Barley Anther and Meiocyte Transcriptome Dynamics in Meiotic Prophase I

In flowering plants, successful germinal cell development and meiotic recombination depend upon a combination of environmental and genetic factors. To gain insights into this specialized reproductive development program we used short- and long-read RNA-sequencing (RNA-seq) to study the temporal dynamics of transcript abundance in immuno-cytologically staged barley (Hordeum vulgare) anthers and meiocytes. We show that the most significant transcriptional changes in anthers occur at the transition from pre-meiosis to leptotene–zygotene, which is followed by increasingly stable transcript abundance throughout prophase I into metaphase I–tetrad. Our analysis reveals that the pre-meiotic anthers are enriched in long non-coding RNAs (lncRNAs) and that entry to meiosis is characterized by their robust and significant down regulation. Intriguingly, only 24% of a collection of putative meiotic gene orthologs showed differential transcript abundance in at least one stage or tissue comparison. Argonautes, E3 ubiquitin ligases, and lys48 specific de-ubiquitinating enzymes were enriched in prophase I meiocyte samples. These developmental, time-resolved transcriptomes demonstrate remarkable stability in transcript abundance in meiocytes throughout prophase I after the initial and substantial reprogramming at meiosis entry and the complexity of the regulatory networks involved in early meiotic processes.

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Time-resolved transcriptome of barley anthers and meiocytes reveals robust and largely stable gene expression changes at meiosis entry

10.1101/2020.04.20.051425 ◽

2020 ◽

Author(s):

Abdellah Barakate ◽

Jamie Orr ◽

Miriam Schreiber ◽

Isabelle Colas ◽

Dominika Lewandowska ◽

...

Keyword(s):

Gene Expression ◽

Regulatory Networks ◽

Temporal Dynamics ◽

Transcript Abundance ◽

Future Research ◽

Stable Gene ◽

Rna Seq ◽

Post Translational Modification ◽

Time Resolved ◽

Prophase I

ABSTRACTIn flowering plants, successful germinal cell development and meiotic recombination depend upon a combination of environmental and genetic factors. To gain insights into this specialised reproductive development programme we used short- and long-read RNA-sequencing (RNA-seq) to study the temporal dynamics of transcript abundance in immuno-cytologically staged barley (Hordeum vulgare) anthers and meiocytes. We show that the most significant transcriptional changes occur at the transition from pre-meiosis to leptotene–zygotene, which is followed by largely stable transcript abundance throughout prophase I. Our analysis reveals that the developing anthers and meiocytes are enriched in long non-coding RNAs (lncRNAs) and that entry to meiosis is characterized by their robust and significant down regulation. Intriguingly, only 24% of a collection of putative meiotic gene orthologues showed differential transcript abundance in at least one stage or tissue comparison. Changes in the abundance of numerous transcription factors, representatives of the small RNA processing machinery, and post-translational modification pathways highlight the complexity of the regulatory networks involved. These developmental, time-resolved, and dynamic transcriptomes increase our understanding of anther and meiocyte development and will help guide future research.One sentence summaryAnalysis of RNA-seq data from meiotically staged barley anthers and meiocytes highlights the role of lncRNAs within a complex network of transcriptional and post-transcriptional regulation accompanied by a hiatus in differential gene expression during prophase I.The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Robbie Waugh ([email protected])

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Chromatin regulatory dynamics of early human small intestinal development using a directed differentiation model

10.1101/2019.12.18.881219 ◽

2019 ◽

Author(s):

Yu-Han Hung ◽

Sha Huang ◽

Michael K. Dame ◽

Qianhui Yu ◽

Qing Cissy Yu ◽

...

Keyword(s):

Regulatory Networks ◽

Temporal Dynamics ◽

Developmental Time ◽

Immune Defense ◽

Human Model ◽

Directed Differentiation ◽

Enhancer Activity ◽

Chromatin Dynamics ◽

Hox Gene ◽

Small Intestinal

AbstractBackgroundThe establishment of the small intestinal (SI) lineage during human embryogenesis is critical for the proper development of neonatal gut functions, including nutrient absorption and immune defense. The chromatin dynamics and regulatory networks that drive human SI lineage formation and regional patterning are essentially unknown. To fill this knowledge void, we apply a cutting-edge genomic technology to a state-of-the-art human model of early SI development. Specifically, we leverage chromatin run-on sequencing (ChRO-seq) to define the landscape of active promoters, enhancers, super enhancers, and gene bodies across distinct stages of directed differentiation of human pluripotent stem cells (hPSCs) into SI spheroids with regional specification.ResultsThrough comprehensive ChRO-seq analysis we identify candidate stage-specific chromatin activity states, novel markers, and enhancer hotspots during the directed differentiation process. Moreover, we propose a detailed transcriptional network associated with SI lineage formation or initial regional patterning. Among our findings is a unique pattern of enhancer activity and transcription at HOX gene loci that is previously undescribed. Analysis of single cell RNA-seq data from human fetal SI at early developmental time points shed further light on the unique HOX gene temporal dynamics that underlies SI regional patterning.ConclusionsOverall, the results lead to a new proposed working model for the regulatory underpinnings of human SI lineage formation and regional patterning, thereby adding a novel dimension to the literature that has thus far relied almost exclusively on non-human models.

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High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer

Nature Communications ◽

10.1038/s41467-021-23213-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sarah E. Pierce ◽

Jeffrey M. Granja ◽

William J. Greenleaf

Keyword(s):

Transcription Factor ◽

Single Cell ◽

Cancer Cells ◽

High Throughput ◽

Regulatory Networks ◽

Temporal Dynamics ◽

Chromatin Accessibility ◽

Regulatory Regions ◽

Factor Binding ◽

Genome Wide

AbstractChromatin accessibility profiling can identify putative regulatory regions genome wide; however, pooled single-cell methods for assessing the effects of regulatory perturbations on accessibility are limited. Here, we report a modified droplet-based single-cell ATAC-seq protocol for perturbing and evaluating dynamic single-cell epigenetic states. This method (Spear-ATAC) enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at once. Spear-ATAC profiling of 104,592 cells representing 414 sgRNA knock-down populations reveals the temporal dynamics of epigenetic responses to regulatory perturbations in cancer cells and the associations between transcription factor binding profiles.

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IMiDs prime myeloma cells for daratumumab-mediated cytotoxicity through loss of Ikaros and Aiolos

Blood ◽

10.1182/blood-2018-05-850727 ◽

2018 ◽

Vol 132 (20) ◽

pp. 2166-2178 ◽

Cited By ~ 18

Author(s):

Pasquale L. Fedele ◽

Simon N. Willis ◽

Yang Liao ◽

Michael S. Low ◽

Jai Rautela ◽

...

Keyword(s):

Cell Death ◽

Regulatory Networks ◽

Nk Cell ◽

Surface Expression ◽

Nucleosome Remodeling ◽

Interferon Stimulated Genes ◽

Cd38 Expression ◽

Cycle Arrest ◽

Transcriptional Changes ◽

Insight Into

Abstract Recent studies have demonstrated that the immunomodulatory drugs (IMiDs) lead to the degradation of the transcription factors Ikaros and Aiolos. However, why their loss subsequently leads to multiple myeloma (MM) cell death remains unclear. Using CRISPR-Cas9 genome editing, we have deleted IKZF1/Ikaros and IKZF3/Aiolos in human MM cell lines to gain further insight into their downstream gene regulatory networks. Inactivation of either factor alone recapitulates the cell intrinsic action of the IMiDs, resulting in cell cycle arrest and induction of apoptosis. Furthermore, evaluation of the transcriptional changes resulting from their loss demonstrates striking overlap with lenalidomide treatment. This was not dependent on reduction of the IRF4-MYC “axis,” as neither protein was consistently downregulated, despite cell death occurring, and overexpression of either factor failed to rescue for Ikaros loss. Importantly, Ikaros and Aiolos repress the expression of interferon-stimulated genes (ISGs), including CD38, and their loss led to the activation of an interferon-like response, contributing to MM cell death. Ikaros/Aiolos repressed CD38 expression through interaction with the nucleosome remodeling and deacetylase complex in MM. IMiD-induced loss of Ikaros or treatment with interferon resulted in an upregulation of CD38 surface expression on MM cells, priming for daratumumab-induced NK cell-mediated antibody-dependent cellular cytotoxicity. These results give further insight into the mechanism of action of the IMiDs and provide mechanistic rationale for combination with anti-CD38 monoclonal antibodies.

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Identification of drugs for leukaemia differentiation therapy by network pharmacology

10.1101/676106 ◽

2019 ◽

Author(s):

Eleni G Christodoulou ◽

Lin Ming Lee ◽

Kian Leong Lee ◽

Tsz Kan Fung ◽

Eric So ◽

...

Keyword(s):

Gene Regulatory Networks ◽

Regulatory Networks ◽

Cellular Reprogramming ◽

Network Pharmacology ◽

Differentiation Therapy ◽

Disease States ◽

Small Molecule Drugs ◽

Transcriptional Changes ◽

Gene Regulatory ◽

Computational Platform

AbstractAcute leukaemias differ from their normal haematopoietic counterparts in their inability to differentiate. This phenomenon is thought to be the result of aberrant cellular reprogramming involving transcription factors (TFs). Here we leveraged on Mogrify, a network-based algorithm, to identify TFs and their gene regulatory networks that drive differentiation of the acute promyelocytic leukaemia (APL) cell line NB4 in response to ATRA (all-transretinoic acid). We further integrated the detected TF regulatory networks with the Connectivity Map (CMAP) repository and recovered small molecule drugs which induce similar transcriptional changes. Our method outperformed standard approaches, retrieving ATRA as the top hit. Of the other drug hits, dimaprit and mebendazole enhanced ATRA-mediated differentiation in both parental NB4 and ATRA-resistant NB4-MR2 cells. Thus, we provide proof-of-principle of our network-based computational platform for drug discovery and repositioning in leukaemia differentiation therapy, which can be extended to other dysregulated disease states.

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Massively parallel, time-resolved single-cell RNA sequencing with scNT-Seq

10.1101/2019.12.19.882050 ◽

2019 ◽

Cited By ~ 2

Author(s):

Qi Qiu ◽

Peng Hu ◽

Kiya W. Govek ◽

Pablo G. Camara ◽

Hao Wu

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Temporal Dynamics ◽

Embryonic Stem ◽

Droplet Microfluidics ◽

Massively Parallel ◽

Specific Gene ◽

Time Resolved ◽

Single Cell Rna Sequencing ◽

Rna Biogenesis

ABSTRACTSingle-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal dynamics of RNA biogenesis and decay. Here we present single-cell new transcript tagging sequencing (scNT-Seq), a method for massively parallel analysis of newly-transcribed and pre-existing RNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking metabolically labeled newly-transcribed RNAs with T-to-C substitutions. By simultaneously measuring new and old transcriptomes, scNT-Seq reveals neuronal subtype-specific gene regulatory networks and time-resolved RNA trajectories in response to brief (minutes) versus sustained (hours) neuronal activation. Integrating scNT-Seq with genetic perturbation reveals that DNA methylcytosine dioxygenases may inhibit stepwise transition from pluripotent embryonic stem cell state to intermediate and totipotent two-cell-embryo-like (2C-like) states by promoting global RNA biogenesis. Furthermore, pulse-chase scNT-Seq enables transcriptome-wide measurements of RNA stability in rare 2C-like cells. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms.

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Comparative transcriptomics identifies differences in the regulation of the floral transition between Arabidopsis and Brassica rapa cultivars

10.1101/2020.08.26.266494 ◽

2020 ◽

Author(s):

Alexander Calderwood ◽

Jo Hepworth ◽

Shannon Woodhouse ◽

Lorelei Bilham ◽

D. Marc Jones ◽

...

Keyword(s):

Gene Expression ◽

Brassica Rapa ◽

Regulatory Networks ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Expression Profiles ◽

Detailed Comparison ◽

Developmental Time ◽

Floral Transition ◽

Gene Regulatory

AbstractThe timing of the floral transition affects reproduction and yield, however its regulation in crops remains poorly understood. Here, we use RNA-Seq to determine and compare gene expression dynamics through the floral transition in the model species Arabidopsis thaliana and the closely related crop Brassica rapa. A direct comparison of gene expression over time between species shows little similarity, which could lead to the inference that different gene regulatory networks are at play. However, these differences can be largely resolved by synchronisation, through curve registration, of gene expression profiles. We find that different registration functions are required for different genes, indicating that there is no common ‘developmental time’ to which Arabidopsis and B. rapa can be mapped through gene expression. Instead, the expression patterns of different genes progress at different rates. We find that co-regulated genes show similar changes in synchronisation between species, suggesting that similar gene regulatory sub-network structures may be active with different wiring between them. A detailed comparison of the regulation of the floral transition between Arabidopsis and B. rapa, and between two B. rapa accessions reveals different modes of regulation of the key floral integrator SOC1, and that the floral transition in the B. rapa accessions is triggered by different pathways, even when grown under the same environmental conditions. Our study adds to the mechanistic understanding of the regulatory network of flowering time in rapid cycling B. rapa under long days and highlights the importance of registration methods for the comparison of developmental gene expression data.

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Transcriptome Profiling Reveals Spatial-temporal Dynamics of Gene Expression Essential for Soybean Seed Development

10.21203/rs.3.rs-128593/v1 ◽

2020 ◽

Author(s):

Hengyou Zhang ◽

Zhenbin Hu ◽

Yuming Yang ◽

Xiaoqian Liu ◽

Haiyan Lv ◽

...

Keyword(s):

Seed Development ◽

Temporal Dynamics ◽

Expression Patterns ◽

Soybean Seed ◽

Transcriptome Profiling ◽

Transcript Abundance ◽

Specific Gene ◽

Oilseed Crop ◽

Seed Formation ◽

Developing Seeds

Abstract Background: Seeds are the economic basis of oilseed crops, especially for soybean, thus far the most widely cultivated oilseed crop worldwide. Seed development is accompanied with a multitude of diverse cellular processes and revealing the underlying regulatory activities is critical for seed improvement. Results: Here, we profiled transcriptomes of developing seeds (20, 25, 30, 40 days after flowering) representing key points of seed development from early to full development. We identified a set of highly-abundant genes and highlighted the importance of these genes to support nutrient accumulation and transcriptional regulation in developing seeds. We identified 8,925 differentially expressed genes that exhibited temporal expression patterns over the course and had expression specificities in distinct tissues including seeds and non-seed tissues (roots, stems, leaves). Genes with specificities to non-seed tissues have tissue-specialized roles while remain relatively low transcript abundance in developing seeds, exhibiting their supportive roles spatially for seed development. Co-expression network analysis identified several under-explored genes in soybean that bridge tissue-specific gene modules. Conclusions: Our study provides a global view of gene activities and biological processes critical for seed formation in soybean and prioritizes a set of genes for further study. The results shed insight into the mechanism controlling seed development and storage reserves.

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Emergence of neuronal diversity during vertebrate brain development

10.1101/839860 ◽

2019 ◽

Author(s):

Bushra Raj ◽

Jeffrey A. Farrell ◽

Aaron McKenna ◽

Jessica L. Leslie ◽

Alexander F. Schier

Keyword(s):

Brain Development ◽

Single Cell ◽

Time Course ◽

Temporal Dynamics ◽

Cell Types ◽

Developmental Time ◽

Neural Progenitors ◽

Neuronal Diversity ◽

Gene Markers ◽

Vertebrate Brain

ABSTRACTNeurogenesis in the vertebrate brain comprises many steps ranging from the proliferation of progenitors to the differentiation and maturation of neurons. Although these processes are highly regulated, the landscape of transcriptional changes and progenitor identities underlying brain development are poorly characterized. Here, we describe the first developmental single-cell RNA-seq catalog of more than 200,000 zebrafish brain cells encompassing 12 stages from 12 hours post-fertilization to 15 days post-fertilization. We characterize known and novel gene markers for more than 800 clusters across these timepoints. Our results capture the temporal dynamics of multiple neurogenic waves from embryo to larva that expand neuronal diversity from ∼20 cell types at 12 hpf to ∼100 cell types at 15 dpf. We find that most embryonic neural progenitor states are transient and transcriptionally distinct from long-lasting neural progenitors of post-embryonic stages. Furthermore, we reconstruct cell specification trajectories for the retina and hypothalamus, and identify gene expression cascades and novel markers. Our analysis reveal that late-stage retinal neural progenitors transcriptionally overlap cell states observed in the embryo, while hypothalamic neural progenitors become progressively distinct with developmental time. These data provide the first comprehensive single-cell transcriptomic time course for vertebrate brain development and suggest distinct neurogenic regulatory paradigms between different stages and tissues.

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Metabolic and immune markers for precise monitoring of COVID-19 severity and treatment

10.1101/2021.09.05.21263141 ◽

2021 ◽

Author(s):

André F. Rendeiro ◽

Charles Kyriakos Vorkas ◽

Jan Krumsiek ◽

Harjot Singh ◽

Shashi Kapatia ◽

...

Keyword(s):

Temporal Dynamics ◽

Severe Disease ◽

Deep Understanding ◽

Response To Therapy ◽

High Dimensional ◽

Time Resolved ◽

Novel Approach ◽

Metabolomic Data ◽

Clinical Action ◽

Rich Information

AbstractDeep understanding of the SARS-CoV-2 effects on host molecular pathways is paramount for the discovery of early biomarkers of outcome of coronavirus disease 2019 (COVID-19) and the identification of novel therapeutic targets. In that light, we generated metabolomic data from COVID-19 patient blood using high-throughput targeted nuclear magnetic resonance (NMR) spectroscopy and high-dimensional flow cytometry. We find considerable changes in serum metabolome composition of COVID-19 patients associated with disease severity, and response to tocilizumab treatment. We built a clinically annotated, biologically-interpretable space for precise time-resolved disease monitoring and characterize the temporal dynamics of metabolomic change along the clinical course of COVID-19 patients and in response to therapy. Finally, we leverage joint immuno-metabolic measurements to provide a novel approach for patient stratification and early prediction of severe disease. Our results show that high-dimensional metabolomic and joint immune-metabolic readouts provide rich information content for elucidation of the host’s response to infection and empower discovery of novel metabolic-driven therapies, as well as precise and efficient clinical action.

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