This proposal will investigate RNA-directed regulation of transcription by the RNA- binding protein FUS. FUS has been shown to be translocated in liposarcomas, sarcomas and acute myeloid leukemia (AML). Also, several point mutations in FUS have been strongly associated with ALS and frontal temporal dementia. The discovery that mutations in FUS cause an estimated 5% of cases of familial type ALS has been regarded by the ALS association as "one of the most significant breakthroughs in recent history of ALS research". Despite excitement over the discovery, poor understanding of the role that FUS plays in cells and the nucleic acid sequences and structures that FUS binds has limited understanding about the possible role mutated FUS proteins may play in these diseases. My preliminary investigations have provide support to the model that FUS regulates transcription. This model proposed in a 2008 paper from the Rosenfeld lab, suggested that FUS is recruited to chromatin by noncoding RNA transcripts and inhibits histone acetylation in gene promoters, thereby inhibiting transcription. My preliminary microarray and sequencing data also supports the model that FUS is an inhibitor of transcription and associates with chromatin. I will use high-throughput sequencing techniques to discover genes in human cells that are regulated by direct interactions with FUS. I will use molecular techniques in cells where FUS is expressed or silenced to confirm that these genes are inhibited by FUS associating with RNAs transcribed from their promoters. I will also use sequencing and molecular techniques to determine whether FUS regulates transcription by affecting histone acetylation or through some other mechanism. For a selection of the genes I have discovered are regulated by FUS in human cells, I will clone the RNA targets of FUS and use purified FUS protein and RNA to study the sequence and structures required for this interaction. I will elucidate the minimum sequence and structure required for high affinity FUS binding. I will then test the ability of the FUS:noncoding RNA complex to inhibit histone acetylation with an in vitro assay using purified components. Finally, will develop mutants of FUS that are either unable to inhibit histone acetylation or to bind nuclei acid. I will reintroduce this mutated protein into cells to test whether the mutated FUS protein can regulate expression of its target genes. Completion of this study will provide a list of genes regulated by FUS, solidify a mechanism for the RNA-directed recruitment of FUS and demonstrate how these interactions are required for FUS activity in cells.
This project will discover genes whose expression is regulated by the protein FUS, an RNA- binding protein that is mutated in several cancers, ALS, and frontal temporal dementia. I will determine the nucleic acid sequence and structure required for FUS binding and test the role that these nucleic acids play in FUS'ability to regulate gene expression. This study will provide important insight into the role of FUS in cells and the mechanism by which FUS works, so that more direct hypotheses can be developed about the role of FUS in diseases such as cancer and ALS.
|Schwartz, Jacob C; Podell, Elaine R; Han, Steve S W et al. (2014) FUS is sequestered in nuclear aggregates in ALS patient fibroblasts. Mol Biol Cell 25:2571-8|