanovic, A. Harvey Millar, Michiel Vermeulen, Ryan Lister","doi":"10.1101/gr.279379.124","DOIUrl":"https://doi.org/10.1101/gr.279379.124","url":null,"abstract":"In plants, cytosine DNA methylation (mC) is largely associated with transcriptional repression of transposable elements, but it can also be found in the body of expressed genes, referred to as gene body methylation (gbM). gbM is correlated with ubiquitously expressed genes; however, its function, or absence thereof, is highly debated. The different outputs that mC can have raise questions as to how it is interpreted—or read—differently in these sequence and genomic contexts. To screen for potential mC-binding proteins, we performed an unbiased DNA affinity pull-down assay combined with quantitative mass spectrometry using methylated DNA probes for each DNA sequence context. All mC readers known to date preferentially bind to the methylated probes, along with a range of new mC-binding protein candidates. Functional characterization of these mC readers, focused on the MBD and SUVH families, was undertaken by ChIP-seq mapping of genome-wide binding sites, their protein interactors, and the impact of high-order mutations on transcriptomic and epigenomic profiles. Together, these results highlight specific context preferences for these proteins, and in particular the ability of MBD2 to bind predominantly to gbM. This comprehensive analysis of <em>Arabidopsis</em> mC readers emphasizes the complexity and interconnectivity between DNA methylation and chromatin remodeling processes in plants.","PeriodicalId":12678,"journal":{"name":"Genome research","volume":"28 1","pages":""},"PeriodicalIF":7.0,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142776758","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Irmgard U. Haussmann, Thomas C. Dix, David W.J. McQuarrie, Veronica Dezi, Abdullah I. Hans, Roland Arnold, Matthias Soller
A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable variation among different prediction tools when applied to different data sets. To aid selection of efficient sgRNAs, we developed web-based PlatinumCRISPr, an sgRNA design tool to evaluate base-pairing and sequence composition parameters for optimal design of highly efficient sgRNAs for Cas9 genome editing. We applied this tool to select sgRNAs to efficiently generate gene deletions in Drosophila Ythdc1 and Ythdf, that bind to N6 methylated adenosines (m6A) in mRNA. However, we discovered that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions can be removed by standard genetic recombination and chromosome exchange. These new insights into sgRNA design and the mechanisms of CRISPR–Cas9 genome editing advance the efficient use of this technique for safer applications in humans.
{"title":"Structure-optimized sgRNA selection with PlatinumCRISPr for efficient Cas9 generation of knockouts","authors":"Irmgard U. Haussmann, Thomas C. Dix, David W.J. McQuarrie, Veronica Dezi, Abdullah I. Hans, Roland Arnold, Matthias Soller","doi":"10.1101/gr.279479.124","DOIUrl":"https://doi.org/10.1101/gr.279479.124","url":null,"abstract":"A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable variation among different prediction tools when applied to different data sets. To aid selection of efficient sgRNAs, we developed web-based PlatinumCRISPr, an sgRNA design tool to evaluate base-pairing and sequence composition parameters for optimal design of highly efficient sgRNAs for Cas9 genome editing. We applied this tool to select sgRNAs to efficiently generate gene deletions in <em>Drosophila Ythdc1</em> and <em>Ythdf</em>, that bind to <em>N</em><sup>6</sup> methylated adenosines (m<sup>6</sup>A) in mRNA. However, we discovered that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions can be removed by standard genetic recombination and chromosome exchange. These new insights into sgRNA design and the mechanisms of CRISPR–Cas9 genome editing advance the efficient use of this technique for safer applications in humans.","PeriodicalId":12678,"journal":{"name":"Genome research","volume":"2 1","pages":""},"PeriodicalIF":7.0,"publicationDate":"2024-12-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142763467","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Eugenio López-Cortegano, Jobran Chebib, Anika Jonas, Anastasia Vock, Sven Künzel, Peter D. Keightley, Diethard Tautz
All forms of genetic variation originate from new mutations, making it crucial to understand their rates and mechanisms. Here, we use long-read PacBio sequencing to investigate de novo mutations that accumulated in 12 inbred mouse lines derived from three commonly used inbred strains (C3H, C57BL/6, and FVB) maintained for 8-15 generations in a mutation accumulation (MA) experiment. We built chromosome-level genome assemblies based on the MA line founders' genomes, and then employed a combination of read and assembly-based methods to call the complete spectrum of new mutations. On average, there are ~45 mutations per haploid genome per generation, about half of which (54%) are insertions and deletions shorter than 50 bp (indels). The remainder are single nucleotide mutations (SNMs, 44%) and large structural mutations (SMs, 2%). We found that the degree of DNA repetitiveness is positively correlated with SNM and indel rates, and that a substantial fraction of SMs can be explained by homology-dependent mechanisms associated with repeat sequences. Most (90%) indels can be attributed to microsatellite contractions and expansions, and there is a marked bias towards 4 bp indels. Among the different types of SMs, tandem repeat mutations have the highest mutation rate, followed by insertions of transposable elements (TEs). We uncover a rich landscape of active TEs, and notable differences in their spectrum among MA lines and strains, and a high rate of gene retroposition. Our study offers novel insights into mammalian genome evolution, and highlights the importance of repetitive elements in shaping genomic diversity.
所有形式的遗传变异都源于新的突变，因此了解它们的速率和机制至关重要。在这里，我们使用长读PacBio测序研究了在突变积累（MA）实验中积累在12个自交系（C3H， C57BL/6和FVB）中积累8-15代的新生突变。我们基于MA系创始人的基因组构建了染色体水平的基因组组装，然后结合基于读取和组装的方法来调用新突变的完整谱。平均每个单倍体基因组每代约有45个突变，其中约一半（54%）是短于50 bp （indel）的插入和缺失。其余为单核苷酸突变（SNMs, 44%）和大结构突变（SMs, 2%）。我们发现DNA的重复程度与SNM和indel率呈正相关，并且相当一部分的SMs可以通过与重复序列相关的同源依赖机制来解释。大多数（90%）指数可归因于微卫星收缩和扩张，并且明显偏向于4 bp指数。在不同类型的SMs中，串联重复突变的突变率最高，其次是转座因子插入。我们发现了丰富的活性te景观，在MA系和菌株之间的光谱差异显著，基因逆转录率高。我们的研究为哺乳动物基因组进化提供了新的见解，并强调了重复元素在塑造基因组多样性中的重要性。
{"title":"The rate and spectrum of new mutations in mice inferred by long-read sequencing","authors":"Eugenio López-Cortegano, Jobran Chebib, Anika Jonas, Anastasia Vock, Sven Künzel, Peter D. Keightley, Diethard Tautz","doi":"10.1101/gr.279982.124","DOIUrl":"https://doi.org/10.1101/gr.279982.124","url":null,"abstract":"All forms of genetic variation originate from new mutations, making it crucial to understand their rates and mechanisms. Here, we use long-read PacBio sequencing to investigate de novo mutations that accumulated in 12 inbred mouse lines derived from three commonly used inbred strains (C3H, C57BL/6, and FVB) maintained for 8-15 generations in a mutation accumulation (MA) experiment. We built chromosome-level genome assemblies based on the MA line founders' genomes, and then employed a combination of read and assembly-based methods to call the complete spectrum of new mutations. On average, there are ~45 mutations per haploid genome per generation, about half of which (54%) are insertions and deletions shorter than 50 bp (indels). The remainder are single nucleotide mutations (SNMs, 44%) and large structural mutations (SMs, 2%). We found that the degree of DNA repetitiveness is positively correlated with SNM and indel rates, and that a substantial fraction of SMs can be explained by homology-dependent mechanisms associated with repeat sequences. Most (90%) indels can be attributed to microsatellite contractions and expansions, and there is a marked bias towards 4 bp indels. Among the different types of SMs, tandem repeat mutations have the highest mutation rate, followed by insertions of transposable elements (TEs). We uncover a rich landscape of active TEs, and notable differences in their spectrum among MA lines and strains, and a high rate of gene retroposition. Our study offers novel insights into mammalian genome evolution, and highlights the importance of repetitive elements in shaping genomic diversity.","PeriodicalId":12678,"journal":{"name":"Genome research","volume":"45 1","pages":""},"PeriodicalIF":7.0,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142760310","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Fabien Wehbe, Levi Adams, Jordan Babadoudou, Samantha Yuen, Yoon-Seong Kim, Yoshiaki Tanaka
Application of single-cell/nucleus genomic sequencing to patient-derived tissues offers potential solutions to delineate disease mechanisms in human. However, individual cells in patient-derived tissues are in different pathological stages, and hence such cellular variability impedes subsequent differential gene expression analyses. To overcome such heterogeneity issue, we present a novel deep learning approach, scIDST, that infers disease progressive levels of individual cells with weak supervision framework. The inferred disease progressive cells displayed significant differential expression of disease-relevant genes, which could not be detected by comparative analysis between patients and healthy donors. In addition, we demonstrated that pretrained models by scIDST are applicable to multiple independent data resources, and advantageous to infer cells related to certain disease risks and comorbidities. Taken together, scIDST offers a new strategy of single-cell sequencing analysis to identify bona fide disease-associated molecular features.
{"title":"Inferring disease progressive stages in single-cell transcriptomics using a weakly-supervised deep learning approach","authors":"Fabien Wehbe, Levi Adams, Jordan Babadoudou, Samantha Yuen, Yoon-Seong Kim, Yoshiaki Tanaka","doi":"10.1101/gr.278812.123","DOIUrl":"https://doi.org/10.1101/gr.278812.123","url":null,"abstract":"Application of single-cell/nucleus genomic sequencing to patient-derived tissues offers potential solutions to delineate disease mechanisms in human. However, individual cells in patient-derived tissues are in different pathological stages, and hence such cellular variability impedes subsequent differential gene expression analyses. To overcome such heterogeneity issue, we present a novel deep learning approach, scIDST, that infers disease progressive levels of individual cells with weak supervision framework. The inferred disease progressive cells displayed significant differential expression of disease-relevant genes, which could not be detected by comparative analysis between patients and healthy donors. In addition, we demonstrated that pretrained models by scIDST are applicable to multiple independent data resources, and advantageous to infer cells related to certain disease risks and comorbidities. Taken together, scIDST offers a new strategy of single-cell sequencing analysis to identify bona fide disease-associated molecular features.","PeriodicalId":12678,"journal":{"name":"Genome research","volume":"32 1","pages":""},"PeriodicalIF":7.0,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142760320","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Thiago L. Knittel, Brooke E. Montgomery, Alex J. Tate, Ennis W. Deihl, Anastasia S. Nawrocki, Frederic J. Hoerndli, Taiowa A. Montgomery
Canonical small interfering RNAs (siRNAs) are processed from double-stranded RNA (dsRNA) by Dicer and associate with Argonautes to direct RNA silencing. In Caenorhabditis elegans, 22G-RNAs and 26G-RNAs are often referred to as siRNAs but display distinct characteristics. For example, 22G-RNAs do not originate from dsRNA and do not depend on Dicer, whereas 26G-RNAs require Dicer but derive from an atypical RNA duplex and are produced exclusively antisense to their messenger RNA (mRNA) templates. To identify canonical siRNAs in C. elegans, we first characterized the siRNAs produced via the exogenous RNA interference (RNAi) pathway. During RNAi, dsRNA is processed into ∼23 nt duplexes with ∼2 nt, 3′-overhangs, ultimately yielding siRNAs devoid of 5′G-containing sequences that bind with high affinity to the Argonaute RDE-1, but also to the microRNA (miRNA) pathway Argonaute, ALG-1. Using these characteristics, we searched for their endogenous counterparts and identified thousands of endogenous loci representing dozens of unique elements that give rise to mostly low to moderate levels of siRNAs, called 23H-RNAs. These loci include repetitive elements, putative coding genes, pseudogenes, noncoding RNAs, and unannotated features, many of which adopt hairpin (hp) structures reminiscent of the hpRNA/RNAi pathway in flies and mice. RDE-1 competes with other Argonautes for binding to 23H-RNAs. When RDE-1 is depleted, these siRNAs are enriched in ALG-1 and ALG-2 complexes. Our results expand the known repertoire of C. elegans small RNAs and their Argonaute interactors, and demonstrate that key features of the endogenous siRNA pathway are relatively unchanged in animals.
{"title":"A low-abundance class of Dicer-dependent siRNAs produced from a variety of features in C. elegans","authors":"Thiago L. Knittel, Brooke E. Montgomery, Alex J. Tate, Ennis W. Deihl, Anastasia S. Nawrocki, Frederic J. Hoerndli, Taiowa A. Montgomery","doi":"10.1101/gr.279083.124","DOIUrl":"https://doi.org/10.1101/gr.279083.124","url":null,"abstract":"Canonical small interfering RNAs (siRNAs) are processed from double-stranded RNA (dsRNA) by Dicer and associate with Argonautes to direct RNA silencing. In <em>Caenorhabditis elegans</em>, 22G-RNAs and 26G-RNAs are often referred to as siRNAs but display distinct characteristics. For example, 22G-RNAs do not originate from dsRNA and do not depend on Dicer, whereas 26G-RNAs require Dicer but derive from an atypical RNA duplex and are produced exclusively antisense to their messenger RNA (mRNA) templates. To identify canonical siRNAs in <em>C. elegans</em>, we first characterized the siRNAs produced via the exogenous RNA interference (RNAi) pathway. During RNAi, dsRNA is processed into ∼23 nt duplexes with ∼2 nt, 3′-overhangs, ultimately yielding siRNAs devoid of 5′G-containing sequences that bind with high affinity to the Argonaute RDE-1, but also to the microRNA (miRNA) pathway Argonaute, ALG-1. Using these characteristics, we searched for their endogenous counterparts and identified thousands of endogenous loci representing dozens of unique elements that give rise to mostly low to moderate levels of siRNAs, called 23H-RNAs. These loci include repetitive elements, putative coding genes, pseudogenes, noncoding RNAs, and unannotated features, many of which adopt hairpin (hp) structures reminiscent of the hpRNA/RNAi pathway in flies and mice. RDE-1 competes with other Argonautes for binding to 23H-RNAs. When RDE-1 is depleted, these siRNAs are enriched in ALG-1 and ALG-2 complexes. Our results expand the known repertoire of <em>C. elegans</em> small RNAs and their Argonaute interactors, and demonstrate that key features of the endogenous siRNA pathway are relatively unchanged in animals.","PeriodicalId":12678,"journal":{"name":"Genome research","volume":"45 1","pages":""},"PeriodicalIF":7.0,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142760361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Howard Womersley, Daniel Muliaditan, Ramanuj DasGupta, Lih Feng Cheow
Interrogating regulatory epigenetic alterations during tumor progression at the resolution of single cells has remained an understudied area of research. Here we developed a highly sensitive single-nucleus CUT&RUN (snCUT&RUN) assay to profile histone modifications in isogenic primary, metastatic, and cisplatin-resistant head and neck squamous cell carcinoma (HNSCC) patient-derived tumor cell lines. We find that the epigenome can be involved in diverse modes to contribute towards HNSCC progression. First, we demonstrate that gene expression changes during HNSCC progression can be comodulated by alterations in both copy number and chromatin activity, driving epigenetic rewiring of cell states. Furthermore, intratumour epigenetic heterogeneity (ITeH) may predispose subclonal populations within the primary tumour to adapt to selective pressures and foster the acquisition of malignant characteristics. In conclusion, snCUT&RUN serves as a valuable addition to the existing toolkit of single-cell epigenomic assays and can be used to dissect the functionality of the epigenome during cancer progression.
{"title":"Single-nucleus CUT&RUN elucidates the function of intrinsic and genomics-driven epigenetic heterogeneity in head and neck cancer progression","authors":"Howard Womersley, Daniel Muliaditan, Ramanuj DasGupta, Lih Feng Cheow","doi":"10.1101/gr.279105.124","DOIUrl":"https://doi.org/10.1101/gr.279105.124","url":null,"abstract":"Interrogating regulatory epigenetic alterations during tumor progression at the resolution of single cells has remained an understudied area of research. Here we developed a highly sensitive single-nucleus CUT&RUN (snCUT&RUN) assay to profile histone modifications in isogenic primary, metastatic, and cisplatin-resistant head and neck squamous cell carcinoma (HNSCC) patient-derived tumor cell lines. We find that the epigenome can be involved in diverse modes to contribute towards HNSCC progression. First, we demonstrate that gene expression changes during HNSCC progression can be comodulated by alterations in both copy number and chromatin activity, driving epigenetic rewiring of cell states. Furthermore, intratumour epigenetic heterogeneity (ITeH) may predispose subclonal populations within the primary tumour to adapt to selective pressures and foster the acquisition of malignant characteristics. In conclusion, snCUT&RUN serves as a valuable addition to the existing toolkit of single-cell epigenomic assays and can be used to dissect the functionality of the epigenome during cancer progression.","PeriodicalId":12678,"journal":{"name":"Genome research","volume":"13 1","pages":""},"PeriodicalIF":7.0,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142760655","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Amy R Vandiver, Allen Herbst, Paul Stothard, Jonathan Wanagat
While it is well understood that mitochondrial DNA (mtDNA) deletion mutations cause incurable diseases and contribute to aging, little is known about the transcriptional products that arise from these DNA structural variants. We hypothesized that mitochondrial genomes containing deletion mutations express chimeric mitochondrial RNAs. To test this, we analyzed human and rat RNA sequencing data to identify, quantitate, and characterize chimeric mitochondrial RNAs. We observed increased chimeric mitochondrial RNA frequency in samples from patients with mitochondrial genetic diseases and in samples from aged humans. The spectrum of chimeric mitochondrial transcripts reflected the known pattern of mtDNA deletion mutations. To test the hypothesis that mtDNA deletions induce chimeric RNA transcripts, we treated 18 mo and 34 mo rats with guanidinopropionic acid to induce high levels of skeletal muscle mtDNA deletion mutations. With mtDNA deletion induction, we demonstrate that the chimeric mitochondrial transcript frequency also increased and correlated strongly with an orthogonal DNA-based mutation assay performed on identical samples. Further, we show that the frequency of chimeric mitochondrial transcripts predicts expression of both nuclear and mitochondrial genes central to mitochondrial function, demonstrating the utility of these events as metrics of age-induced metabolic change. Mapping and quantitation of chimeric mitochondrial RNAs provides an accessible, orthogonal approach to DNA-based mutation assays, offers a potential method for identifying mitochondrial pathology in widely accessible datasets, and opens a new area of study in mitochondrial genetics and transcriptomics.
尽管线粒体 DNA（mtDNA）缺失突变会导致无法治愈的疾病并导致衰老，但人们对这些 DNA 结构变异产生的转录产物却知之甚少。我们假设，含有缺失突变的线粒体基因组会表达嵌合线粒体 RNA。为了验证这一假设，我们分析了人类和大鼠的 RNA 测序数据，以识别、定量和描述嵌合线粒体 RNA。我们观察到，在线粒体遗传疾病患者的样本和老年人的样本中，嵌合线粒体 RNA 的频率有所增加。嵌合线粒体转录本的频谱反映了已知的 mtDNA 缺失突变模式。为了验证 mtDNA 缺失会诱导嵌合 RNA 转录本的假设，我们用胍基丙酸处理了 18 个月和 34 个月的大鼠，以诱导高水平的骨骼肌 mtDNA 缺失突变。随着 mtDNA 缺失的诱导，我们发现嵌合线粒体转录本的频率也在增加，并且与在相同样本上进行的基于 DNA 的正交突变检测密切相关。此外，我们还发现嵌合线粒体转录本的频率可以预测线粒体功能的核心核基因和线粒体基因的表达情况，从而证明了这些事件作为年龄诱导的代谢变化指标的实用性。嵌合线粒体 RNA 的制图和定量为基于 DNA 的突变检测提供了一种便捷、正交的方法，为在广泛获取的数据集中识别线粒体病理学提供了一种潜在的方法，并为线粒体遗传学和转录组学的研究开辟了一个新的领域。
{"title":"Chimeric mitochondrial RNA transcripts predict mitochondrial genome deletion mutations in mitochondrial genetic diseases and aging","authors":"Amy R Vandiver, Allen Herbst, Paul Stothard, Jonathan Wanagat","doi":"10.1101/gr.279072.124","DOIUrl":"https://doi.org/10.1101/gr.279072.124","url":null,"abstract":"While it is well understood that mitochondrial DNA (mtDNA) deletion mutations cause incurable diseases and contribute to aging, little is known about the transcriptional products that arise from these DNA structural variants. We hypothesized that mitochondrial genomes containing deletion mutations express chimeric mitochondrial RNAs. To test this, we analyzed human and rat RNA sequencing data to identify, quantitate, and characterize chimeric mitochondrial RNAs. We observed increased chimeric mitochondrial RNA frequency in samples from patients with mitochondrial genetic diseases and in samples from aged humans. The spectrum of chimeric mitochondrial transcripts reflected the known pattern of mtDNA deletion mutations. To test the hypothesis that mtDNA deletions induce chimeric RNA transcripts, we treated 18 mo and 34 mo rats with guanidinopropionic acid to induce high levels of skeletal muscle mtDNA deletion mutations. With mtDNA deletion induction, we demonstrate that the chimeric mitochondrial transcript frequency also increased and correlated strongly with an orthogonal DNA-based mutation assay performed on identical samples. Further, we show that the frequency of chimeric mitochondrial transcripts predicts expression of both nuclear and mitochondrial genes central to mitochondrial function, demonstrating the utility of these events as metrics of age-induced metabolic change. Mapping and quantitation of chimeric mitochondrial RNAs provides an accessible, orthogonal approach to DNA-based mutation assays, offers a potential method for identifying mitochondrial pathology in widely accessible datasets, and opens a new area of study in mitochondrial genetics and transcriptomics.","PeriodicalId":12678,"journal":{"name":"Genome research","volume":"25 1","pages":""},"PeriodicalIF":7.0,"publicationDate":"2024-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142718243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shuai Guo, Xiaoqian Liu, Xuesen Cheng, Yujie Jiang, Shuangxi Ji, Qingnan Liang, Andrew Koval, Yumei Li, Leah A. Owen, Ivana K. Kim, Ana Aparicio, Sanghoon Lee, Anil K. Sood, Scott Kopetz, John Paul Shen, John N. Weinstein, Margaret M. DeAngelis, Rui Chen, Wenyi Wang
Bulk deconvolution with single-cell/nucleus RNA-seq data is critical for understanding heterogeneity in complex biological samples, yet the technological discrepancy across sequencing platforms limits deconvolution accuracy. To address this, we utilize an experimental design to match inter-platform biological signals, hence revealing the technological discrepancy, and then develop a deconvolution framework called DeMixSC using this well-matched, i.e., benchmark, data. Built upon a novel weighted nonnegative least-squares framework, DeMixSC identifies and adjusts genes with high technological discrepancy and aligns the benchmark data with large patient cohorts of matched-tissue-type for large-scale deconvolution. Our results using two benchmark datasets of healthy retinas and ovarian cancer tissues suggest much-improved deconvolution accuracy. Leveraging tissue-specific benchmark datasets, we applied DeMixSC to a large cohort of 453 age-related macular degeneration patients and a cohort of 30 ovarian cancer patients with various responses to neoadjuvant chemotherapy. Only DeMixSC successfully unveiled biologically meaningful differences across patient groups, demonstrating its broad applicability in diverse real-world clinical scenarios. Our findings reveal the impact of technological discrepancy on deconvolution performance and underscore the importance of a well-matched dataset to resolve this challenge. The developed DeMixSC framework is generally applicable for accurately deconvolving large cohorts of disease tissues, including cancers, when a well-matched benchmark dataset is available.
{"title":"A deconvolution framework that uses single-cell sequencing plus a small benchmark dataset for accurate analysis of cell type ratios in complex tissue samples","authors":"Shuai Guo, Xiaoqian Liu, Xuesen Cheng, Yujie Jiang, Shuangxi Ji, Qingnan Liang, Andrew Koval, Yumei Li, Leah A. Owen, Ivana K. Kim, Ana Aparicio, Sanghoon Lee, Anil K. Sood, Scott Kopetz, John Paul Shen, John N. Weinstein, Margaret M. DeAngelis, Rui Chen, Wenyi Wang","doi":"10.1101/gr.278822.123","DOIUrl":"https://doi.org/10.1101/gr.278822.123","url":null,"abstract":"Bulk deconvolution with single-cell/nucleus RNA-seq data is critical for understanding heterogeneity in complex biological samples, yet the technological discrepancy across sequencing platforms limits deconvolution accuracy. To address this, we utilize an experimental design to match inter-platform biological signals, hence revealing the technological discrepancy, and then develop a deconvolution framework called DeMixSC using this well-matched, i.e., benchmark, data. Built upon a novel weighted nonnegative least-squares framework, DeMixSC identifies and adjusts genes with high technological discrepancy and aligns the benchmark data with large patient cohorts of matched-tissue-type for large-scale deconvolution. Our results using two benchmark datasets of healthy retinas and ovarian cancer tissues suggest much-improved deconvolution accuracy. Leveraging tissue-specific benchmark datasets, we applied DeMixSC to a large cohort of 453 age-related macular degeneration patients and a cohort of 30 ovarian cancer patients with various responses to neoadjuvant chemotherapy. Only DeMixSC successfully unveiled biologically meaningful differences across patient groups, demonstrating its broad applicability in diverse real-world clinical scenarios. Our findings reveal the impact of technological discrepancy on deconvolution performance and underscore the importance of a well-matched dataset to resolve this challenge. The developed DeMixSC framework is generally applicable for accurately deconvolving large cohorts of disease tissues, including cancers, when a well-matched benchmark dataset is available.","PeriodicalId":12678,"journal":{"name":"Genome research","volume":"35 1","pages":""},"PeriodicalIF":7.0,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142712790","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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