Assessing sufficiency of the Tip60/p400 complex in inducing transcription of Merkel Cell Polyomavirus Small T antigen target genes.
Mentor: James DeCaprio, MD (Dana Farber Cancer Institute)
Merkel Cell Polyomavirus (MCPyV) is an etiological agent of virus-positive Merkel Cell Carcinoma, a rare and highly aggressive skin cancer. MCPyV contains genes that code for the tumor antigens, or T antigens, known as small T antigen (ST) and large T antigen (LT). While LT is known to inactivate retinoblastoma protein and inhibit its tumor suppressor function, the function of ST is less well understood. It has been demonstrated through immunoprecipitation experiments that ST binds specifically to L-Myc and the Tip60/p400 complex, a 15-component chromatin modifying complex . The resulting ST-containing complex binds to a plethora of gene promoters to activate gene expression, and these changes in transcriptional regulation may contribute to tumorigenesis. The mechanism of ST-transcriptional activation has not been determined, and is the subject of current research. We hypothesize that ST redirects the EP400 complex to L-myc binding sites. To test this hypothesis, we will determine if redirecting the EP400 complex to L-myc binding sites in the absence of ST can result in similar gene activation. This will be accomplished using the rapamycin-dependent dimerization of Fkbp and Frb to induce binding between a chromatin remodeling complex tagged with Frb and a dCas9-MS2 anchor fused with Fkbp (Figure 2) . We will use different sgRNAs to target the Tip60/p400 complex to previously identified L-myc and ST target genes and observe changes in gene expression. We expect that rapamycin treatment will recruit the V5-Frb-tagged Tip60/p400 complex to the promoters targeted by the sgRNAs. We expect that in the absence of ST the expression level of the target genes will be significantly increased with rapamycin treatment in cells expressing the Tip60/p400 subunit-Frb fusion protein and the VP64-Frb fusion protein (positive control), but not in those expressing the GFP-Frb fusion protein.