Pub Date : 2024-09-09DOI: 10.1101/2024.09.09.612068
Amy Longtin, Marina M Watowich, Baptiste Sadoughi, Rachel Petersen, Sarah F Brosnan, Kenneth Buetow, Qiuyin Cai, Michael D Gurven, Heather M Highland, Yi-Ting Huang, Hillard Kaplan, Thomas S Kraft, Yvonne A L Lim, Jirong Long, Amanda D Melin, Jamie Roberson, Kee-Seong Ng, Jonathan Stieglitz, Benjamin C Trumble, Vivek V Venkataraman, Ian J Wallace, Jie Wu, Noah Snyder-Mackler, Angela Jones, Alexander G Bick, Amanda Lea
Characterizing DNA methylation patterns is important for addressing key questions in evolutionary biology, geroscience, and medical genomics. While costs are decreasing, whole-genome DNA methylation profiling remains prohibitively expensive for most population-scale studies, creating a need for cost-effective, reduced representation approaches (i.e., assays that rely on microarrays, enzyme digests, or sequence capture to target a subset of the genome). Most common whole genome and reduced representation techniques rely on bisulfite conversion, which can damage DNA resulting in DNA loss and sequencing biases. Enzymatic methyl sequencing (EM-seq) was recently proposed to overcome these issues, but thorough benchmarking of EM-seq combined with cost-effective, reduced representation strategies has not yet been performed. To do so, we optimized Targeted Methylation Sequencing protocol (TMS)-which profiles ~4 million CpG sites-for miniaturization, flexibility, and multispecies use at a cost of ~$80. First, we tested modifications to increase throughput and reduce cost, including increasing multiplexing, decreasing DNA input, and using enzymatic rather than mechanical fragmentation to prepare DNA. Second, we compared our optimized TMS protocol to commonly used techniques, specifically the Infinium MethylationEPIC BeadChip (n=55 paired samples) and whole genome bisulfite sequencing (n=6 paired samples). In both cases, we found strong agreement between technologies (R-squared = 0.97 and 0.99, respectively). Third, we tested the optimized TMS protocol in three non-human primate species (rhesus macaques, geladas, and capuchins). We captured a high percentage (mean=77.1%) of targeted CpG sites and produced methylation level estimates that agreed with those generated from reduced representation bisulfite sequencing (R-squared = 0.98). Finally, we applied our protocol to profile age-associated DNA methylation variation in two subsistence-level populations-the Tsimane of lowland Bolivia and the Orang Asli of Peninsular Malaysia-and found age-methylation patterns that were strikingly similar to those reported in high income cohorts, despite known differences in age-health relationships between lifestyle contexts. Altogether, our optimized TMS protocol will enable cost-effective, population-scale studies of genome-wide DNA methylation levels across human and non-human primate species.
描述 DNA 甲基化模式对于解决进化生物学、遗传科学和医学基因组学中的关键问题非常重要。虽然成本在不断降低，但全基因组 DNA 甲基化分析对于大多数群体规模的研究来说仍然过于昂贵，因此需要具有成本效益的缩减表示方法（即依赖微阵列、酶消化或序列捕获以基因组子集为目标的检测方法）。大多数常见的全基因组和缩减表征技术都依赖于亚硫酸氢盐转换，而亚硫酸氢盐转换会损伤 DNA，导致 DNA 丢失和测序偏差。最近提出的酶促甲基测序（EM-seq）克服了这些问题，但尚未对 EM-seq 与具有成本效益的缩减表示策略相结合进行全面的基准测试。为此，我们对靶向甲基化测序协议（TMS）进行了优化，该协议以约 80 美元的成本剖析了约 400 万个 CpG 位点，实现了微型化、灵活性和多物种使用。首先，我们测试了提高通量和降低成本的方法，包括增加多路复用、减少 DNA 输入量以及使用酶而不是机械破碎法制备 DNA。其次，我们将优化的 TMS 方案与常用技术进行了比较，特别是 Infinium MethylationEPIC BeadChip（55 个配对样本）和全基因组亚硫酸氢盐测序（6 个配对样本）。在这两种情况下，我们发现技术之间的一致性很强（R-平方分别为 0.97 和 0.99）。第三，我们在三个非人灵长类物种（猕猴、狮尾狒和卷尾猴）中测试了优化的 TMS 方案。我们捕获了很高比例（平均=77.1%）的目标 CpG 位点，并得出了甲基化水平估计值，该估计值与还原表征亚硫酸氢盐测序得出的估计值一致（R 平方=0.98）。最后，我们应用我们的方案分析了两个自给自足人群（玻利维亚低地的 Tsimane 人和马来西亚半岛的 Orang Asli 人）中与年龄相关的 DNA 甲基化变异，发现年龄甲基化模式与高收入人群中报告的模式惊人地相似，尽管已知不同生活方式背景下的年龄-健康关系存在差异。总之，我们优化的 TMS 方案将能够对人类和非人灵长类物种的全基因组 DNA 甲基化水平进行具有成本效益的群体规模研究。
{"title":"Cost-effective solutions for high-throughput enzymatic DNA methylation sequencing","authors":"Amy Longtin, Marina M Watowich, Baptiste Sadoughi, Rachel Petersen, Sarah F Brosnan, Kenneth Buetow, Qiuyin Cai, Michael D Gurven, Heather M Highland, Yi-Ting Huang, Hillard Kaplan, Thomas S Kraft, Yvonne A L Lim, Jirong Long, Amanda D Melin, Jamie Roberson, Kee-Seong Ng, Jonathan Stieglitz, Benjamin C Trumble, Vivek V Venkataraman, Ian J Wallace, Jie Wu, Noah Snyder-Mackler, Angela Jones, Alexander G Bick, Amanda Lea","doi":"10.1101/2024.09.09.612068","DOIUrl":"https://doi.org/10.1101/2024.09.09.612068","url":null,"abstract":"Characterizing DNA methylation patterns is important for addressing key questions in evolutionary biology, geroscience, and medical genomics. While costs are decreasing, whole-genome DNA methylation profiling remains prohibitively expensive for most population-scale studies, creating a need for cost-effective, reduced representation approaches (i.e., assays that rely on microarrays, enzyme digests, or sequence capture to target a subset of the genome). Most common whole genome and reduced representation techniques rely on bisulfite conversion, which can damage DNA resulting in DNA loss and sequencing biases. Enzymatic methyl sequencing (EM-seq) was recently proposed to overcome these issues, but thorough benchmarking of EM-seq combined with cost-effective, reduced representation strategies has not yet been performed. To do so, we optimized Targeted Methylation Sequencing protocol (TMS)-which profiles ~4 million CpG sites-for miniaturization, flexibility, and multispecies use at a cost of ~$80. First, we tested modifications to increase throughput and reduce cost, including increasing multiplexing, decreasing DNA input, and using enzymatic rather than mechanical fragmentation to prepare DNA. Second, we compared our optimized TMS protocol to commonly used techniques, specifically the Infinium MethylationEPIC BeadChip (n=55 paired samples) and whole genome bisulfite sequencing (n=6 paired samples). In both cases, we found strong agreement between technologies (R-squared = 0.97 and 0.99, respectively). Third, we tested the optimized TMS protocol in three non-human primate species (rhesus macaques, geladas, and capuchins). We captured a high percentage (mean=77.1%) of targeted CpG sites and produced methylation level estimates that agreed with those generated from reduced representation bisulfite sequencing (R-squared = 0.98). Finally, we applied our protocol to profile age-associated DNA methylation variation in two subsistence-level populations-the Tsimane of lowland Bolivia and the Orang Asli of Peninsular Malaysia-and found age-methylation patterns that were strikingly similar to those reported in high income cohorts, despite known differences in age-health relationships between lifestyle contexts. Altogether, our optimized TMS protocol will enable cost-effective, population-scale studies of genome-wide DNA methylation levels across human and non-human primate species.","PeriodicalId":501161,"journal":{"name":"bioRxiv - Genomics","volume":"67 5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214116","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 : 2024-09-09DOI: 10.1101/2024.09.06.611585
Yanhai Gong, Qintao Wang, Li Wei, Lianhong Wang, Nana Lv, Xuefeng Du, Chen Shen, Yi Xin, Luyang Sun, Jian Xu
Despite their ecological and physiological significance, how carbon-concentrating mechanisms (CCM) are regulated in microalgae remains elusive. Here in the model industrial microalga Nannochloropsis oceanica, we uncovered an epigenetic regulatory mechanism for CCM via comprehensive, multi-dimensional epigenomic analyses. Our integrated study reveals the complex interplay among histone modifications, dynamic nucleosome positioning, and 3D chromatin structure in regulating gene expression during low CO2 adaptation, despite minimal DNA methylation. Histone modifications, including lysine acetylation (H3K9ac and H3K27ac), crotonylation (Kcr), and methylation (H3K4me2), were associated with active chromatin states. Significantly altered ChIP-Seq peaks were linked to 43.1% of the differentially expressed genes (DEGs). Notably, H3K4me2 exhibited a distinct dual-peak profile around the transcription start site (TSS), which is unique among microalgae and plants. Chromatin compartment dynamics were correlated with gene expression and histone modifications, particularly H3K4me2, while differentially positioned nucleosomes were associated with key CCM-related genes and transcription factors. To further elucidate the role of H3K4me2, we knocked out its methyltransferase, resulting in genome-wide H3K4me2 peak shifts, slower growth, and reduced photosynthesis. These changes were accompanied by differential expression of key genes of NoHINT and NoPMA2, whose subsequent deletion and overexpression revealed their subtle yet significant impacts on growth and photosynthetic efficiency under low CO2 conditions, with NoHINT regulating growth and NoPMA2 influencing photosynthesis. Finally, we proposed a comprehensive model for epigenetic regulation of CCM in N. oceanica, which established a foundation for enhancing microalgal productivity through targeted epigenetic modifications.
{"title":"Dynamic epigenomic landscape of carbon-concentrating mechanisms in the model industrial oleaginous microalga Nannochloropsis oceanica","authors":"Yanhai Gong, Qintao Wang, Li Wei, Lianhong Wang, Nana Lv, Xuefeng Du, Chen Shen, Yi Xin, Luyang Sun, Jian Xu","doi":"10.1101/2024.09.06.611585","DOIUrl":"https://doi.org/10.1101/2024.09.06.611585","url":null,"abstract":"Despite their ecological and physiological significance, how carbon-concentrating mechanisms (CCM) are regulated in microalgae remains elusive. Here in the model industrial microalga <em>Nannochloropsis oceanica</em>, we uncovered an epigenetic regulatory mechanism for CCM via comprehensive, multi-dimensional epigenomic analyses. Our integrated study reveals the complex interplay among histone modifications, dynamic nucleosome positioning, and 3D chromatin structure in regulating gene expression during low CO<sub>2</sub> adaptation, despite minimal DNA methylation. Histone modifications, including lysine acetylation (H3K9ac and H3K27ac), crotonylation (Kcr), and methylation (H3K4me2), were associated with active chromatin states. Significantly altered ChIP-Seq peaks were linked to 43.1% of the differentially expressed genes (DEGs). Notably, H3K4me2 exhibited a distinct dual-peak profile around the transcription start site (TSS), which is unique among microalgae and plants. Chromatin compartment dynamics were correlated with gene expression and histone modifications, particularly H3K4me2, while differentially positioned nucleosomes were associated with key CCM-related genes and transcription factors. To further elucidate the role of H3K4me2, we knocked out its methyltransferase, resulting in genome-wide H3K4me2 peak shifts, slower growth, and reduced photosynthesis. These changes were accompanied by differential expression of key genes of NoHINT and NoPMA2, whose subsequent deletion and overexpression revealed their subtle yet significant impacts on growth and photosynthetic efficiency under low CO2 conditions, with NoHINT regulating growth and NoPMA2 influencing photosynthesis. Finally, we proposed a comprehensive model for epigenetic regulation of CCM in <em>N. oceanica</em>, which established a foundation for enhancing microalgal productivity through targeted epigenetic modifications.","PeriodicalId":501161,"journal":{"name":"bioRxiv - Genomics","volume":"36 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142227015","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 : 2024-09-09DOI: 10.1101/2024.09.09.612085
Basheer Becerra, Sandra Wittibschlager, Zain Munir Patel, Ana Kutschat, Justin Delano, Anzhelika Karjalainen, Ting Wu, Marlena Starrs, Martin Jankowiak, Daniel Bauer, Davide Seruggia, Luca Pinello
CRISPR tiling screens have advanced the identification and characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing. This study introduces CRISPR-CLEAR, an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. This approach enables the dissection of regulatory elements at nucleotide resolution, facilitating a direct assessment of genotype-phenotype effects.
{"title":"CRISPR-CLEAR: Nucleotide-Resolution Mapping of Regulatory Elements via Allelic Readout of Tiled Base Editing","authors":"Basheer Becerra, Sandra Wittibschlager, Zain Munir Patel, Ana Kutschat, Justin Delano, Anzhelika Karjalainen, Ting Wu, Marlena Starrs, Martin Jankowiak, Daniel Bauer, Davide Seruggia, Luca Pinello","doi":"10.1101/2024.09.09.612085","DOIUrl":"https://doi.org/10.1101/2024.09.09.612085","url":null,"abstract":"CRISPR tiling screens have advanced the identification and characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing. This study introduces CRISPR-CLEAR, an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. This approach enables the dissection of regulatory elements at nucleotide resolution, facilitating a direct assessment of genotype-phenotype effects.","PeriodicalId":501161,"journal":{"name":"bioRxiv - Genomics","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214119","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 : 2024-09-09DOI: 10.1101/2024.09.09.611954
Luis Cabrera-Sosa, Mahdi Safarpour, Johanna H Kattenberg, Roberson Ramirez, Joseph M. Vinetz, Anna Rosanas-Urgell, Dionicia Gamboa, Christopher Delgado-Ratto
Malaria molecular surveillance (MMS) can provide insights into transmission dynamics, guiding national control/elimination programs. Considering the genetic differences among parasites from different areas in the Peruvian Amazon, we previously designed SNP barcode panels for Plasmodium vivax (Pv) and P. falciparum (Pf), integrated into AmpliSeq assays, to provide population genetics estimates of malaria parasites. These AmpliSeq assays are ideal for MMS: multiplexing different traits of interest, applicable to many use cases, and high throughput for large numbers of samples. The present study compares the genetic resolution of the SNP barcode panels in the AmpliSeq assays with widely used microsatellite (MS) panels to investigate Amazonian malaria parasites. Malaria samples collected in remote areas of the Peruvian Amazon (51 Pv & 80 Pf samples) were characterized using the Ampliseq assays and MS. Population genetics estimates (complexity of infection, genetic diversity and differentiation, and population structure) were compared using the SNP barcodes (Pv: 40 SNPs & Pf: 28 SNPs) and MS panels (Pv: 16 MS & Pf: 7 MS). The genetic diversity of Pv (expected heterozygosity, He) was similar across the subpopulations for both makers: HeMS = 0.68 – 0.78 (p = 0.23) and HeSNP = 0.36 – 0.38 (p = 0.80). Pairwise genetic differentiation (fixation index, FST) was also comparable: FST-MS = 0.04 – 0.14 and FST-SNP = 0.03 – 0.12 (p = 0.34 – 0.85). No geographic clustering was observed with any panel. In addition, Pf genetic diversity trends (HeMS = 0 – 0.48 p = 0.03 – 1; HeSNP = 0 – 0.09, p = 0.03 – 1) and pairwise FST comparisons (FST-MS = 0.14 – 0.65, FST-SNP = 0.19 – 0.61, p = 0.24 – 0.83) were concordant between the panels. Similar population structure clustering was observed with both SNP and MS, highlighting one Pf subpopulation in an indigenous community. The SNP barcodes in the Pv AmpliSeq v2 Peru and Pf AmpliSeq v1 Peru assays offer comparable results to MS panels when investigating population genetics in Pv and Pv populations. Therefore, the AmpliSeq assays can efficiently characterize malaria transmission dynamics and population structure and support malaria elimination efforts in Peru.
{"title":"Comparing newly developed SNP barcode panels with microsatellites to explore population genetics of malaria parasites in the Peruvian Amazon","authors":"Luis Cabrera-Sosa, Mahdi Safarpour, Johanna H Kattenberg, Roberson Ramirez, Joseph M. Vinetz, Anna Rosanas-Urgell, Dionicia Gamboa, Christopher Delgado-Ratto","doi":"10.1101/2024.09.09.611954","DOIUrl":"https://doi.org/10.1101/2024.09.09.611954","url":null,"abstract":"Malaria molecular surveillance (MMS) can provide insights into transmission dynamics, guiding national control/elimination programs. Considering the genetic differences among parasites from different areas in the Peruvian Amazon, we previously designed SNP barcode panels for <em>Plasmodium vivax</em> (Pv) and <em>P. falciparum</em> (Pf), integrated into AmpliSeq assays, to provide population genetics estimates of malaria parasites. These AmpliSeq assays are ideal for MMS: multiplexing different traits of interest, applicable to many use cases, and high throughput for large numbers of samples. The present study compares the genetic resolution of the SNP barcode panels in the AmpliSeq assays with widely used microsatellite (MS) panels to investigate Amazonian malaria parasites. Malaria samples collected in remote areas of the Peruvian Amazon (51 Pv & 80 Pf samples) were characterized using the Ampliseq assays and MS. Population genetics estimates (complexity of infection, genetic diversity and differentiation, and population structure) were compared using the SNP barcodes (Pv: 40 SNPs & Pf: 28 SNPs) and MS panels (Pv: 16 MS & Pf: 7 MS). The genetic diversity of Pv (expected heterozygosity, He) was similar across the subpopulations for both makers: He<sub>MS</sub> = 0.68 – 0.78 (p = 0.23) and He<sub>SNP</sub> = 0.36 – 0.38 (p = 0.80). Pairwise genetic differentiation (fixation index, F<sub>ST</sub>) was also comparable: F<sub>ST-MS</sub> = 0.04 – 0.14 and F<sub>ST-SNP</sub> = 0.03 – 0.12 (p = 0.34 – 0.85). No geographic clustering was observed with any panel. In addition, Pf genetic diversity trends (He<sub>MS</sub> = 0 – 0.48 p = 0.03 – 1; He<sub>SNP</sub> = 0 – 0.09, p = 0.03 – 1) and pairwise FST comparisons (F<sub>ST-MS</sub> = 0.14 – 0.65, F<sub>ST-SNP</sub> = 0.19 – 0.61, p = 0.24 – 0.83) were concordant between the panels. Similar population structure clustering was observed with both SNP and MS, highlighting one Pf subpopulation in an indigenous community. The SNP barcodes in the Pv AmpliSeq v2 Peru and Pf AmpliSeq v1 Peru assays offer comparable results to MS panels when investigating population genetics in Pv and Pv populations. Therefore, the AmpliSeq assays can efficiently characterize malaria transmission dynamics and population structure and support malaria elimination efforts in Peru.","PeriodicalId":501161,"journal":{"name":"bioRxiv - Genomics","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214117","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 : 2024-09-08DOI: 10.1101/2024.09.06.611737
Michael Kosicki, Boyang Zhang, Anusri Pampari, Jennifer A Akiyama, Ingrid Playzer-Frick, Catherine S Novak, Stella Tran, Yiwen Zhu, Momoe Kato, Riana D Hunter, Kianna von Maydell, Sarah Barton, Erik Beckman, Anshul Kundaje, Diane E Dickel, Axel Visel, Len A Pennacchio
Distant-acting enhancers are central to human development. However, our limited understanding of their functional sequence features prevents the interpretation of enhancer mutations in disease. Here, we determined the functional sensitivity to mutagenesis of human developmental enhancers in vivo. Focusing on seven enhancers active in the developing brain, heart, limb and face, we created over 1700 transgenic mice for over 260 mutagenized enhancer alleles. Systematic mutation of 12-basepair blocks collectively altered each sequence feature in each enhancer at least once. We show that 69% of all blocks are required for normal in vivo activity, with mutations more commonly resulting in loss (60%) than in gain (9%) of function. Using predictive modeling, we annotated critical nucleotides at base-pair resolution. The vast majority of motifs predicted by these machine learning models (88%) coincided with changes to in vivo function, and the models showed considerable sensitivity, identifying 59% of all functional blocks. Taken together, our results reveal that human enhancers contain a high density of sequence features required for their normal in vivo function and provide a rich resource for further exploration of human enhancer logic.
{"title":"Mutagenesis Sensitivity Mapping of Human Enhancers In Vivo","authors":"Michael Kosicki, Boyang Zhang, Anusri Pampari, Jennifer A Akiyama, Ingrid Playzer-Frick, Catherine S Novak, Stella Tran, Yiwen Zhu, Momoe Kato, Riana D Hunter, Kianna von Maydell, Sarah Barton, Erik Beckman, Anshul Kundaje, Diane E Dickel, Axel Visel, Len A Pennacchio","doi":"10.1101/2024.09.06.611737","DOIUrl":"https://doi.org/10.1101/2024.09.06.611737","url":null,"abstract":"Distant-acting enhancers are central to human development. However, our limited understanding of their functional sequence features prevents the interpretation of enhancer mutations in disease. Here, we determined the functional sensitivity to mutagenesis of human developmental enhancers in vivo. Focusing on seven enhancers active in the developing brain, heart, limb and face, we created over 1700 transgenic mice for over 260 mutagenized enhancer alleles. Systematic mutation of 12-basepair blocks collectively altered each sequence feature in each enhancer at least once. We show that 69% of all blocks are required for normal in vivo activity, with mutations more commonly resulting in loss (60%) than in gain (9%) of function. Using predictive modeling, we annotated critical nucleotides at base-pair resolution. The vast majority of motifs predicted by these machine learning models (88%) coincided with changes to in vivo function, and the models showed considerable sensitivity, identifying 59% of all functional blocks. Taken together, our results reveal that human enhancers contain a high density of sequence features required for their normal in vivo function and provide a rich resource for further exploration of human enhancer logic.","PeriodicalId":501161,"journal":{"name":"bioRxiv - Genomics","volume":"38 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142213863","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 : 2024-09-08DOI: 10.1101/2024.09.04.611279
Jing Dong, Michael T Zimmermann, Neshatul Haque, Shahram Arsang-Jang, Wael Saber, Raul A Urrutia
Mitochondria are essential cellular organelles that play critical roles in hematological disorders. Recurrent mutations in mitochondrial DNA (mtDNA) have been identified in patients with myelodysplastic syndromes (MDS) and serve as significant prognostic indicators for their outcomes following allogeneic hematopoietic stem-cell transplantation (allo-HCT). However, the biological mechanisms of mtDNA mutations remain unclear. The current study utilizes computational structural genomics to improve our understanding of pathogenic variants in mitochondria-encoded genes. This emerging genomics discipline employs structural models, molecular mechanic calculations, and accelerated molecular dynamic simulations to analyze gene products, focusing on their structures and motions that determine their function. We applied this methodology to perform deep variant phenotyping of entire mitochondria-encoded protein complexes associated with the pathobiology of MDS and their prognosis after HCT. Our results demonstrate that this approach significantly outperforms conventional analytical methods, providing enhanced and more accurate information to support the potential pathogenicity of these variants and better infer their dysfunctional mechanisms. We conclude that the adoption and further expansion of computational structural genomics approaches, as applied to the mitochondrial genome, have the potential to significantly increase our understanding of molecular mechanisms underlying the disease. Our study lays a foundation for translating mitochondrial biology into clinical applications, which is of significant mechanistic and biomedical relevance and should be considered in modern biomedical research.
{"title":"Advancing Variant Phenotyping in Myelodysplastic Syndromes via Computational Genomics of Mitochondrial Enzyme Complexes","authors":"Jing Dong, Michael T Zimmermann, Neshatul Haque, Shahram Arsang-Jang, Wael Saber, Raul A Urrutia","doi":"10.1101/2024.09.04.611279","DOIUrl":"https://doi.org/10.1101/2024.09.04.611279","url":null,"abstract":"Mitochondria are essential cellular organelles that play critical roles in hematological disorders. Recurrent mutations in mitochondrial DNA (mtDNA) have been identified in patients with myelodysplastic syndromes (MDS) and serve as significant prognostic indicators for their outcomes following allogeneic hematopoietic stem-cell transplantation (allo-HCT). However, the biological mechanisms of mtDNA mutations remain unclear. The current study utilizes computational structural genomics to improve our understanding of pathogenic variants in mitochondria-encoded genes. This emerging genomics discipline employs structural models, molecular mechanic calculations, and accelerated molecular dynamic simulations to analyze gene products, focusing on their structures and motions that determine their function. We applied this methodology to perform deep variant phenotyping of entire mitochondria-encoded protein complexes associated with the pathobiology of MDS and their prognosis after HCT. Our results demonstrate that this approach significantly outperforms conventional analytical methods, providing enhanced and more accurate information to support the potential pathogenicity of these variants and better infer their dysfunctional mechanisms. We conclude that the adoption and further expansion of computational structural genomics approaches, as applied to the mitochondrial genome, have the potential to significantly increase our understanding of molecular mechanisms underlying the disease. Our study lays a foundation for translating mitochondrial biology into clinical applications, which is of significant mechanistic and biomedical relevance and should be considered in modern biomedical research.","PeriodicalId":501161,"journal":{"name":"bioRxiv - Genomics","volume":"71 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142213865","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 : 2024-09-08DOI: 10.1101/2024.09.06.611599
Jolet Yvette Mimpen, Mathew J Baldwin, Claudia Paul, Lorenzo Ramos-Mucci, Alina Kurjan, Carla J Cohen, Shreeya Sharma, Marie S.N. Chevalier Florquin, Philippa A Hulley, John McMaster, Andrew Titchener, Alexander Martin, Matthew L Costa, Stephen E Gwilym, Adam P Cribbs, Sarah J.B. Snelling
Tendon ruptures in humans have regularly been studied during the chronic phase of injury. However, the early response to injury remains less investigated. Quadriceps tendons, which require prompt surgical treatment, offer a model to investigate this early response. Therefore, this study aimed to explore the early cellular changes in ruptured compared to healthy human quadriceps tendons. Quadriceps tendon samples were collected from patients undergoing tibial shaft fracture repair (healthy) or tendon repair surgery for complete rupture (collected 7-8 days post-injury). Nuclei were isolated for single-nucleus RNA sequencing, and comprehensive transcriptomic analysis was conducted to profile cellular changes. The transcriptomes of 12,808 nuclei were profiled, including 7,268 nuclei from healthy and 5,540 nuclei from ruptured quadriceps tendons, revealing 12 major cell types and several cell subtypes and states. Rupture samples showed increased expression of genes related to extracellular matrix organisation and cell cycle signalling, and a decrease in expression of genes in lipid metabolism pathways. These changes were driven predominantly by gene expression changes in the fibroblast, vascular endothelial cells (VECs), mural cell, and macrophage populations: fibroblasts shift to an activated phenotype upon rupture and there is an increase in proportion of capillary and dividing VECs, suggesting an angiogenic response. A diverse immune environment was observed, with a shift from homeostatic to activated macrophages following rupture. Cell-cell interactions increased in rupture, both in their number and diversity, and primarily involving fibroblast and endothelial cell populations. Collectively, this transcriptomic analysis suggests that fibroblasts and endothelial cells are key orchestrators of the early injury response within ruptured quadriceps tendon.
{"title":"Exploring cellular changes in ruptured human quadriceps tendons at single-cell resolution","authors":"Jolet Yvette Mimpen, Mathew J Baldwin, Claudia Paul, Lorenzo Ramos-Mucci, Alina Kurjan, Carla J Cohen, Shreeya Sharma, Marie S.N. Chevalier Florquin, Philippa A Hulley, John McMaster, Andrew Titchener, Alexander Martin, Matthew L Costa, Stephen E Gwilym, Adam P Cribbs, Sarah J.B. Snelling","doi":"10.1101/2024.09.06.611599","DOIUrl":"https://doi.org/10.1101/2024.09.06.611599","url":null,"abstract":"Tendon ruptures in humans have regularly been studied during the chronic phase of injury. However, the early response to injury remains less investigated. Quadriceps tendons, which require prompt surgical treatment, offer a model to investigate this early response. Therefore, this study aimed to explore the early cellular changes in ruptured compared to healthy human quadriceps tendons. Quadriceps tendon samples were collected from patients undergoing tibial shaft fracture repair (healthy) or tendon repair surgery for complete rupture (collected 7-8 days post-injury). Nuclei were isolated for single-nucleus RNA sequencing, and comprehensive transcriptomic analysis was conducted to profile cellular changes. The transcriptomes of 12,808 nuclei were profiled, including 7,268 nuclei from healthy and 5,540 nuclei from ruptured quadriceps tendons, revealing 12 major cell types and several cell subtypes and states. Rupture samples showed increased expression of genes related to extracellular matrix organisation and cell cycle signalling, and a decrease in expression of genes in lipid metabolism pathways. These changes were driven predominantly by gene expression changes in the fibroblast, vascular endothelial cells (VECs), mural cell, and macrophage populations: fibroblasts shift to an activated phenotype upon rupture and there is an increase in proportion of capillary and dividing VECs, suggesting an angiogenic response. A diverse immune environment was observed, with a shift from homeostatic to activated macrophages following rupture. Cell-cell interactions increased in rupture, both in their number and diversity, and primarily involving fibroblast and endothelial cell populations. Collectively, this transcriptomic analysis suggests that fibroblasts and endothelial cells are key orchestrators of the early injury response within ruptured quadriceps tendon.","PeriodicalId":501161,"journal":{"name":"bioRxiv - Genomics","volume":"46 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142227016","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 : 2024-09-08DOI: 10.1101/2024.09.04.611281
Xingzhao Wen, Sheng Zhong
RNA-chromatin interactions play crucial roles in gene regulation and genome organization, but the interaction landscape remains poorly understood. In this study, we conducted an in-depth analysis of a previously published dataset on RNase-treated in situ mapping of the RNA–genome interactome in human embryonic stem cells. This dataset globally profiles RNase-insensitive RNA-chromatin interactions. Our analysis revealed that RNase treatment selectively preserved long-range RNA-chromatin interactions while removing promiscuous interactions resulting from the local diffusion of nascent transcripts. RNase-insensitive chromatin-associated RNAs (RI-caRNAs) exhibited high sequence conservation and preferentially localized to functional genomic regions, including promoters, transcription factor binding sites, and regions with specific histone modifications. Interestingly, coding and non-coding RNA transcripts showed distinct sensitivities to RNase, with lncRNAs and disease-associated transcripts being enriched among RI-caRNAs. Furthermore, we identified specific caRNA classes associated with individual transcription factors and histone modifications. Altogether, our findings reveal a RNase-inaccessible regulatory RNA-chromatin interactome and provide a resource for understanding RNA-mediated chromatin regulation.
{"title":"Genome-wide identification of stable RNA-chromatin interactions","authors":"Xingzhao Wen, Sheng Zhong","doi":"10.1101/2024.09.04.611281","DOIUrl":"https://doi.org/10.1101/2024.09.04.611281","url":null,"abstract":"RNA-chromatin interactions play crucial roles in gene regulation and genome organization, but the interaction landscape remains poorly understood. In this study, we conducted an in-depth analysis of a previously published dataset on RNase-treated in situ mapping of the RNA–genome interactome in human embryonic stem cells. This dataset globally profiles RNase-insensitive RNA-chromatin interactions. Our analysis revealed that RNase treatment selectively preserved long-range RNA-chromatin interactions while removing promiscuous interactions resulting from the local diffusion of nascent transcripts. RNase-insensitive chromatin-associated RNAs (RI-caRNAs) exhibited high sequence conservation and preferentially localized to functional genomic regions, including promoters, transcription factor binding sites, and regions with specific histone modifications. Interestingly, coding and non-coding RNA transcripts showed distinct sensitivities to RNase, with lncRNAs and disease-associated transcripts being enriched among RI-caRNAs. Furthermore, we identified specific caRNA classes associated with individual transcription factors and histone modifications. Altogether, our findings reveal a RNase-inaccessible regulatory RNA-chromatin interactome and provide a resource for understanding RNA-mediated chromatin regulation.","PeriodicalId":501161,"journal":{"name":"bioRxiv - Genomics","volume":"95 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142213862","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 : 2024-09-07DOI: 10.1101/2024.09.04.611151
Rob J Dekker, Wim C de Leeuw, Marina van Olst, Wim A Ensink, Selina van Leeuwen, Timo M Breit, Martijs J Jonker
This study reports the discovery of a new Phenuivirus species, named Lachenalia Phenuivirus-1 (LacPhV-1), from Lachenalia plants in an urban botanic garden in Amsterdam. Using a combination of smallRNA-seq, RNA-seq, and advanced bioinformatics, we identified a segmented negative-strand RNA virus in the Phenuiviridae family. Our findings show significant divergence between this new virus and known Phenuiviruses, such as Tulip Streak Virus (TuSV) and Lactuca Big Vein associated Phlebovirus (LBVaPV), supporting its classification as a distinct species. Notably, the sequence differences found in the conserved 5' and 3' ends of these segments suggest potential co-evolution. Despite the observed genomic distances, there is significant conservation in the RdRp subdomain, underscoring evolutionary relationships among LacPhV-1, TuSV, and LBVaPV. Our findings expand the known global virome and highlight the importance of exploring plant viromes in diverse ecological settings to better understand virus evolution and diversity.
{"title":"Discovery of a new Phenuivirus species in Lachenalia plants reveals possible co-evolution between 5' and 3' RNA sequence motifs","authors":"Rob J Dekker, Wim C de Leeuw, Marina van Olst, Wim A Ensink, Selina van Leeuwen, Timo M Breit, Martijs J Jonker","doi":"10.1101/2024.09.04.611151","DOIUrl":"https://doi.org/10.1101/2024.09.04.611151","url":null,"abstract":"This study reports the discovery of a new Phenuivirus species, named Lachenalia Phenuivirus-1 (LacPhV-1), from Lachenalia plants in an urban botanic garden in Amsterdam. Using a combination of smallRNA-seq, RNA-seq, and advanced bioinformatics, we identified a segmented negative-strand RNA virus in the Phenuiviridae family. Our findings show significant divergence between this new virus and known Phenuiviruses, such as Tulip Streak Virus (TuSV) and Lactuca Big Vein associated Phlebovirus (LBVaPV), supporting its classification as a distinct species. Notably, the sequence differences found in the conserved 5' and 3' ends of these segments suggest potential co-evolution. Despite the observed genomic distances, there is significant conservation in the RdRp subdomain, underscoring evolutionary relationships among LacPhV-1, TuSV, and LBVaPV. Our findings expand the known global virome and highlight the importance of exploring plant viromes in diverse ecological settings to better understand virus evolution and diversity.","PeriodicalId":501161,"journal":{"name":"bioRxiv - Genomics","volume":"107 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142213866","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 : 2024-09-07DOI: 10.1101/2024.09.03.611012
Natalia Shylo, Andrew Price, Sofia Robb, Richard Kupronis, Irán Andira Guzmán Méndez, Dustin DeGraffenreid, Tony Gamble, Paul Trainor
The family Chamaeleonidae comprises 228 species, boasting an extensive geographic spread and an array of evolutionary novelties and adaptations, but a paucity of genetic and molecular analyses. Veiled chameleon (Chamaeleo calyptratus) has emerged as a tractable research organism for the study of squamate early development and evolution. Here we report a chromosomal-level assembly and annotation of the veiled chameleon genome. We note a remarkable chromosomal conservation across squamates, but comparisons to more distant genomes reveal GC peaks correlating with ancestral chromosome fusion events. We subsequently identified the XX/XY region on chromosome 5, confirming environmental-independent sex determination in veiled chameleons. Furthermore, our analysis of the Hox gene family indicates that veiled chameleons possess the most complete set of 41 Hox genes, retained from an amniote ancestor. Lastly, the veiled chameleon genome has retained both ancestral paralogs of the Nodal gene, but is missing Dand5 and several other genes, recently associated with the loss of motile cilia during the establishment of left-right patterning. Thus, a complete veiled chameleon genome provides opportunities for novel insights into the evolution of reptilian genomes and the molecular mechanisms driving phenotypic variation and ecological adaptation.
{"title":"Chamaeleo calyptratus (veiled chameleon) chromosome-scale genome assembly and annotation provides insights into the evolution of reptiles and developmental mechanisms.","authors":"Natalia Shylo, Andrew Price, Sofia Robb, Richard Kupronis, Irán Andira Guzmán Méndez, Dustin DeGraffenreid, Tony Gamble, Paul Trainor","doi":"10.1101/2024.09.03.611012","DOIUrl":"https://doi.org/10.1101/2024.09.03.611012","url":null,"abstract":"The family Chamaeleonidae comprises 228 species, boasting an extensive geographic spread and an array of evolutionary novelties and adaptations, but a paucity of genetic and molecular analyses. Veiled chameleon (<em>Chamaeleo calyptratus</em>) has emerged as a tractable research organism for the study of squamate early development and evolution. Here we report a chromosomal-level assembly and annotation of the veiled chameleon genome. We note a remarkable chromosomal conservation across squamates, but comparisons to more distant genomes reveal GC peaks correlating with ancestral chromosome fusion events. We subsequently identified the XX/XY region on chromosome 5, confirming environmental-independent sex determination in veiled chameleons. Furthermore, our analysis of the <em>Hox</em> gene family indicates that veiled chameleons possess the most complete set of 41 <em>Hox</em> genes, retained from an amniote ancestor. Lastly, the veiled chameleon genome has retained both ancestral paralogs of the <em>Nodal</em> gene, but is missing <em>Dand5</em> and several other genes, recently associated with the loss of motile cilia during the establishment of left-right patterning. Thus, a complete veiled chameleon genome provides opportunities for novel insights into the evolution of reptilian genomes and the molecular mechanisms driving phenotypic variation and ecological adaptation.","PeriodicalId":501161,"journal":{"name":"bioRxiv - Genomics","volume":"40 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142213876","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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