There are a number of nuclear factors that play an integral role in transcriptional elongation. Once such factor is hIws1, a nuclear protein first characterized to bind the histone H3 chaperone, Spt6. Our lab previously showed that Spt6 binds directly to the phosphorylated C-terminal tail of RNA polymerase II (RNAPII), and recruits Iws1 to control specific events that occur during elongation, including mRNA processing, mRNA export, and histone H3K36 methylation. hIws1 interacts directly with the histone H3K36 methyltransferase, Hypb/Set2, as well as the mRNA export adaptor protein, REF1/Aly. Iws1 is reportedly an essential mammalian protein, but little is know about its structure or functional domains.
In the present study, I constructed GST tagged hIws1 protein constructs that were subsequently used in pulldown assays to evaluate its nuclear binding partner proteins. Pulldown assays were performed with either HeLa whole cell extract or HeLa nuclear extract. The N-terminal region of Iws1 (residues 1 through 495) was found to associate with the LEDGF/p75, a protein that has been previously identified to take part in lentiviral genome integration. In addition, I found that the C-terminal region of Iws (residues 522 through 819) binds to the E74-like factor 1 (Elf1). Interestingly, the hIws1 C-terminus contains a novel domain called the LW motif, which is highly conserved in Iws1 proteins from other species, and is also present in two transcription factors, Med26 and TFIIS. To test the role of the LW domain, I created three GST-Iws1 point mutant proteins, which were purified and used in GST-pulldown assays. Two residues (tyrosine-665 and tryptophan-685) contained within the LW motif were found to be crucial for the hIws1-Spt6 interaction. By contrast, Med26 was found to also associate with Iws1, but this interaction was not mediated by the LW motif, and Iws1 also binds to the LEDGF factor through a region outside of the LW sequence. Thus these findings identify a new domain that is likely to be critical for binding of Iws1 to Spt6 and assembly of the RNAPII elongation complex.
|Advisor:||Jones, Katherine A.|
|Commitee:||Johnson, Tracy, Jones, Katherine A., Kadonaga, James T.|
|School:||University of California, San Diego|
|School Location:||United States -- California|
|Source:||MAI 48/01M, Masters Abstracts International|
|Keywords:||Iws, LW motif, Spt6, Tfiis, hIws1|
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