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. 2006 Mar 20;34(5):1653-65.
doi: 10.1093/nar/gkl052. Print 2006.

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

Fanny Turlure  1 , Goedele Maertens, Shaila Rahman, Peter Cherepanov, Alan Engelman
Affiliations

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

Fanny Turlure et al. Nucleic Acids Res. .

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.

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Figures

Figure 1
Figure 1
LEDGF/p75 binds to various DNA sequences in vitro. (A) Sequences of 21 bp substrates. The HSE sequence, containing two 5′-GAA motifs (underlined), has a G/C content of 47%. Mut1 (5% G/C rich) and Mut2 (24% G/C rich) were derived from HSE by mutating G or C residues to A or T (17). Altered residues are in bold type. (B) Binding of LEDGF/p75 to DNA as determined by SPR. The response for each substrate is plotted as a function of time. Values obtained from a blank streptavidin-coated surface were subtracted from DNA-dependent signals. Similar results were obtained over multiple (more than a dozen) independent experiments. (C) A subset of the LEDGF proteins used in this study. Approximately 2 µg of the following purified proteins were detected by staining with Coomassie blue following polyacrylamide gel electrophoresis: lane 1, non-tagged wild-type LEDGF/p75; lane 2, PreScission Protease-treated wild-type LEDGF/p75; lane 3, 347–530; lane 4, 1–116; lane 5, 93–226; lane 6, MutL1; lane 7, MutL3; lane 8, MutL4; lane 9, MutL5. The migration positions of molecular mass standards in kDa are shown next to the gel. (D) LEDGF/p75 DNA-binding as determined by EMSA. The reactions in lanes 2, 9 and 16 contained 125 nM LEDGF/p75. Lanes 3, 10 and 17 contained 250 nM LEDGF/p75; lanes 4, 11 and 18, 500 nM; lanes 5, 12 and 19, 1 µM; lanes 6, 13 and 20, 2 µM; lanes 7, 14 and 21, 4 µM. LEDGF/p75 was omitted from the reactions in lanes 1, 8 and 15. The migration positions of starting substrates, shifted complexes and slowly-migrating high molecular weight (HMW) complexes are indicated. Similar results were obtained following multiple independent experiments. (E) Supershift experiment. LEDGF/p75 was omitted from the reaction in lane 1. Lane 2 contained 2 µM LEDGF/p75 and 100 nM Mut1 DNA. Lane 3, anti-LEDGF/p75 antibody was added 5 min after LEDGF/p75 and DNA. Lane 4, same as lane 3 except control IgG1 was substituted for the specific antibody. Results are representative of five independent supershift experiments.
Figure 2
Figure 2
LEDGF/p75 residues 93–226 harbor DNA-binding activity. (A) Schematic representation of wild-type LEDGF/p75 and deletion mutant proteins. Conserved sequence motifs/functional domains are indicated above the protein, with boundaries indicated below. NLS, nuclear localization signal; IBD, integrase-binding domain. (B) DNA-binding activities of deletion mutant proteins. DNA (100 nM Mut1) was reacted with the following protein concentrations: 250 nM (lanes 2, 5, 8, 11 and 14), 2 µM (lanes 3, 6, 9, 12 and 15) or 5 µM (lanes 4, 7, 10, 13 and 16). LEDGF was omitted from the reaction in lane 1. The results are representative of four independent gel shift experiments. The migration positions of free DNA and nucleoprotein complexes are indicated.
Figure 3
Figure 3
The NLS and both AT-hooks mediate DNA-binding. (A) Alignment of invariant amino acids within human LEDGF/p75 AT2, AT1 and NLS sequences to the AT-hook consensus motif. Of the five residues that comprise the motif, four (RGRP) are invariant (Inv) (37). Residues in LEDGF/p75 that match the consensus sequence are underlined. Lys-150 within the NLS (marked by an asterisk) is essential for nuclear import (19). (B) Residues altered in MutL1–MutL5. Asterisks indicate amino acid residues that are identical among human, chicken and frog LEDGF/p75 orthologs (15). Invariant NLS and AT-hook residues are underlined with continuous and dotted lines, respectively. Mutated residues are in bold type. (C) MutL1–MutL5 activities, expressed as percent of wild-type DNA-binding. Error bars represent the variation obtained from duplicate measurements.
Figure 4
Figure 4
Distribution of wild-type and mutant LEDGF/p75 fusion proteins in live cells. (A) Wild-type (WT) LEDGF/p75. Note the characteristic irregular nucleoplasmic distribution and exclusion from nucleoli (9,11). These two cells, not adjacent in the original image, were brought together to highlight the wild-type phenotype. (B–F) Intracellular/nuclear distribution of the indicated LEDGF mutant proteins. Two cells expressing relatively low levels of EGFP-MutL7 were highlighted in panel C; part of a cell expressing higher levels of mutant protein is seen at the upper edge in the first and third frames. The other panels depict two to three cells that are representative of the total population of transfected cells.
Figure 5
Figure 5
The PWWP domain, NLS and AT-hooks contribute to chromatin interactions in vivo. (A–F) Cells undergoing mitosis were analyzed for expression of the indicated EGFP fusion protein and DNA content.
Figure 6
Figure 6
The role of LEDGF/p75 DNA-binding in stimulating HIV-1 integration in vitro. (A) Efficient use of pTZ18U/PL as an integration target. Reactions in lanes 2, 4–8, 10 and 12–16 contained 0.6 µM integrase, whereas reactions in lanes 3–8 and 11–16 contained 0.2 µM of the indicated protein. Reactions in lanes 9–16 additionally contained pTZ18U/PL. The migration positions of mini-HIV substrate DNA, supercoiled (s.c.) pTZ18U/PL and strand transfer reaction products are indicated. The migration pattern of the plasmid on its own is shown on the right; molecular mass standards are to the left. (B) Integrase (IN) alone, integration reaction conducted with integrase alone. Other reactions additionally contained 0.2 µM of the indicated protein. Relative levels of integration activity were quantified by real-time PCR; IN alone activity was arbitrarily set to 1.0. Error bars are variation obtained from duplicate real-time assays. Similar levels of wild-type and mutant LEDGF/p75 activities were observed following independent sets of integration reactions.
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