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Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions. ​

Literature Information ​

DOI10.1016/j.cell.2015.04.004
PMID25959774
JournalCell
Impact Factor42.5
JCR QuartileQ1
Publication Year2015
Times Cited1116
Keywordstopological chromatin domains, gene-enhancer interactions, CRISPR/Cas genome editing, human limb malformations, structural variants
Literature TypeJournal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.
ISSN0092-8674
Pages1012-1025
Issue161(5)
AuthorsDarío G Lupiáñez, Katerina Kraft, Verena Heinrich, Peter Krawitz, Francesco Brancati, Eva Klopocki, Denise Horn, Hülya Kayserili, John M Opitz, Renata Laxova, Fernando Santos-Simarro, Brigitte Gilbert-Dussardier, Lars Wittler, Marina Borschiwer, Stefan A Haas, Marco Osterwalder, Martin Franke, Bernd Timmermann, Jochen Hecht, Malte Spielmann, Axel Visel, Stefan Mundlos

TL;DR ​

This study reveals that disruptions in topologically associated domains (TADs) can lead to pathological limb malformations in humans by altering the regulatory architecture of the WNT6/IHH/EPHA4/PAX3 locus. By employing CRISPR/Cas genome editing in mice, the researchers demonstrated that such structural changes cause aberrant gene interactions and enhancer misplacement, highlighting the crucial role of TADs in gene expression regulation and providing insights into predicting the pathogenicity of non-coding genomic variants.

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topological chromatin domains · gene-enhancer interactions · CRISPR/Cas genome editing · human limb malformations · structural variants

Abstract ​

Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.

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Primary Questions Addressed ​

  1. How do specific structural changes in TADs influence gene expression in other genomic regions?
  2. What are the potential therapeutic strategies to correct the pathogenic effects of disrupted TADs?
  3. How do different types of structural variants (deletions, inversions, duplications) uniquely affect gene-enhancer interactions?
  4. What role do non-coding regions play in the overall function of TADs and gene regulation?
  5. How can advancements in genome editing technologies improve our understanding of TAD-related diseases?

Key Findings ​

Research Background and Objectives ​

The study investigates how disruptions in topologically associated domains (TADs) can lead to pathogenic alterations in gene-enhancer interactions, contributing to various limb malformations. The primary aim is to elucidate the mechanisms by which structural variants affect chromatin architecture and gene expression, focusing on specific cases of congenital disorders.

Main Methods/Materials/Experimental Design ​

The researchers employed a combination of genetic, molecular, and genomic techniques, including:

  • Identification of Structural Variants: Utilized array comparative genome hybridization (CGH) and whole-exome sequencing to detect structural variants associated with limb malformations in patients.
  • CRISPR/Cas Genome Editing: Generated mouse models mimicking human structural variants to study their effects on limb development.
  • Chromatin Interaction Studies: Employed 4C-seq (circular chromosome conformation capture sequencing) to analyze chromatin interactions in mouse limbs and human fibroblasts.
  • RNA Sequencing: Assessed gene expression changes in limb tissues from both mouse models and human patients.

The overall workflow can be summarized in the following mermaid code:

Mermaid diagram

Key Results and Findings ​

  1. Disruption of TAD Structure: Identified that structural variants at the EPHA4 locus disrupt TAD boundaries, leading to ectopic enhancer-promoter interactions and misexpression of developmental genes such as Pax3, Wnt6, and Ihh.
  2. Phenotypic Correlation: The study linked specific structural variants to distinct limb malformations in both human patients and mouse models, demonstrating a direct connection between genomic alterations and phenotypic outcomes.
  3. Ectopic Interactions: Found that disruptions caused by deletions, inversions, and duplications led to new interactions between enhancers and target genes, significantly altering the expression landscape in limb development.

Main Conclusions/Significance/Innovation ​

The findings highlight the critical role of TADs and their boundary elements in maintaining proper gene regulation during development. The study presents a framework for understanding how structural variants can lead to congenital disorders through the disruption of chromatin architecture. It emphasizes the importance of considering three-dimensional genome organization when assessing the pathogenicity of structural variations.

Research Limitations and Future Directions ​

  • Limitations: The study primarily focused on a specific genomic region (EPHA4 locus) and a limited number of phenotypes, which may not fully represent the complexity of structural variant effects across the genome.
  • Future Directions: Further research is needed to explore the effects of structural variations in other genomic contexts and to develop predictive models for the pathogenicity of structural variants in various diseases. Expanding the analysis to other TADs and their associated phenotypes could enhance understanding of the broader implications of TAD disruptions in human health.

References ​

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  3. Topology of mammalian developmental enhancers and their regulatory landscapes. - Wouter de Laat;Denis Duboule - Nature (2013)
  4. Structural variations, the regulatory landscape of the genome and their alteration in human disease. - Malte Spielmann;Stefan Mundlos - BioEssays : news and reviews in molecular, cellular and developmental biology (2013)
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  6. Deletions, Inversions, Duplications: Engineering of Structural Variants using CRISPR/Cas in Mice. - Katerina Kraft;Sinje Geuer;Anja J Will;Wing Lee Chan;Christina Paliou;Marina Borschiwer;Izabela Harabula;Lars Wittler;Martin Franke;Daniel M Ibrahim;Bjørt K Kragesteen;Malte Spielmann;Stefan Mundlos;Darío G Lupiáñez;Guillaume Andrey - Cell reports (2015)
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Literatures Citing This Work ​

  1. A CRISPR Connection between Chromatin Topology and Genetic Disorders. - Bing Ren;Jesse R Dixon - Cell (2015)
  2. Architectural proteins, transcription, and the three-dimensional organization of the genome. - Caelin Cubeñas-Potts;Victor G Corces - FEBS letters (2015)
  3. Genome organization: Disorder — from chromatin to limb development. - Bryony Jones - Nature reviews. Genetics (2015)
  4. Non-coding genetic variants in human disease. - Feng Zhang;James R Lupski - Human molecular genetics (2015)
  5. The oncogenic BRD4-NUT chromatin regulator drives aberrant transcription within large topological domains. - Artyom A Alekseyenko;Erica M Walsh;Xin Wang;Adlai R Grayson;Peter T Hsi;Peter V Kharchenko;Mitzi I Kuroda;Christopher A French - Genes & development (2015)
  6. An Overview of Genome Organization and How We Got There: from FISH to Hi-C. - James Fraser;Iain Williamson;Wendy A Bickmore;Josée Dostie - Microbiology and molecular biology reviews : MMBR (2015)
  7. Three Dimensional Organization of the Nucleus: adding DNA sequences to the big picture. - David M Gilbert;Peter Fraser - Genome biology (2015)
  8. Analysis methods for studying the 3D architecture of the genome. - Ferhat Ay;William S Noble - Genome biology (2015)
  9. Chromatin Insulators and Topological Domains: Adding New Dimensions to 3D Genome Architecture. - Navneet K Matharu;Sajad H Ahanger - Genes (2015)
  10. Structural and functional diversity of Topologically Associating Domains. - Job Dekker;Edith Heard - FEBS letters (2015)

... (1106 more literatures)

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