Characterising open chromatin identifies novel cis-regulatory elements important for paraxial mesoderm formation and axis extension

SUMMARYThe development of multicellular organisms is exquisitely regulated through differential gene activity, which governs cell differentiation programs. However, many details of spatiotemporal control of gene regulation are still poorly understood. We used the accessibility of chick embryos to examine genome-wide signatures characterizing the progressive differentiation of paraxial mesoderm along the head-to-tail axis. Paraxial mesoderm becomes organized into repetitive units, termed somites, the hallmark of the segmented vertebrate body plan. New somite pairs form periodically as the axis extends at the posterior end. This process generates a developmental gradient within a single embryo, with anterior somites more advanced in their differentiation compared to posterior somites. Following somite formation, cell rearrangements generate compartments, comprising lineages of the musculoskeletal system, including cartilage of the vertebral column and ribs, and skeletal muscle cells of the trunk and limbs. To examine how paraxial mesoderm becomes regionalized and patterned to eventually generate these discrete lineages, we investigated dynamic changes of the transcriptome and of chromatin accessibility using RNA-seq and ATAC-seq across a spatiotemporal series along the embryonic axis. Footprint analysis uncovers differential coverage of binding sites for a number of key transcription factors known to be involved in axial patterning and differentiation, including HOX genes. Furthermore, associating accessible chromatin with nearby expressed genes identifies candidate cis-regulatory elements (CRE). As exemplars we use TCF15 and MEOX1, which are crucial for somite formation and differentiation, to experimentally validate CREs in vivo using fluorescent reporters. Time-lapse microscopy reveals CRE spatiotemporal activity and mutation analysis uncovers necessary upstream regulators. The CRE for MEOX1 is conserved and recognized in Xenopus. In addition, a human element is active in chicken. In vivo epigenome editing of TCF15 and MEOX1 CREs disrupts gene expression regulation and recapitulates phenotypic abnormalities of anterior-posterior axis extension.

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Characterising open chromatin in chick embryos identifies cis-regulatory elements important for paraxial mesoderm formation and axis extension

Nature Communications ◽

10.1038/s41467-021-21426-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Gi Fay Mok ◽

Leighton Folkes ◽

Shannon A. Weldon ◽

Eirini Maniou ◽

Victor Martinez-Heredia ◽

...

Keyword(s):

Regulatory Networks ◽

Integrated Approach ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Paraxial Mesoderm ◽

Open Chromatin ◽

Regulatory Circuits ◽

Axis Extension ◽

Footprint Analysis

AbstractSomites arising from paraxial mesoderm are a hallmark of the segmented vertebrate body plan. They form sequentially during axis extension and generate musculoskeletal cell lineages. How paraxial mesoderm becomes regionalised along the axis and how this correlates with dynamic changes of chromatin accessibility and the transcriptome remains unknown. Here, we report a spatiotemporal series of ATAC-seq and RNA-seq along the chick embryonic axis. Footprint analysis shows differential coverage of binding sites for several key transcription factors, including CDX2, LEF1 and members of HOX clusters. Associating accessible chromatin with nearby expressed genes identifies cis-regulatory elements (CRE) for TCF15 and MEOX1. We determine their spatiotemporal activity and evolutionary conservation in Xenopus and human. Epigenome silencing of endogenous CREs disrupts TCF15 and MEOX1 gene expression and recapitulates phenotypic abnormalities of anterior–posterior axis extension. Our integrated approach allows dissection of paraxial mesoderm regulatory circuits in vivo and has implications for investigating gene regulatory networks.

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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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Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements

Development ◽

10.1242/dev.125.22.4349 ◽

1998 ◽

Vol 125 (22) ◽

pp. 4349-4358 ◽

Cited By ~ 4

Author(s):

J. Charite ◽

W. de Graaff ◽

D. Consten ◽

M.J. Reijnen ◽

J. Korving ◽

...

Keyword(s):

Binding Sites ◽

Hox Genes ◽

Regulatory Element ◽

Ectopic Expression ◽

Positional Information ◽

Regulatory Elements ◽

Gene Products ◽

Anteroposterior Patterning

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

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Systematic dissection of genomic features determining transcription factor binding and enhancer function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1621150114 ◽

2017 ◽

Vol 114 (7) ◽

pp. E1291-E1300 ◽

Cited By ~ 74

Author(s):

Sharon R. Grossman ◽

Xiaolan Zhang ◽

Li Wang ◽

Jesse Engreitz ◽

Alexandre Melnikov ◽

...

Keyword(s):

Regulatory Elements ◽

Chromatin Accessibility ◽

Binding Assays ◽

Genomic Locus ◽

Enhancer Activity ◽

Genomic Features ◽

Motif Site ◽

Reporter Assays ◽

Enhancer Function

Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function—including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.

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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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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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A human cell atlas of fetal chromatin accessibility

Science ◽

10.1126/science.aba7612 ◽

2020 ◽

Vol 370 (6518) ◽

pp. eaba7612 ◽

Cited By ~ 1

Author(s):

Silvia Domcke ◽

Andrew J. Hill ◽

Riza M. Daza ◽

Junyue Cao ◽

Diana R. O’Day ◽

...

Keyword(s):

Gene Expression ◽

Human Cell ◽

Single Cells ◽

Complex Trait ◽

Cell Types ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Cell Type ◽

Cell Type Specific

The chromatin landscape underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of chromatin accessibility and gene expression in fetal tissues. For chromatin accessibility, we devised a three-level combinatorial indexing assay and applied it to 53 samples representing 15 organs, profiling ~800,000 single cells. We leveraged cell types defined by gene expression to annotate these data and cataloged hundreds of thousands of candidate regulatory elements that exhibit cell type–specific chromatin accessibility. We investigated the properties of lineage-specific transcription factors (such as POU2F1 in neurons), organ-specific specializations of broadly distributed cell types (such as blood and endothelial), and cell type–specific enrichments of complex trait heritability. These data represent a rich resource for the exploration of in vivo human gene regulation in diverse tissues and cell types.

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Mechanism and functional role of the interaction between CP190 and the architectural protein Pita in Drosophila melanogaster

Epigenetics & Chromatin ◽

10.1186/s13072-021-00391-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Marat Sabirov ◽

Olga Kyrchanova ◽

Galina V. Pokholkova ◽

Artem Bonchuk ◽

Natalia Klimenko ◽

...

Keyword(s):

Regulatory Elements ◽

Open Chromatin ◽

Wild Type ◽

Long Distance ◽

Regulatory Domains ◽

Time Interaction ◽

Architectural Proteins

AbstractBackgroundPita is required for Drosophila development and binds specifically to a long motif in active promoters and insulators. Pita belongs to the Drosophila family of zinc-finger architectural proteins, which also includes Su(Hw) and the conserved among higher eukaryotes CTCF. The architectural proteins maintain the active state of regulatory elements and the long-distance interactions between them. In particular, Pita is involved in the formation of several boundaries between regulatory domains that controlled the expression of threehoxgenes in the Bithorax complex (BX-C). The CP190 protein is recruited to chromatin through interaction with the architectural proteins.ResultsUsing in vitro pull-down analysis, we precisely mapped two unstructured regions of Pita that interact with the BTB domain of CP190. Then we constructed transgenic lines expressing the Pita protein of thewild-typeand mutant variants lacking CP190-interacting regions. We have demonstrated that CP190-interacting region of the Pita can maintain nucleosome-free open chromatin and is critical for Pita-mediated enhancer blocking activity in BX-C. At the same time, interaction with CP190 is not required for the in vivo function of the mutant Pita protein, which binds to the same regions of the genome as the wild-type protein. Unexpectedly, we found that CP190 was still associated with the most of genome regions bound by the mutant Pita protein, which suggested that other architectural proteins were continuing to recruit CP190 to these regions.ConclusionsThe results directly demonstrate role of CP190 in insulation and support a model in which the regulatory elements are composed of combinations of binding sites that interact with several architectural proteins with similar functions.

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Dynamics of Cardiomyocyte Transcriptome and Chromatin Landscape Demarcates Key Events of Heart Development

10.1101/488593 ◽

2018 ◽

Author(s):

Michal Pawlak ◽

Katarzyna Z. Kedzierska ◽

Maciej Migdal ◽

Karim Abu Nahia ◽

Jordan A. Ramilowski ◽

...

Keyword(s):

Heart Development ◽

Regulatory Networks ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Proximal Promoter ◽

Global Changes ◽

Open Chromatin ◽

Sequence Motifs ◽

Loss Of Function ◽

Heart Looping

ABSTRACTThe development of an organ involves dynamic regulation of gene transcription and complex multipathway interactions. To better understand transcriptional regulatory mechanism driving heart development and the consequences of its disruption, we isolated cardiomyocytes (CMs) from wild-type zebrafish embryos at 24, 48 and 72 hours post fertilization corresponding to heart looping, chamber formation and heart maturation, and from mutant lines carrying loss-of-function mutations in gata5, tbx5a and hand2, transcription factors (TFs) required for proper heart development. The integration of CM transcriptomics (RNA-seq) and genome-wide chromatin accessibility maps (ATAC-seq) unravelled dynamic regulatory networks driving crucial events of heart development. These networks contained key cardiac TFs including Gata5/6, Nkx2.5, Tbx5/20, and Hand2, and are associated with open chromatin regions enriched for DNA sequence motifs belonging to the family of the corresponding TFs. These networks were disrupted in cardiac TF mutants, indicating their importance in proper heart development. The most prominent gene expression changes, which correlated with chromatin accessibility modifications within their proximal promoter regions, occurred between heart looping and chamber formation, and were associated with metabolic and hematopoietic/cardiac switch during CM maturation. Furthermore, loss of function of cardiac TFs Gata5, Tbx5a, and Hand2 affected the cardiac regulatory networks and caused global changes in chromatin accessibility profile. Among regions with differential chromatin accessibility in mutants were highly conserved non-coding elements which represent putative cis regulatory elements with potential role in heart development and disease. Altogether, our results revealed the dynamic regulatory landscape at key stages of heart development and identified molecular drivers of heart morphogenesis.

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