Purchase this article with an account.
D.P. Singh, E. Kubo, T. Shinohara, L.T. Chylack,Jr, N. Fatma; DNA Binding Domains of LEDGF: Contribution of the Two Helix– Turn–Helix (HTH) and One Zinc Finger–Like Domains to the Trans–activation potential of LEDGF . Invest. Ophthalmol. Vis. Sci. 2004;45(13):393.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose:LEDGF, a nuclear protein, binds to consensus core DNA sequences of nAGGn or nGAAn repeats, and trans–activates stress response genes, and thus provides cytoprotection. To understand how LEDGF protein coordinates and regulates a diverse set of genes, will require the comprehensive knowledge of its functional domains. We characterized these domains in LEDGF by analyzing mutated proteins in DNA binding and transactivation experiments. In addition, we identified the contributions of the transactivation potential of each domain. Methods: LEDGF proteins; LEDGF–F (amino acids (aa);1– 530), NI (1–187), NII (1–135), NIII (1–101), CI (199–530), CII (302–530), CIII (322–530), CIV (348–530), and CV (418–530) were tagged with green fluorescent protein (GFP) or HA by cloning into the TOPO–GFP and/or TOPO–HA vectors and expressed in eukaryotic and prokaryotic cells, respectively. Expression was analyzed by fluorescence microscopy and by western blotting. EMSA and CAT–ELISA assays were used to analyze the functional DNA binding domains. Site–directed mutagenesis was carried out to define DNA binding domains. Hsp27 and αB–crystallin promoters were used to monitor the transactivation–potentials of each LEDGF truncated construct. Results: LEDGF contained three DNA binding domains. N–terminal LEDGF, a stretch of 70 amino–acids (5–70) displays A/TGGGGA/T binding and repressive activity. The C–terminus of LEDGF contains two HTH domains at positions; 421–442 and 471–492. These bind to nGAA and contribute in a cooperative manner to the transactivation potential of the LEDGF. Deletion of either HTH domain significantly reduces the transactivation potential. In addition, deletion of the N–terminal LEDGF (aa;1–187) greatly enhances the transactivation potential of LEDGF. Conclusions: Collectively, our results demonstrate that LEDGF proteins contains three distinct functional domains and that may regulate gene expression and modify the physiological condition of cells.
This PDF is available to Subscribers Only