Abstract
Abstract: :
Purpose: LEDGF, a growth and survival factor, localizes to the nucleus, binds to, and activates the heat shock element (HSE) and the stress-related element (STRE). LEDGF contains 7 potential nuclear localization signals (NLS), four potential DNA-binding domains, one basic leucine zipper (Zip), and three helix-turn-helix domains (HTH1-3). We have identified a potential Tat-domain that may enable trafficking of LEDGF through plasma membrane. LEDGF lacks a signal peptide, yet appears to be transported from extracellular space into nucleus. Methods: Mutagenesis and electrophoretic mobility shift assays (EMSA) were used to assess LEDGF-DNA binding. Truncated GFT-LEDGFs were used to identify functional significance of each NLS and the role of Tat domain in trafficking. Results: The HTH1 and Zip sequences in the N-terminusl overlap. The HTH2 and HTH3 sequences are in close proximity and located in the C-terminus. A partial LEDGF fragment in the N-terminus containing HTH1+Zip binds to STRE, but not to HSE. The C-terminal region of LEDGF containing HTH2 and HTH3 binds to HSE, but not to STRE. EMSA studies using mutant DNA probes confirmed these bindings. Sequences of HTH2 and HTH3 are highly homologous with HTH1 and HTH2 in the DNA binding domain of heat shock regulatory factor 1. The functional NLS was found to be the NLS2 site. That LEDGF has a consensus Tat domain is consistent with the finding that LEDGF moves in and out of cells without a signal peptide. We found GFP-LEDGF in culture medium of LECs over-expressing GFP-LEDGF, and in nuclei of normal cells cultured in medium containing GFP-LEDGF. Conclusion: We defined a functional NLS and defined a possible Tat domain in LEDGF. Even though LEDGF lacks a signal peptide, it is transported from the extra-cellular space to the nucleus. It is a bipartite trans-activator binding to two different promoter elements, HSE and STRE, to activate genes having these elements.
Keywords: 417 gene/expression • 423 growth factors/growth factor receptors • 476 molecular biology