|dc.description.abstract||Chromatin remodeling is a widespread nuclear function that impacts almost every aspect of nuclear physiology. Thus, the action of SWI/SNF chromatin remodelers can impact a broad range of cellular processes. SWI/SNF remodelers traditionally have been thought to localize to euchromatin via bromodomains to activate transcription. However, SWI/SNF remodelers are also involved in a broad range of other functions including transcriptional repression, the DNA damage response, and specification of boundary elements.
We developed a method of examining the binding of the mammalian SWI/SNF remodeler, PBAF, to chromatin with single particle precision. This involves tagging the BAF180 subunit within PBAF with a modified bacterial haloalkane dehalogenase: the halo tag. This allows BAF180 to be conjugated to bright organic fluorophores. When imaged at slow acquisition times, molecules that are freely diffusing will blur into the background and stationary molecules are thus able to be resolved with single particle precision. We were able to further localize PBAF binding activity by performing two-color imaging between Halo-BAF180 and chromatin structural proteins tagged with the SNAP tag: HP1a and H3.3. We added the SNAP tag to H3.3, which is associated with transcriptionally active euchromatin, and HP1a, which is associated with transcriptionally repressed heterochromatin. We also found that PBAF resided in the transcriptionally active euchromatin for less time than it did in transcriptionally repressed heterochromatin, which we think is linked with chromatin stability. Furthermore, we found that for PBAF, the BAF180 bromodomains didn't have an appreciable effect on the amount of time that PBAF resided within euchromatin. Surprisingly, we found that the BAF180 bromodomains did affect the amount of time that PBAF resided in heterochromatin, suggesting that it targets an unknown acetylated factor in heterochromatin.
Apart from its extensive role in various aspects of cellular physiology, PBAF is also highly mutated across a range of cancers. These PBAF mutations were thought to be associated exclusively with tumor suppressive functions. However, recent literature has shown that certain PBAF subunits can also be over-expressed in neoplastic processes and that this overexpression can drive tumorigenesis. We found that overexpression of a BAF180WT transgenic construct in U2-OS cells leads to increased cellular migration in a wound healing assay, decreased cellular adhesion, and increased colony size in colony formation assays. These are all traits that are associated with tumor progression. We examined expression of a cancer-derived missense mutation within the second bromodomain of BAF180 (BAF180-T232P) and found that it leads to truncation of the protein after the third bromodomain, which likely inactivates PBAF function. We find that Halo-fBAF180-T232P didn't appear to have these same effects on wound healing, cellular adhesion, and colony formation. Thus ectopic expression of a Halo-fBAF180WT construct appeared to increase tumorigenic behavior in U2-OS cells and expression of a point mutation in a bromodomain (Halo-fBAF180-T232P) reversed this tumorigenic function. We further performed ChIP-exo of both of these constructs and found that Halo-fBAF180WT localized to genomic regions associated with focal adhesion, WNT signaling, and TGF(3 signaling. Finally, we performed dynamic fluorescent cellular imaging of cells expressing either Halo-fBAF180WT or HalofBAF180-T232P. We found that in FRAP assays, Halo-fBAF180-T232P exhibited rapid recovery dynamics when compared to Halo-fBAF180WT. When performing single particle slow-diffusion imaging however, we found that the amount of time that HalofBAF180WT attached to chromatin was comparable to Halo-fBAF180-T232P. Thus, Halo-fBAF180-T232P appears to have a defect in association with chromatin, but once bound it doesn't appear to have a deficit in residence on chromatin.
Our work thus describes the development of a system that can survey chromatin residence times and bromodomain-dependent interactions. We have also defined a potential tumorigenic function of BAF180 in U2-OS cells, and determined that mutations within a single bromodomain could reduce this tumorigenic function.||en_US