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The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering.

Hendrix J, Gijsbers R, De Rijck J, Voet A, Hotta J, McNeely M, Hofkens J, Debyser Z, Engelborghs Y - Nucleic Acids Res. (2010)

Bottom Line: After interaction with HIV-1 integrase via its IBD, a general protein-protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin.The PWWP is crucial for locking the complex on chromatin.We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Biomolecular Dynamics, University of Leuven, Leuven, Flanders, B-3000, Belgium.

ABSTRACT
Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein-protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting.

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The PWWP domain of LEDGF/p75 contributes to high affinity chromatin binding and is crucial for chromatin tethering of HIV-1 integrase. (A–C) Confocal fluorescence images of HeLa cells expressing (A) eGFP-LEDGF/p75 K56D, (B) R74D and (C) K56D-R74D. (D) sFRAP experiment. (E) CP experiment of eGFP-LEDGF/p75 K56D-R74D. The CP curves of eGFP and eGFP-LEDGF/p75 are shown as a reference. (F) FCS experiment of eGFP-LEDGF/p75 mutants. The ACFs of eGFP and eGFP-LEDGF/p75 are shown as a reference. Error bars = SD.
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Figure 5: The PWWP domain of LEDGF/p75 contributes to high affinity chromatin binding and is crucial for chromatin tethering of HIV-1 integrase. (A–C) Confocal fluorescence images of HeLa cells expressing (A) eGFP-LEDGF/p75 K56D, (B) R74D and (C) K56D-R74D. (D) sFRAP experiment. (E) CP experiment of eGFP-LEDGF/p75 K56D-R74D. The CP curves of eGFP and eGFP-LEDGF/p75 are shown as a reference. (F) FCS experiment of eGFP-LEDGF/p75 mutants. The ACFs of eGFP and eGFP-LEDGF/p75 are shown as a reference. Error bars = SD.

Mentions: We set out to verify the previously documented contribution of the PWWP domain to the in vivo chromatin binding properties of LEDGF/p75 (15,16). To affect the overall protein structure as little as possible, we sought to specifically alter the affinity of this domain for chromatin. Based on the molecular model of the PWWP domain of HDGF in complex with DNA (54), we predicted that positively charged residues K56 and R74 in LEDGF/p75 are most likely interacting with the phosphates of the host DNA. Next, we constructed and purified two single mutant proteins of LEDGF/p75, K56D and R74D and the double mutant K56D-R74D. Correct overall folding was corroborated with circular dichroism spectroscopy (Supplementary Table S2). Next, eGFP-fusions of the same proteins were transiently expressed in HeLa cells. The proteins were characterized by a more diffuse nuclear localization (Figure 5A–C), compared with the typical heterogeneous nuclear distribution of the wild-type protein (22) (Figure 1C), suggesting that chromatin binding properties were affected. Next, we used sFRAP, CP and FCS to study the dynamics of the mutants. All mutants were considerably faster (4- to 8-fold) than wild-type LEDGF/p75 (Figure 5D and F and Table 2). The R74D mutation had a more pronounced effect than the K56D mutation (Figure 5D and F green and orange, respectively) while the double mutant did not show an additive effect compared with the R74D single mutation (Figure 5D and F, gray). All mutants were still considerably slower than expected for free diffusion, indicating they still have some residual interactions with chromatin. Importantly, the difference in cellular dynamics between the K56D and R74D mutants could not be inferred from a differential distribution of the proteins, but was only evidenced by our sFRAP and FCS measurements, demonstrating the sensitivity of these techniques. Finally, the ACF of eGFP-LEDGF/p75 K56D-R74D still varied with the focus size, implying that this protein also moves by diffusion (Figure 3E). In summary, by mutating a single amino acid residue in LEDGF/p75-PWWP, R74D, chromatin binding of the protein was severely impaired, as evidenced by an up to 8-fold increase in dynamics with respect to wild-type LEDGF/p75. The PWWP domain of LEDGF/p75 is thus important but not sufficient for in vivo chromatin binding.Figure 5.


The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering.

Hendrix J, Gijsbers R, De Rijck J, Voet A, Hotta J, McNeely M, Hofkens J, Debyser Z, Engelborghs Y - Nucleic Acids Res. (2010)

The PWWP domain of LEDGF/p75 contributes to high affinity chromatin binding and is crucial for chromatin tethering of HIV-1 integrase. (A–C) Confocal fluorescence images of HeLa cells expressing (A) eGFP-LEDGF/p75 K56D, (B) R74D and (C) K56D-R74D. (D) sFRAP experiment. (E) CP experiment of eGFP-LEDGF/p75 K56D-R74D. The CP curves of eGFP and eGFP-LEDGF/p75 are shown as a reference. (F) FCS experiment of eGFP-LEDGF/p75 mutants. The ACFs of eGFP and eGFP-LEDGF/p75 are shown as a reference. Error bars = SD.
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Figure 5: The PWWP domain of LEDGF/p75 contributes to high affinity chromatin binding and is crucial for chromatin tethering of HIV-1 integrase. (A–C) Confocal fluorescence images of HeLa cells expressing (A) eGFP-LEDGF/p75 K56D, (B) R74D and (C) K56D-R74D. (D) sFRAP experiment. (E) CP experiment of eGFP-LEDGF/p75 K56D-R74D. The CP curves of eGFP and eGFP-LEDGF/p75 are shown as a reference. (F) FCS experiment of eGFP-LEDGF/p75 mutants. The ACFs of eGFP and eGFP-LEDGF/p75 are shown as a reference. Error bars = SD.
Mentions: We set out to verify the previously documented contribution of the PWWP domain to the in vivo chromatin binding properties of LEDGF/p75 (15,16). To affect the overall protein structure as little as possible, we sought to specifically alter the affinity of this domain for chromatin. Based on the molecular model of the PWWP domain of HDGF in complex with DNA (54), we predicted that positively charged residues K56 and R74 in LEDGF/p75 are most likely interacting with the phosphates of the host DNA. Next, we constructed and purified two single mutant proteins of LEDGF/p75, K56D and R74D and the double mutant K56D-R74D. Correct overall folding was corroborated with circular dichroism spectroscopy (Supplementary Table S2). Next, eGFP-fusions of the same proteins were transiently expressed in HeLa cells. The proteins were characterized by a more diffuse nuclear localization (Figure 5A–C), compared with the typical heterogeneous nuclear distribution of the wild-type protein (22) (Figure 1C), suggesting that chromatin binding properties were affected. Next, we used sFRAP, CP and FCS to study the dynamics of the mutants. All mutants were considerably faster (4- to 8-fold) than wild-type LEDGF/p75 (Figure 5D and F and Table 2). The R74D mutation had a more pronounced effect than the K56D mutation (Figure 5D and F green and orange, respectively) while the double mutant did not show an additive effect compared with the R74D single mutation (Figure 5D and F, gray). All mutants were still considerably slower than expected for free diffusion, indicating they still have some residual interactions with chromatin. Importantly, the difference in cellular dynamics between the K56D and R74D mutants could not be inferred from a differential distribution of the proteins, but was only evidenced by our sFRAP and FCS measurements, demonstrating the sensitivity of these techniques. Finally, the ACF of eGFP-LEDGF/p75 K56D-R74D still varied with the focus size, implying that this protein also moves by diffusion (Figure 3E). In summary, by mutating a single amino acid residue in LEDGF/p75-PWWP, R74D, chromatin binding of the protein was severely impaired, as evidenced by an up to 8-fold increase in dynamics with respect to wild-type LEDGF/p75. The PWWP domain of LEDGF/p75 is thus important but not sufficient for in vivo chromatin binding.Figure 5.

Bottom Line: After interaction with HIV-1 integrase via its IBD, a general protein-protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin.The PWWP is crucial for locking the complex on chromatin.We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Biomolecular Dynamics, University of Leuven, Leuven, Flanders, B-3000, Belgium.

ABSTRACT
Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein-protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting.

Show MeSH
Related in: MedlinePlus