TLS stands for translocated in liposarcoma in which the gene is fused to CHOP due to a reciprocal chromosome translocation between chr. 12 and 16. A similar molecular alteration was also found in a subset of leukemia. This finding suggests an important role for TLS in cancer. The TLS gene contains an RNA recognition motif (RRM), a Zn finger, and a transcriptional activation domain indicating that it may play a role in transcription and RNA processing. The TLS protein colocalizes with splicing factors in the nucleus, consistent with its involvement in RNA splicing. In keeping with the lab's long-term interest in understanding the potential coupling between transcription and splicing, I proposed to take advantage of a set of new technologies recently developed in the Fu lab. My goal is to determine (1) whether TLS directly interacts with DNA and RNA, and if so, what are its direct targets; (2) how TLS is involved in transcription and splicing or both, and (3) whether the protein uses its RNA binding domain to couple transcription and splicing and/or uses RNA as ligands to regulate its activity in transcription.

My current studies involve two areas of new data. One concerns the finding that TLS binding to RNA is regulated by PKC. The other shows the identification of gene promoters that are directly bound by Drosha, a key component for processing microRNAs. The reason I did the promoter array experiment with Drosha is because TLS was recently reported to form a complex with Drosha. My findings now suggest a new paradigm that TLS may function together with Drosha for co-transcriptional processing of microRNAs, which may in turn serve as ligands to regulate the activity of TLS. Because of our shared interest in this important gene with the Glass and Rosenfeld labs at UCSD, we decided to jointly pursue this project in the near future.

In addition to previously mentioned future experiments, I am interested in testing truncation mutants and particularly the oncogenic fusions of TLS, and determining the varied effects in vivo. I have designed various constructs and plan to deliver them by retrovirus-mediated transduction to TLS null MEFs to determine the role of various domains in TLS-mediated transcription. In particular, I am interested in determining the requirement for the RNA recognition motif (RRM) in regulated transcription. I plan to perform Chip-DSL, splicing array, and transcriptional assays with the MEFs complemented by wt and mutant TLS proteins, including the oncogenic TLS-CHOP fusion protein. I believe that this will reveal how these proteins lead to cancer and potentially expose therapeutic targets.

PUBLICATIONS (resulting from this training)

Dinakar R, Fu XD. (2007) TLS/FUS regulates both transcription and splicing in vivo through complete tranversion of hyperacetylated coding regions. In Preparation.