The zebrafish (Danio rerio) has been widely used in the study of human disease and development, and about 70% of the protein-coding genes are conserved between the two species
. However, studies in ...zebrafish remain constrained by the sparse annotation of functional control elements in the zebrafish genome. Here we performed RNA sequencing, assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing, whole-genome bisulfite sequencing, and chromosome conformation capture (Hi-C) experiments in up to eleven adult and two embryonic tissues to generate a comprehensive map of transcriptomes, cis-regulatory elements, heterochromatin, methylomes and 3D genome organization in the zebrafish Tübingen reference strain. A comparison of zebrafish, human and mouse regulatory elements enabled the identification of both evolutionarily conserved and species-specific regulatory sequences and networks. We observed enrichment of evolutionary breakpoints at topologically associating domain boundaries, which were correlated with strong histone H3 lysine 4 trimethylation (H3K4me3) and CCCTC-binding factor (CTCF) signals. We performed single-cell ATAC-seq in zebrafish brain, which delineated 25 different clusters of cell types. By combining long-read DNA sequencing and Hi-C, we assembled the sex-determining chromosome 4 de novo. Overall, our work provides an additional epigenomic anchor for the functional annotation of vertebrate genomes and the study of evolutionarily conserved elements of 3D genome organization.
Structural variants (SVs) can contribute to oncogenesis through a variety of mechanisms. Despite their importance, the identification of SVs in cancer genomes remains challenging. Here, we present a ...framework that integrates optical mapping, high-throughput chromosome conformation capture (Hi-C), and whole-genome sequencing to systematically detect SVs in a variety of normal or cancer samples and cell lines. We identify the unique strengths of each method and demonstrate that only integrative approaches can comprehensively identify SVs in the genome. By combining Hi-C and optical mapping, we resolve complex SVs and phase multiple SV events to a single haplotype. Furthermore, we observe widespread structural variation events affecting the functions of noncoding sequences, including the deletion of distal regulatory sequences, alteration of DNA replication timing, and the creation of novel three-dimensional chromatin structural domains. Our results indicate that noncoding SVs may be underappreciated mutational drivers in cancer genomes.
Inherited noncoding genetic variants confer significant disease susceptibility to childhood acute lymphoblastic leukemia (ALL) but the molecular processes linking germline polymorphisms with somatic ...lesions in this cancer are poorly understood. Through targeted sequencing in 5,008 patients, we identified a key regulatory germline variant in GATA3 associated with Philadelphia chromosome-like ALL (Ph-like ALL). Using CRISPR-Cas9 editing and samples from patients with Ph-like ALL, we showed that this variant activated a strong enhancer that upregulated GATA3 transcription. This, in turn, reshaped global chromatin accessibility and three-dimensional genome organization, including regions proximal to the ALL oncogene CRLF2. Finally, we showed that GATA3 directly regulated CRLF2 and potentiated the JAK-STAT oncogenic effects during leukemogenesis. Taken together, we provide evidence for a distinct mechanism by which a germline noncoding variant contributes to oncogene activation, epigenetic regulation and three-dimensional genome reprogramming.
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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