The proposal goal is to create a toolkit in which endogenous nuclear bodies (NBs) and chromatin compartments can be reversibly disrupted and endogenous genes can be moved among compartments. This toolkit will allow study of the functional interactions between NBs and chromatin and will be made widely available to the scientific community through the 4D Nucleome consortium. It is clear that chromatin reorganizes during differentiation, yet it is not well-understood how NBs interact with chromatin and influence this reorganization. For example, it has been difficult to study how organization of the repressive chromatin compartment might be seeded or affected by NBs such as the nucleolus because current tools are primarily limited to the study of exogenously expressed components and/or are not rapidly reversible. This toolkit will be developed in mouse embryonic stem cells (mESCs) because they are primary cells that can be differentiated into multiple cell types by end users.
In Specific Aim 1, auxin inducible degrons (AIDs) will be employed to construct systems in which NBs and chromatin compartments are rapidly and reversibly disrupted. The AID sequence will be stably integrated into both alleles of endogenous genes coding for A) proteins essential for the integrity of the nucleolus (as a test NB) and B) lamina proteins that anchor peripheral heterochromatin (PH, as a test compartment). In contrast to current RNAi knockdown or cre/lox-based knockout techniques, this approach will allow rapid degradation of target proteins (minutes/hrs vs days), is not dependent on the cell cycle and is rapidly reversible.
In Specific Aim 2, a novel modified TetO/TetR system will be used to tether specific proteins of interest to individual genes and move genes among compartments. Tethering of specific nucleolar and laminar structural proteins to both active and inactive mESC genes will be used as test cases and proteins that most effectively move genes among compartments will be identified. This tethering system will be more useful than current systems because it will not affect the chromatin context of the target region (as do large repetitive arrays) nor risk off-target effects (as in nuclease deficient dCas9-based approaches). Labeling unique genes is also simpler and quicker as compared to the transcription activator-like effector (TALE) approach, where TALEs targeting multiple sequences must be synthesized.
In Specific Aim 3 we describe proof of principle experiments in differentiating cells to test the redundancy of the heterochromatic compartment and characterize microdomains within the PH. We demonstrate by collaboration that other researchers are interested in these tools. Successful implementation of these tools by the general scientific community would permit detailed study of the role that NBs play in chromatin organization and provide tethering tools to study how genes move between nuclear compartments during both development and disease.
Tools to study how nuclear bodies and compartments affect or potentially seed chromatin organization are currently lacking. This proposal describes creation of a toolkit containing a battery of genetically modified mouse embryonic cells in which 1) nuclear bodies and compartments can be rapidly and reversibly disrupted and 2) different proteins of interest can be tethered to marked genes to study effects on their activity, chromatin state and intranuclear location. This complete toolkit will be made available to the general scientific community for study of nuclear reorganization in both development and disease.
| Casasola, Andrea; Scalzo, David; Nandakumar, Vivek et al. (2016) Prelamin A processing, accumulation and distribution in normal cells and laminopathy disorders. Nucleus 7:84-102 |
| Politz, Joan C Ritland; Scalzo, David; Groudine, Mark (2016) The redundancy of the mammalian heterochromatic compartment. Curr Opin Genet Dev 37:1-8 |
