? PROJECT 4: UW-CNOF BIOLOGICAL MODEL DEVELOPMENT AND DATA GENERATION Projects 1-3 develop new experimental and computational methods for mapping and modeling genome architecture and then validate these methods against established benchmarks in molecular cytogenetics. As these technologies come into place, Project 4 focuses on establishing biological models for studying nuclear architecture and assessing how this architecture evolves during development, disease, or in response to environmental stress. We will use our well-developed system of differentiating human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) into cardiomyocytes and endothelial cells. This system has been used successfully as part of the ENCODE project to identify dynamic chromatin signatures that mark cardiovascular developmental regulators. Recent studies suggest that differences in nuclear architecture are established early in development, but we know little about how stable these differences are or whether structurally interacting domains can identify new regulatory pathways. Similarly, we know almost nothing about how nuclear architecture evolves during disease or whether such changes are drivers or passengers in disease pathogenesis. Our goals are to perform a key series of experiments that will be enabled by DNase Hi- C and other mapping approaches in our program.
In Aim 1, we will define the dynamics of nuclear architecture during the differentiation of nave hESCs into cardiomyocytes and endothelial cells. This study utilizes the newly derived ELF1 cell line, a nave hESC at the earliest stage of development, and tracks the dynamics of nuclear architecture using bulk and single-cell DNase Hi-C. We will study differentiation into primed hESCs, mesoderm, cardiovascular progenitors, and definitive cardiomyocytes and endothelium. Comparison with established transcription factor and epigenetic networks will identify spatial clusters of coordinately activated and repressed genes that regulate heart development.
In Aim 2, we will test the hypothesis that cardiomyopathy-inducing mutations in the nuclear scaffolding protein, lamin A/C (LMNA), are associated with derangements in cardiomyocyte nuclear architecture. We will study hiPSCs from patients with LMNA-induced dilated cardiomyopathy and genetically repaired, isogenic controls to determine if LMNA mutations unfavorably change nuclear architecture in cardiomyocytes. Additionally, we will test the hypothesis that sub-lethal mechanical stress exacerbates this deranged architecture.
In Aim 3, we will determine the changes in nuclear architecture induced by trisomy 21 (Down Syndrome). Down Syndrome is the most common cause of congenital heart disease, and we hypothesize that the additional chromosome 21 results in disease-causing alterations in nuclear structure. We will study isogenic lines of hiPSCs with and without trisomy 21 in bulk and at the single-cell level to determine how nuclear architecture is perturbed by an additional chromosome 21. Interactions that are gained or lost will identify candidate loci for causing congenital heart disease.
| Ma, Wenxiu; Bonora, Giancarlo; Berletch, Joel B et al. (2018) X-Chromosome Inactivation and Escape from X Inactivation in Mouse. Methods Mol Biol 1861:205-219 |
| Ma, Wenxiu; Ay, Ferhat; Lee, Choli et al. (2018) Using DNase Hi-C techniques to map global and local three-dimensional genome architecture at high resolution. Methods 142:59-73 |
| Wray-Dutra, Michelle N; Chawla, Raghav; Thomas, Kerri R et al. (2018) Activated CARD11 accelerates germinal center kinetics, promoting mTORC1 and terminal differentiation. J Exp Med 215:2445-2461 |
| Bonora, G; Deng, X; Fang, H et al. (2018) Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome. Nat Commun 9:1445 |
| Kim, Seungsoo; Liachko, Ivan; Brickner, Donna G et al. (2017) The dynamic three-dimensional organization of the diploid yeast genome. Elife 6: |
| Ramani, Vijay; Deng, Xinxian; Qiu, Ruolan et al. (2017) Massively multiplex single-cell Hi-C. Nat Methods 14:263-266 |
| Eser, Umut; Chandler-Brown, Devon; Ay, Ferhat et al. (2017) Form and function of topologically associating genomic domains in budding yeast. Proc Natl Acad Sci U S A 114:E3061-E3070 |
| Bonora, Giancarlo; Disteche, Christine M (2017) Structural aspects of the inactive X chromosome. Philos Trans R Soc Lond B Biol Sci 372: |
| Cao, Junyue; Packer, Jonathan S; Ramani, Vijay et al. (2017) Comprehensive single-cell transcriptional profiling of a multicellular organism. Science 357:661-667 |
| Qiu, Xiaojie; Hill, Andrew; Packer, Jonathan et al. (2017) Single-cell mRNA quantification and differential analysis with Census. Nat Methods 14:309-315 |
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