Chromatin Accessibility Predetermines Odontoblast Terminal Differentiation

Embryonic development and stem cell differentiation are orchestrated by changes in sequential binding of regulatory transcriptional factors to their motifs. These processes are invariably accompanied by the alternations in chromatin accessibility, conformation, and histone modification. Odontoblast lineage originates from cranial neural crest cells and is crucial in dentinogenesis. Our previous work revealed several transcription factors (TFs) that promote odontoblast differentiation. However, it remains elusive as to whether chromatin accessibility affects odontoblast terminal differentiation. Herein, integration of single-cell RNA-seq and bulk RNA-seq revealed that in vitro odontoblast differentiation using dental papilla cells at E18.5 was comparable to the crown odontoblast differentiation trajectory of OC (osteocalcin)-positive odontogenic lineage. Before in vitro odontoblast differentiation, ATAC-seq and H3K27Ac CUT and Tag experiments demonstrated high accessibility of chromatin regions adjacent to genes associated with odontogenic potential. However, following odontoblastic induction, regions near mineralization-related genes became accessible. Integration of RNA-seq and ATAC-seq results further revealed that the expression levels of these genes were correlated with the accessibility of nearby chromatin. Time-course ATAC-seq experiments further demonstrated that odontoblast terminal differentiation was correlated with the occupation of the basic region/leucine zipper motif (bZIP) TF family, whereby we validated the positive role of ATF5 in vitro. Collectively, this study reports a global mapping of open chromatin regulatory elements during dentinogenesis and illustrates how these regions are regulated via dynamic binding of different TF families, resulting in odontoblast terminal differentiation. The findings also shed light on understanding the genetic regulation of dentin regeneration using dental mesenchymal stem cells.

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EPCO-31. EPIGENOMIC INTRATUMORAL HETEROGENEITY OF GLIOBLASTOMA IN THREE-DIMENSIONAL SPACE

Neuro-Oncology ◽

10.1093/neuonc/noaa215.310 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii76-ii76

Author(s):

Radhika Mathur ◽

Sriranga Iyyanki ◽

Stephanie Hilz ◽

Chibo Hong ◽

Joanna Phillips ◽

...

Keyword(s):

Spatial Organization ◽

Dimensional Space ◽

Three Dimensional ◽

Spatial Location ◽

Therapy Resistance ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Intratumoral Heterogeneity ◽

Tumor Evolution ◽

Open Chromatin

Abstract Treatment failure in glioblastoma is often attributed to intratumoral heterogeneity (ITH), which fosters tumor evolution and generation of therapy-resistant clones. While ITH in glioblastoma has been well-characterized at the genomic and transcriptomic levels, the extent of ITH at the epigenomic level and its biological and clinical significance are not well understood. In collaboration with neurosurgeons, neuropathologists, and biomedical imaging experts, we have established a novel topographical approach towards characterizing epigenomic ITH in three-dimensional (3-D) space. We utilize pre-operative MRI scans to define tumor volume and then utilize 3-D surgical neuro-navigation to intra-operatively acquire 10+ samples representing maximal anatomical diversity. The precise spatial location of each sample is mapped by 3-D coordinates, enabling tumors to be visualized in 360-degrees and providing unprecedented insight into their spatial organization and patterning. For each sample, we conduct assay for transposase-accessible chromatin using sequencing (ATAC-Seq), which provides information on the genomic locations of open chromatin, DNA-binding proteins, and individual nucleosomes at nucleotide resolution. We additionally conduct whole-exome sequencing and RNA sequencing for each spatially mapped sample. Integrative analysis of these datasets reveals distinct patterns of chromatin accessibility within glioblastoma tumors, as well as their associations with genetically defined clonal expansions. Our analysis further reveals how differences in chromatin accessibility within tumors reflect underlying transcription factor activity at gene regulatory elements, including both promoters and enhancers, and drive expression of particular gene expression sets, including neuronal and immune programs. Collectively, this work provides the most comprehensive characterization of epigenomic ITH to date, establishing its importance for driving tumor evolution and therapy resistance in glioblastoma. As a resource for further investigation, we have provided our datasets on an interactive data sharing platform – The 3D Glioma Atlas – that enables 360-degree visualization of both genomic and epigenomic ITH.

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Identification and classification of cis-regulatory elements in the amphipod crustacean Parhyale hawaiensis

10.1101/2021.09.16.460328 ◽

2021 ◽

Author(s):

Dennis A Sun ◽

Nipam H Patel

Keyword(s):

Gene Expression ◽

Genome Annotation ◽

Time Course ◽

Regulatory Elements ◽

Rna Seq ◽

Genome Wide ◽

Long Read ◽

Amphipod Crustacean ◽

Accessible Chromatin

AbstractEmerging research organisms enable the study of biology that cannot be addressed using classical “model” organisms. The development of novel data resources can accelerate research in such animals. Here, we present new functional genomic resources for the amphipod crustacean Parhyale hawaiensis, facilitating the exploration of gene regulatory evolution using this emerging research organism. We use Omni-ATAC-Seq, an improved form of the Assay for Transposase-Accessible Chromatin coupled with next-generation sequencing (ATAC-Seq), to identify accessible chromatin genome-wide across a broad time course of Parhyale embryonic development. This time course encompasses many major morphological events, including segmentation, body regionalization, gut morphogenesis, and limb development. In addition, we use short- and long-read RNA-Seq to generate an improved Parhyale genome annotation, enabling deeper classification of identified regulatory elements. We leverage a variety of bioinformatic tools to discover differential accessibility, predict nucleosome positioning, infer transcription factor binding, cluster peaks based on accessibility dynamics, classify biological functions, and correlate gene expression with accessibility. Using a Minos transposase reporter system, we demonstrate the potential to identify novel regulatory elements using this approach, including distal regulatory elements. This work provides a platform for the identification of novel developmental regulatory elements in Parhyale, and offers a framework for performing such experiments in other emerging research organisms.Primary Findings-Omni-ATAC-Seq identifies cis-regulatory elements genome-wide during crustacean embryogenesis-Combined short- and long-read RNA-Seq improves the Parhyale genome annotation-ImpulseDE2 analysis identifies dynamically regulated candidate regulatory elements-NucleoATAC and HINT-ATAC enable inference of nucleosome occupancy and transcription factor binding-Fuzzy clustering reveals peaks with distinct accessibility and chromatin dynamics-Integration of accessibility and gene expression reveals possible enhancers and repressors-Omni-ATAC can identify known and novel regulatory elements

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Single-cell landscape of nuclear configuration and gene expression during stem cell differentiation and X inactivation

Genome Biology ◽

10.1186/s13059-021-02432-w ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Giancarlo Bonora ◽

Vijay Ramani ◽

Ritambhara Singh ◽

He Fang ◽

Dana L. Jackson ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Nuclear Structure ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Chromatin Accessibility ◽

X Inactivation ◽

Rna Seq ◽

X Chromosomes ◽

Allele Specific

Abstract Background Mammalian development is associated with extensive changes in gene expression, chromatin accessibility, and nuclear structure. Here, we follow such changes associated with mouse embryonic stem cell differentiation and X inactivation by integrating, for the first time, allele-specific data from these three modalities obtained by high-throughput single-cell RNA-seq, ATAC-seq, and Hi-C. Results Allele-specific contact decay profiles obtained by single-cell Hi-C clearly show that the inactive X chromosome has a unique profile in differentiated cells that have undergone X inactivation. Loss of this inactive X-specific structure at mitosis is followed by its reappearance during the cell cycle, suggesting a “bookmark” mechanism. Differentiation of embryonic stem cells to follow the onset of X inactivation is associated with changes in contact decay profiles that occur in parallel on both the X chromosomes and autosomes. Single-cell RNA-seq and ATAC-seq show evidence of a delay in female versus male cells, due to the presence of two active X chromosomes at early stages of differentiation. The onset of the inactive X-specific structure in single cells occurs later than gene silencing, consistent with the idea that chromatin compaction is a late event of X inactivation. Single-cell Hi-C highlights evidence of discrete changes in nuclear structure characterized by the acquisition of very long-range contacts throughout the nucleus. Novel computational approaches allow for the effective alignment of single-cell gene expression, chromatin accessibility, and 3D chromosome structure. Conclusions Based on trajectory analyses, three distinct nuclear structure states are detected reflecting discrete and profound simultaneous changes not only to the structure of the X chromosomes, but also to that of autosomes during differentiation. Our study reveals that long-range structural changes to chromosomes appear as discrete events, unlike progressive changes in gene expression and chromatin accessibility.

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CellWalker integrates single-cell and bulk data to resolve regulatory elements across cell types in complex tissues

10.1101/847657 ◽

2019 ◽

Cited By ~ 1

Author(s):

Pawel F. Przytycki ◽

Katherine S. Pollard

Keyword(s):

Single Cell ◽

Cell Types ◽

Regulatory Elements ◽

Cell Labeling ◽

Open Chromatin ◽

Specific Cell ◽

Rna Seq ◽

Data Types ◽

Bulk Data ◽

Cell Type Specific

Single-cell and bulk genomics assays have complementary strengths and weaknesses, and alone neither strategy can fully capture regulatory elements across the diversity of cells in complex tissues. We present CellWalker, a method that integrates single-cell open chromatin (scATAC-seq) data with gene expression (RNA-seq) and other data types using a network model that simultaneously improves cell labeling in noisy scATAC-seq and annotates cell-type specific regulatory elements in bulk data. We demonstrate CellWalker’s robustness to sparse annotations and noise using simulations and combined RNA-seq and ATAC-seq in individual cells. We then apply CellWalker to the developing brain. We identify cells transitioning between transcriptional states, resolve enhancers to specific cell types, and observe that autism and other neurological traits can be mapped to specific cell types through their enhancers.

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Improved single-cell ATAC-seq reveals chromatin dynamics of in vitro corticogenesis

10.1101/637256 ◽

2019 ◽

Cited By ~ 5

Author(s):

Ryan M. Mulqueen ◽

Brooke A. DeRosa ◽

Casey A. Thornton ◽

Zeynep Sayar ◽

Kristof A. Torkenczy ◽

...

Keyword(s):

Single Cell ◽

Gene Networks ◽

Regulatory Gene ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Chromatin Dynamics ◽

Cell Library ◽

Fate Decision

AbstractDevelopment is a complex process that requires the precise modulation of regulatory gene networks controlled through dynamic changes in the epigenome. Single-cell-omic technologies provide an avenue for understanding the mechanisms of these processes by capturing the progression of epigenetic cell states during the course of cellular differentiation using in vitro or in vivo models1. However, current single-cell epigenomic methods are limited in the information garnered per individual cell, which in turn limits their ability to measure chromatin dynamics and state shifts. Single-cell combinatorial indexing (sci-) has been applied as a strategy for identifying single-cell-omic originating libraries and removes the necessity of single-cell, single-compartment chemistry2. Here, we report an improved sci-assay for transposase accessible chromatin by sequencing (ATAC-seq), which utilizes the small molecule inhibitor Pitstop 2™ (scip-ATAC-seq)3. We demonstrate that these improvements, which theoretically could be applied to any in situ transposition method for single-cell library preparation, significantly increase the ability of transposase to enter the nucleus and generate highly complex single-cell libraries, without altering biological signal. We applied sci-ATAC-seq and scip-ATAC-seq to characterize the chromatin dynamics of developing forebrain-like organoids, an in vitro model of human corticogenesis4. Using these data, we characterized novel putative regulatory elements, compared the epigenome of the organoid model to human cortex data, generated a high-resolution pseudotemporal map of chromatin accessibility through differentiation, and measured epigenomic changes coinciding with a neurogenic fate decision point. Finally, we combined transcription factor motif accessibility with gene activity (GA) scores to directly observe the dynamics of complex regulatory programs that regulate neurogenesis through developmental pseudotime. Overall, scip-ATAC-seq increases information content per cell and bolsters the potential for future single-cell studies into complex developmental processes.

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Chromatin accessibility and regulatory vocabulary across indicine cattle tissues

Genome Biology ◽

10.1186/s13059-021-02489-7 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Pâmela A. Alexandre ◽

Marina Naval-Sánchez ◽

Moira Menzies ◽

Loan T. Nguyen ◽

Laercio R. Porto-Neto ◽

...

Keyword(s):

Motif Discovery ◽

Target Genes ◽

Developmental Stages ◽

Bos Taurus ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Model Organisms ◽

Open Chromatin ◽

Health And Disease ◽

Collaborative Efforts

Abstract Background Spatiotemporal changes in the chromatin accessibility landscape are essential to cell differentiation, development, health, and disease. The quest of identifying regulatory elements in open chromatin regions across different tissues and developmental stages is led by large international collaborative efforts mostly focusing on model organisms, such as ENCODE. Recently, the Functional Annotation of Animal Genomes (FAANG) has been established to unravel the regulatory elements in non-model organisms, including cattle. Now, we can transition from prediction to validation by experimentally identifying the regulatory elements in tropical indicine cattle. The identification of regulatory elements, their annotation and comparison with the taurine counterpart, holds high promise to link regulatory regions to adaptability traits and improve animal productivity and welfare. Results We generate open chromatin profiles for liver, muscle, and hypothalamus of indicine cattle through ATAC-seq. Using robust methods for motif discovery, motif enrichment and transcription factor binding sites, we identify potential master regulators of the epigenomic profile in these three tissues, namely HNF4, MEF2, and SOX factors, respectively. Integration with transcriptomic data allows us to confirm some of their target genes. Finally, by comparing our results with Bos taurus data we identify potential indicine-specific open chromatin regions and overlaps with indicine selective sweeps. Conclusions Our findings provide insights into the identification and analysis of regulatory elements in non-model organisms, the evolution of regulatory elements within two cattle subspecies as well as having an immediate impact on the animal genetics community in particular for a relevant productive species such as tropical cattle.

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Chromatin accessibility dynamics of Chlamydia-infected epithelial cells

10.1101/681999 ◽

2019 ◽

Cited By ~ 2

Author(s):

Regan J. Hayward ◽

James W. Marsh ◽

Michael S. Humphrys ◽

Wilhelmina M. Huston ◽

Garry S.A. Myers

Keyword(s):

Epithelial Cells ◽

Host Cell ◽

Intracellular Signaling ◽

Chlamydial Infection ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Developmental Cycle ◽

Transmitted Infection ◽

The Impact

AbstractChlamydia are Gram-negative, obligate intracellular bacterial pathogens responsible for a broad spectrum of human and animal diseases. In humans, Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and is the causative agent of trachoma (infectious blindness) in disadvantaged populations. Over the course of its developmental cycle, Chlamydia extensively remodels its intracellular niche and parasitises the host cell for nutrients, with substantial resulting changes to the host cell transcriptome and proteome. However, little information is available on the impact of chlamydial infection on the host cell epigenome and global gene regulation. Regions of open eukaryotic chromatin correspond to nucleosome-depleted regions, which in turn are associated with regulatory functions and transcription factor binding. We applied Formaldehyde-Assisted Isolation of Regulatory Elements enrichment followed by sequencing (FAIRE-Seq) to generate temporal chromatin maps of C. trachomatis-infected human epithelial cells in vitro over the chlamydial developmental cycle. We detected both conserved and distinct temporal changes to genome-wide chromatin accessibility associated with C. trachomatis infection. The observed differentially accessible chromatin regions, including several Clusters of Open Regulatory Elements (COREs) and temporally-enriched sets of transcription factors, may help shape the host cell response to infection. These regions and motifs were linked to genomic features and genes associated with immune responses, re-direction of host cell nutrients, intracellular signaling, cell-cell adhesion, extracellular matrix, metabolism and apoptosis. This work provides another perspective to the complex response to chlamydial infection, and will inform further studies of transcriptional regulation and the epigenome in Chlamydia-infected human cells and tissues

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Chromatin accessibility and transcription dynamics during in vitro astrocyte differentiation of Huntington’s Disease Monkey pluripotent stem cells

Epigenetics & Chromatin ◽

10.1186/s13072-019-0313-6 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Alexandra V. Goodnight ◽

Isaac Kremsky ◽

Sujittra Khampang ◽

Yoon Hee Jung ◽

James M. Billingsley ◽

...

Keyword(s):

Cell Cycle ◽

Huntington's Disease ◽

Huntington’S Disease ◽

Neurodegenerative Disorder ◽

Stem Cell Differentiation ◽

Chromatin Accessibility ◽

Global Changes ◽

Neural Lineage ◽

Astrocyte Differentiation

Abstract Background Huntington’s Disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion, resulting in a mutant huntingtin protein. While it is now clear that astrocytes are affected by HD and significantly contribute to neuronal dysfunction and pathogenesis, the alterations in the transcriptional and epigenetic profiles in HD astrocytes have yet to be characterized. Here, we examine global transcription and chromatin accessibility dynamics during in vitro astrocyte differentiation in a transgenic non-human primate model of HD. Results We found global changes in accessibility and transcription across different stages of HD pluripotent stem cell differentiation, with distinct trends first observed in neural progenitor cells (NPCs), once cells have committed to a neural lineage. Transcription of p53 signaling and cell cycle pathway genes was highly impacted during differentiation, with depletion in HD NPCs and upregulation in HD astrocytes. E2F target genes also displayed this inverse expression pattern, and strong associations between E2F target gene expression and accessibility at nearby putative enhancers were observed. Conclusions The results suggest that chromatin accessibility and transcription are altered throughout in vitro HD astrocyte differentiation and provide evidence that E2F dysregulation contributes to aberrant cell-cycle re-entry and apoptosis throughout the progression from NPCs to astrocytes.

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CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo

eLife ◽

10.7554/elife.32341 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 29

Author(s):

Gabriel N Aughey ◽

Alicia Estacio Gomez ◽

Jamie Thomson ◽

Hang Yin ◽

Tony D Southall

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Differentiation ◽

Ectopic Expression ◽

Stem Cell Differentiation ◽

Cell Types ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Global Changes

During development eukaryotic gene expression is coordinated by dynamic changes in chromatin structure. Measurements of accessible chromatin are used extensively to identify genomic regulatory elements. Whilst chromatin landscapes of pluripotent stem cells are well characterised, chromatin accessibility changes in the development of somatic lineages are not well defined. Here we show that cell-specific chromatin accessibility data can be produced via ectopic expression of E. coli Dam methylase in vivo, without the requirement for cell-sorting (CATaDa). We have profiled chromatin accessibility in individual cell-types of Drosophila neural and midgut lineages. Functional cell-type-specific enhancers were identified, as well as novel motifs enriched at different stages of development. Finally, we show global changes in the accessibility of chromatin between stem-cells and their differentiated progeny. Our results demonstrate the dynamic nature of chromatin accessibility in somatic tissues during stem cell differentiation and provide a novel approach to understanding gene regulatory mechanisms underlying development.

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Smarca5 mediated epigenetic programming facilitates fetal HSPC development in vertebrates

Blood ◽

10.1182/blood.2020005219 ◽

2020 ◽

Author(s):

Yanyan Ding ◽

Wen Wang ◽

Dongyuan Ma ◽

Guixian Liang ◽

Zhixin Kang ◽

...

Keyword(s):

Chromatin Remodeling ◽

Chromatin Accessibility ◽

Rna Seq ◽

Chromatin Remodeler ◽

Dynamic Changes ◽

Functional Assays ◽

Epigenetic Programming

Nascent HSPCs acquire definitive hematopoietic characteristics only when they develop into fetal HSPCs; however, the mechanisms underlying fetal HSPC development are poorly understood. Here, we profiled the chromatin accessibility and transcriptional features of zebrafish nascent- and fetal HSPCs using ATAC-seq and RNA-seq and revealed dynamic changes during HSPC transition. Functional assays demonstrated that chromatin remodeler-mediated epigenetic programming facilitates fetal HSPC development in vertebrates. Systematical screening of chromatin remodeler-related genes identified that smarca5 is responsible for the maintenance of chromatin accessibility at promoters of hematopoiesis-related genes in fetal HSPCs. Mechanistically, Smarca5 interacts with Nucleolin to promote chromatin remodeling, thereby facilitating genomic binding of transcription factors to regulate expression of hematopoietic regulators such as bcl11ab. Our results unravel a new role of epigenetic regulation and reveal that Smarca5-mediated epigenetic programming is responsible for fetal HSPC development, which will provide new insights into the generation of functional HSPCs both in vivo and in vitro.

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