A tripartite DNA-binding element, comprised of the nuclear localization signal and two AT-hook motifs, mediates the association of LEDGF/p75 with chromatin in vivo

摘要

Lens epithelium-derived growth factor p75 (LEDGF/ p75) is a DNA-binding, transcriptionai co-activator that participates in H1V-1 integration site targeting. Using complementary approaches, we determined the mechanisms of LEDGF/p75 DNA-binding in vitroand chromatin-association in living cells. The binding of highfy-puriffed, recombinant protein was assayed by surface piasmon resonance (SPR) and electro-phoretic mobility gel shift. Neither assay revealed evidence for sequence-specific DMA-binding. Residues 146-197 spanning the nuclear localization -signal (NLS) and two AT-hook motifs mediated nonspecific DNA-binding, and DNA-binding deficient mutants retained the ability to efficiently stimulate HIV-1 integrase activity in vitro. Chromatin-association was assessed by visualizing the localization of EGFP fusion proteins in inter phase and mitotic cells. Although a conserved M-terminaS PWWP domain was not required for binding to condensed mitotic chromosomes, its deletion subtly affected the nucleopiasmic distribution of the protein during inter phase. A dual AT-hook mutant associated normally with chromatin, yet when the mutations were combined with NLS changes or deletion of the PWWP domain, chromatin-binding function was lost. As the PWWP domain did not readily bind free DMA in vitro, our results indicate that chromatin-association is primarily affected through DMA-binding, with the PWWP domain likely contributing a protein interaction to the overall affinity of LEDGF/p75 for human chromatin.