Although the human genome encodes about 30 different ATP-dependent chromatin remodeling complexes, their mechanism[s] are largely unknown. In part, this is due to the lack of rapidly acting small molecule inhibitors that could permit precise temporal analysis needed for mechanistic studies. One family of these complexes based on the Brg and Brm ATPases (BAF or mSWI/SNF complexes) are both necessary and in some contexts sufficient to induce the pluripotent state. BAF complex subunits are also tumor suppressors and undergo loss of heterozygosity in certain human malignancies. Finally, they have been shown to be necessary for explicative senescence and their deletion increases the proliferative life of human fibroblasts. Proteomic analysis indicates that the diverse functions of BAF complexes are the result of combinatorial assembly of the complexes from gene families encoding the subunits. Genome wide analysis of the occupancy of BAF complexes in ES cells has revealed that they most commonly suppress their target genes from a distance, suggesting an unanticipated mechanism of action. To understand the poorly defined mechanisms used by SWI/SNF-like BAF complexes to promote pluripotency and suppress tumors, this project aims to identify a comprehensive set of rapidly acting small- molecule inhibitors using a knock-in ESC reporter line. This line contains luciferase inserted into the Bmi1 locus and undergoes rapid activation upon genetic deletion of the ATPase subunit of the BAF complex. The 2 MDa complex contains 13 subunits and has an expected surface area of 0.5 to 2.5 million E2. The extraordinarily large surface of the complex is hypothesized to carry out a sequential series of reactions that can be ordered and probed with small molecules. Once identified, we will define the binding sites of the small molecule inhibitors using established embryonic stem cell lines with null mutations in subunits of the complex. Because the inhibitors could also block regulatory mechanisms impinging upon BAF complexes, we will characterize their effect upon defined post-translational modifications of the complex. The time-of-action of each of the inhibitors in both the repression of Polycomb genes and the activation of important pluripotency targets such as Fgf4 and Bmp4 will help to further define the level of function of each of the rapidly acting inhibitors. Specifically, their effects will be determined on higher-order chromatin structures, long-range interactions, chromatin accessibility, nucleosome positioning, and transcription factor occupancy. This comprehensive and systematic investigation should lead to deeper understanding of the actions of this chromatin-remodeling complex, which plays an essential role in pluripotency, human tumor suppression and cellular senescence. In addition, this valuable toolbox of small molecule probes will be invaluable for other researchers studying chromatin regulation.
We intend to find small molecules that inhibit the activities of a complex of proteins that control the packaging of DNA into the nucleus of a cell. This protein complex plays a critical role in human embryonic stem cells and human tumor suppression. Inhibitors of this complex could lead to improved stem cell therapies and new cancer treatments.
| Dykhuizen, Emily C; Hargreaves, Diana C; Miller, Erik L et al. (2013) BAF complexes facilitate decatenation of DNA by topoisomerase II?. Nature 497:624-7 |
