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

Abstract

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a DNA-binding, transcriptional co-activator that participates in HIV-1 integration site targeting. Using complementary approaches, we determined the mechanisms of LEDGF/p75 DNA-binding in vitro and chromatin-association in living cells. The binding of highly-purified, recombinant protein was assayed by surface plasmon resonance (SPR) and electrophoretic mobility gel shift. Neither assay revealed evidence for sequence-specific DNA-binding. Residues 146–197 spanning the nuclear localization signal (NLS) and two AT-hook motifs mediated non-specific 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 interphase and mitotic cells. Although a conserved N-terminal PWWP domain was not required for binding to condensed mitotic chromosomes, its deletion subtly affected the nucleoplasmic distribution of the protein during interphase. 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 DNA in vitro, our results indicate that chromatin-association is primarily affected through DNA-binding, with the PWWP domain likely contributing a protein interaction to the overall affinity of LEDGF/p75 for human chromatin.

Integrase, LEDGF/p75 and HIV replication

“…In vitro evidence supports an enzymatic cofactor role, since recombinant p75 can augment purified IN protein catalysis (the strand transfer component) with oligonucleotide substrates [73,79,87,89]. The equivalent 3′ processing and strand transfer capacity of PICs isolated from +/+ and −/− MEFs [101] is consistent with the primary effect requiring engagement with chromatin-resident p75.…”

“…The relative contributions of binding to DNA versus protein in chromatin are not known, for this protein or for PWWP domain proteins in general. While sequence-specific in vitro DNA binding of p75 to certain short stress response and heat shock elements was reported [68], this was not verified in a later study [89]. Non-specific in vitro DNA binding activity was detected for the NLS-AT hook region, but deletion of both these elements (or even the PWWP domain in addition) had only minor effects on in vitro integration [89], highlighting further the disconnect between such assays and the true in vivo situation.…”

“…The interaction between p75 and IN is direct [73] and it is confined to only one of the seven retroviral genera, lentivirinae [75,87,100]. A number of laboratories established the role of p75 in chromatin tethering, mapped relevant functional domains, and worked to answer the question of virological relevance [74][75][76][77][78][79][80][81][82][85][86][87][88][89][90][91]93,[95][96][97][100][101][102][103][104][105][108][109][110]. The first indication of the protein's significance was the clarification it provided about the vexing issue of the intracellular trafficking of retroviral integrase proteins.…”

“…Deletion of this domain or just its beta-barrel sub-domain from p75 disrupts microscopically detectable tethering to condensed chromosomes [93]. There is some disagreement, however, since Turlure et al reported chromosome-tethering to be fully preserved after PWWP domain deletion [89]. Analyzed biochemically, deletion of the domain impairs high affinity (Triton X-100-refractory) chromatin binding, and additional deletion of the AT Hook domain abolishes it [93].…”

“…Two additional charged regions (CR2 and CR3) in the N-terminal region also enhance the dominant activity of the PWWP and AT hook domains, with full Triton-resistance only achieved when the Nterminal donor to GFP extends all the way through CR2 [93]. The p75 NLS has been reported to influence p75 chromatin interaction, perhaps through non-specific DNA binding properties [89]. However, the NLS-mutant protein (p75.NLS − ) is cytoplasmic when transiently expressed but becomes efficiently chromatin-trapped as soon as cells transit mitosis; when stably expressed p75.NLS − displays the same constitutively chromatin-bound phenotype as the normal protein, suggesting that the significant role of the NLS is rather to mediate nuclear entry [80].…”