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

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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Barley Anther and Meiocyte Transcriptome Dynamics in Meiotic Prophase I

Frontiers in Plant Science ◽

10.3389/fpls.2020.619404 ◽

2021 ◽

Vol 11 ◽

Author(s):

Abdellah Barakate ◽

Jamie Orr ◽

Miriam Schreiber ◽

Isabelle Colas ◽

Dominika Lewandowska ◽

...

Keyword(s):

Regulatory Networks ◽

Temporal Dynamics ◽

Development Program ◽

Transcript Abundance ◽

Developmental Time ◽

Time Resolved ◽

Prophase I ◽

Long Read ◽

Transcriptional Changes ◽

Gene Orthologs

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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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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The temporal transcription factor E93 controls dynamic enhancer activity and chromatin accessibility during development

10.1101/679753 ◽

2019 ◽

Author(s):

Spencer L. Nystrom ◽

Matthew J. Niederhuber ◽

Daniel J. McKay

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Developmental Time ◽

Chromatin Accessibility ◽

Wild Type ◽

Spatial Cues ◽

Enhancer Activity ◽

Temporal Gene Expression ◽

Developmental Program ◽

Temporal Cues

ABSTRACTHow temporal cues combine with spatial inputs to control gene expression during development is poorly understood. Here, we test the hypothesis that the Drosophila transcription factor E93 controls temporal gene expression by regulating chromatin accessibility. Precocious expression of E93 early in wing development reveals that it can simultaneously activate and deactivate different target enhancers. Notably, the precocious patterns of enhancer activity resemble the wild-type patterns that occur later in development, suggesting that provision of E93 alters the competence of enhancers to respond to spatial cues. Genomic profiling reveals that precocious E93 expression is sufficient to regulate chromatin accessibility at a subset of its targets. These accessibility changes mimic those that normally occur later in development, indicating that precocious E93 accelerates the wild-type developmental program. Further, we find that target enhancers that do not respond to precocious E93 in early wings become responsive after a developmental transition, suggesting that parallel temporal pathways work alongside E93. These findings support a model wherein E93 expression functions as an instructive cue that defines a broad window of developmental time through control of chromatin accessibility.

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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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Chromatin dynamics during hematopoiesis reveal discrete regulatory modules instructing differentiation

10.1101/2020.04.02.022566 ◽

2020 ◽

Author(s):

Grigorios Georgolopoulos ◽

Mineo Iwata ◽

Nikoletta Psatha ◽

Andrew Nishida ◽

Tannishtha Som ◽

...

Keyword(s):

Temporal Dynamics ◽

Ex Vivo ◽

Chromatin Accessibility ◽

Dnase I ◽

Lineage Commitment ◽

Gene Promoters ◽

Chromatin Dynamics ◽

Regulatory Modules ◽

Hypersensitive Sites ◽

Regulatory Landscape

AbstractLineage commitment and differentiation is driven by the concerted action of master transcriptional regulators at their target chromatin sites. Multiple efforts have characterized the key transcription factors (TFs) that determine the various hematopoietic lineages. However, the temporal interactions between individual TFs and their chromatin targets during differentiation and how these interactions dictate lineage commitment remains poorly understood. We performed dense, daily, temporal profiling of chromatin accessibility (DNase I-seq) and gene expression changes (total RNA-seq) along ex vivo human erythropoiesis to comprehensively define developmentally regulated DNase I hypersensitive sites (DHSs) and transcripts. We link both distal DHSs to their target gene promoters and individual TFs to their target DHSs, revealing that the regulatory landscape is organized in distinct sequential regulatory modules that regulate lineage restriction and maturation. Finally, direct comparison of transcriptional dynamics (bulk and single-cell) and lineage potential between erythropoiesis and megakaryopoiesis illuminates the fine-scale temporal dynamics of these regulatory modules during lineage-resolution between these two fates. Collectively, these data provide novel insights into the global regulatory landscape during hematopoiesis.

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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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ASC proneural transcription factors mediate the timely initiation of the neural program during neuroectodermal to neuroblast transition ensuring progeny fidelity

10.1101/2021.11.02.466869 ◽

2021 ◽

Author(s):

Vasiliki Theodorou ◽

Aikaterini Stefanaki ◽

Minas Drakos ◽

Dafne Triantafyllou ◽

Christos Delidakis

Keyword(s):

Transcription Factors ◽

Target Genes ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Cis Elements ◽

Enhancer Activity ◽

Timely Initiation ◽

Chromatin Dynamics ◽

Neural Fate ◽

Neural Specification

Background: ASC/ASCL proneural transcription factors are oncogenic and exhibit impressive reprogramming and pioneer activities. In both Drosophila and mammals, these factors are central in the early specification of the neural fate, where they act in opposition to Notch signalling. However, the role of ASC on the chromatin during CNS neural stem cells birth remains elusive. Results: We investigated the chromatin changes accompanying neural commitment using an integrative genetics and genomics methodology. We found that ASC factors bind equally strongly to two distinct classes of cis-regulatory elements: open regions remodeled earlier during maternal to zygotic transition by Zelda and Zelda-independent, less accessible regions. Both classes cis-elements exhibit enhanced chromatin accessibility during neural specification and correlate with transcriptional regulation of genes involved in many biological processes necessary for neuroblast function. We identified an ASC-Notch regulated TF network that most likely act as the prime regulators of neuroblast function. Using a cohort of ASC target genes, we report that ASC null neuroblasts are defectively specified, remaining initially stalled, lacking expression of many proneural targets and unable to divide. When they eventually start proliferating, they produce compromised progeny. Generation of lacZ reporter lines driven by proneural-bound elements display enhancer activity within neuroblasts and proneural dependency. Therefore, the partial neuroblast identity seen in the absence of ASC genes is driven by other, proneural-independent, cis-elements. Neuroblast impairment and the late differentiation defects of ASC mutants are corrected by ectodermal induction of individual ASC genes but not by individual members of the TF network downstream of ASC. However, in wild type embryos induction of individual members of this network induces CNS hyperplasia, suggesting that they synergize with the activating function of ASC to establish the chromatin dynamics that promote neural specification. Conclusion: ASC factors bind a large number of enhancers to orchestrate the timely activation of the neural chromatin program during neuroectodermal to neuroblast transition. This early chromatin remodeling is crucial for both neuroblast homeostasis as well as future progeny fidelity.

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Transcriptional regulatory networks that promote and restrict identities and functions of intestinal innate lymphoid cells

10.1101/465435 ◽

2018 ◽

Cited By ~ 1

Author(s):

Maria Pokrovskii ◽

Jason A. Hall ◽

David E. Ochayon ◽

Ren Yi ◽

Natalia S. Chaimowitz ◽

...

Keyword(s):

Regulatory Networks ◽

Inflammatory Diseases ◽

Innate Lymphoid Cells ◽

Chromatin Accessibility ◽

Immune Defense ◽

Lymphoid Cells ◽

Transcriptional Regulatory Networks ◽

Transcriptional Regulatory

SummaryInnate lymphoid cells (ILCs) can be subdivided into several distinct cytokine-secreting lineages that promote tissue homeostasis and immune defense but also contribute to inflammatory diseases. Accumulating evidence suggests that ILCs, similarly to other immune populations, are capable of phenotypic and functional plasticity in response to infectious or environmental stimuli. Yet the transcriptional circuits that control ILC identity and function are largely unknown. Here we integrate gene expression and chromatin accessibility data to infer transcriptional regulatory networks within intestinal type 1, 2, and 3 ILCs. We predict the “core” sets of transcription-factor (TF) regulators driving each ILC subset identity, among which only a few TFs were previously known. To assist in the interpretation of these networks, TFs were organized into cooperative clusters, or modules that control gene programs with distinct functions. The ILC network reveals extensive alternative-lineage-gene repression, whose regulation may explain reported plasticity between ILC subsets. We validate new roles for c-MAF and BCL6 as regulators affecting the type 1 and type 3 ILC lineages. Manipulation of TF pathways identified here might provide a novel means to selectively regulate ILC effector functions to alleviate inflammatory disease or enhance host tolerance to pathogenic microbes or noxious stimuli. Our results will enable further exploration of ILC biology, while our network approach will be broadly applicable to identifying key cell state regulators in otherin vivocell populations.

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Bone Remodeling: Histone Modifications as Fate Determinants of Bone Cell Differentiation

International Journal of Molecular Sciences ◽

10.3390/ijms20133147 ◽

2019 ◽

Vol 20 (13) ◽

pp. 3147 ◽

Cited By ~ 8

Author(s):

Sun-Ju Yi ◽

Hyerim Lee ◽

Jisu Lee ◽

Kyubin Lee ◽

Junil Kim ◽

...

Keyword(s):

Regulatory Networks ◽

Bone Cells ◽

Bone Development ◽

Bone Cell ◽

Gene Expressions ◽

Chromatin Dynamics ◽

Multinucleated Cells ◽

Bone Cell Differentiation ◽

Fate Determinants ◽

The Impact

The bone tissue is a dynamic complex that constitutes of several interdependent systems and is continuously remodeled through the concerted actions of bone cells. Osteoblasts are mononucleated cells, derived from mesenchymal stem cells, responsible for bone formation. Osteoclasts are large multinucleated cells that differentiate from hematopoietic progenitors of the myeloid lineage and are responsible for bone resorption. The lineage-specific differentiation of bone cells requires an epigenetic regulation of gene expressions involving chromatin dynamics. The key step for understanding gene regulatory networks during bone cell development lies in characterizing the chromatin modifying enzymes responsible for reorganizing and potentiating particular chromatin structure. This review covers the histone-modifying enzymes involved in bone development, discusses the impact of enzymes on gene expression, and provides future directions and clinical significance in this area.

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A sensitive mNeonGreen reporter system to measure transcriptional dynamics in Drosophila development

Communications Biology ◽

10.1038/s42003-020-01375-5 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Stefano Ceolin ◽

Monika Hanf ◽

Marta Bozek ◽

Andrea Ennio Storti ◽

Nicolas Gompel ◽

...

Keyword(s):

Gene Expression ◽

Fluorescent Protein ◽

Temporal Dynamics ◽

Reconstruction Algorithm ◽

Detection Sensitivity ◽

Axis Formation ◽

Reporter System ◽

Enhancer Activity ◽

Spatio Temporal ◽

Pioneer Transcription Factor

AbstractThe gene regulatory network governing anterior–posterior axis formation in Drosophila is a well-established paradigm to study transcription in developmental biology. The rapid temporal dynamics of gene expression during early stages of development, however, are difficult to track with standard techniques. We optimized the bright and fast-maturing fluorescent protein mNeonGreen as a real-time, quantitative reporter of enhancer expression. We derive enhancer activity from the reporter fluorescence dynamics with high spatial and temporal resolution, using a robust reconstruction algorithm. By comparing our results with data obtained with the established MS2-MCP system, we demonstrate the higher detection sensitivity of our reporter. We used the reporter to quantify the activity of variants of a simple synthetic enhancer, and observe increased activity upon reduction of enhancer–promoter distance or addition of binding sites for the pioneer transcription factor Zelda. Our reporter system constitutes a powerful tool to study spatio-temporal gene expression dynamics in live embryos.

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