Despite extensive correlations, a direct functional relationship has not yet been established between the highest levels of chromatin folding, or large-scale chromatin structure, and regulation of transcription. Similarly, despite extensive correlations of changes in intranuclear gene positioning and association with specific nuclear compartments, we still do not know how genes change intranuclear positions and associations, let alone understand the functional consequences of these changes. Our long-term objectives are to determine the large-scale chromatin folding and intranuclear positioning of specific gene loci, to identify the cis and trans determinants of this folding and intranuclear positioning, and to understand the functional significance of this level of chromatin organization with regard to transcriptional regulation.
The specific aims of this proposal are to: : 1. Determine the relationship between large-scale chromatin compaction versus transcriptional activation. 2. Determine the relationship between nuclear speckle compartmentalization versus transcriptional activity and/or RNA processing. 3. Determine the relationship between localization to the nuclear periphery and specific epigenetic modifications versus transcription activity and induction. This project will exploit a novel system using BAC transgenes to reconstitute key features of endogenous gene loci as well as new methodologies for visualizing specific chromosome loci in live cells and at the ultrastructural level. Our project will critically test both old and new models for how the highest levels of chromatin folding modulate gene expression. Additional impact on a still wider range of basic science and biotechnology will come from our continued development of new technology for visualizing chromosome structure and dynamics and application of these methods for improved transgene and multi-transgene expression.
A major impediment to development of gene therapy methods is our incomplete understanding of how to ensure high and sustained levels of expression from transgenes. Insight from our studies should be useful in guiding the design of future gene constructs and artificial chromosomes used in gene therapy.
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|Chen, Yu; Zhang, Yang; Wang, Yuchuan et al. (2018) Mapping 3D genome organization relative to nuclear compartments using TSA-Seq as a cytological ruler. J Cell Biol 217:4025-4048|
|van Steensel, Bas; Belmont, Andrew S (2017) Lamina-Associated Domains: Links with Chromosome Architecture, Heterochromatin, and Gene Repression. Cell 169:780-791|
|Teng, Kai Wen; Ishitsuka, Yuji; Ren, Pin et al. (2016) Labeling proteins inside living cells using external fluorophores for microscopy. Elife 5:|
|Tajik, Arash; Zhang, Yuejin; Wei, Fuxiang et al. (2016) Transcription upregulation via force-induced direct stretching of chromatin. Nat Mater 15:1287-1296|
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|Kaya-Okur, Hatice S; Belmont, Andrew S (2015) CRISPR EATING on a Low Budget. Dev Cell 34:253-4|
|Khanna, Nimish; Hu, Yan; Belmont, Andrew S (2014) HSP70 transgene directed motion to nuclear speckles facilitates heat shock activation. Curr Biol 24:1138-44|
|Belmont, Andrew S (2014) Large-scale chromatin organization: the good, the surprising, and the still perplexing. Curr Opin Cell Biol 26:69-78|
|Khanna, Nimish; Bian, Qian; Plutz, Matt et al. (2013) BAC manipulations for making BAC transgene arrays. Methods Mol Biol 1042:197-210|
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