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

Laboratory for Biomolecular Dynamics, University of Leuven, Leuven, Flanders, B-3000, Belgium.
Nucleic Acids Research (Impact Factor: 9.11). 10/2010; 39(4):1310-25. DOI: 10.1093/nar/gkq933
Source: PubMed


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.

  • Source
    • "The host protein PSIP1/LEDGF p75 is the major host co-factor for HIV integration [3]–[7]. This protein specifically binds HIV integrase with an integrase-binding domain (IBD) and binds to chromatin via a PWWP domain and two AT hook motifs [8]–[16]. Studies of LEDGF depletion and deletion cells have shown that LEDGF is required for efficient integration to chromatin in vivo [4], [5]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polβ). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5'dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggeted Polβ DNA synthesis activity is not necessary while 5'dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.
    Full-text · Article · Jul 2014 · PLoS ONE
  • Source
    • "Integration of viral genome into human genome is a crucial step in infection by HIV-1. The protein-protein interaction between IN and LEDGF/p75 is crucial for integration[8]. This interaction is detectable as translocation of IN into the nucleus. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Translocation of viral integrase (IN) into the nucleus is a critical precondition of integration during the life cycle of HIV, a causative agent of Acquired Immunodeficiency Syndromes (AIDS). As the first discovered cellular factor to interact with IN, Lens epithelium-derived growth factor (LEDGF/p75) plays an important role in the process of integration. Disruption of the LEDGF/p75-IN interaction has provided a great interest for anti-HIV agent discovery. In this work, we reported that one small molecular compound, 1,4-bis(5-(naphthalen-1-yl)thiophen-2-yl)naphthalene(Compound 15), potently inhibit the IN- LEDGF/p75 interaction and affect the HIV-1 IN nuclear distribution at 1μM. The putative binding mode of Compound 15 was constructed by a molecular docking simulation to provide structural insights into the ligand-binding mechanism. Compound 15 suppressed viral replication by measuring p24 antigen production in HIV-1IIIB acute infected C8166 cells with EC50 value of 11.19μM. Compound 15 might supply useful structural information for further anti-HIV agent discovery.
    Full-text · Article · Jan 2014 · Chemico-biological interactions
  • Source
    • "The PWWP domain is crucial for LEDGF chromatin attachment but the molecular parameters of this interaction are still under investigation [6], [8], [23], [24], [49], [81]. This domain binds to both DNA and nucleosomes and like other PWWP domains, it also interacts with the H3K36me3 histone mark [6], [8], [9]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: PWWP domains are involved in the chromatin attachment of several proteins. They bind to both DNA and proteins and their interaction with specific histone methylation marks define them as a new class of histone code readers. The lens epithelium derived growth factor (LEDGF/p75) contains an N-terminal PWWP domain necessary for its interaction with chromatin but also a C-terminal domain which interacts with several proteins, such as lentiviral integrases. These two domains confer a chromatin-tethering function to LEDGF/p75 and in the case of lentiviral integrases, this tethering participates in the efficiency and site selectivity of integration. Although proteins interacting with LEDGF/p75 C-terminal domain have been extensively studied, no data exist about partners of its PWWP domain regulating its interaction with chromatin. In this study, we report the identification by yeast-two-hybrid of thirteen potential partners of the LEDGF PWWP domain. Five of these interactions were confirmed in mammalian cells, using both a protein complementation assay and co-immunoprecipitation approaches. Three of these partners interact with full length LEDGF/p75, they are specific for PWWP domains of the HDGF family and they require PWWP amino acids essential for the interaction with chromatin. Among them, the transcription activator TOX4 and the splicing cofactor NOVA1 were selected for a more extensive study. These two proteins or their PWWP interacting regions (PIR) colocalize with LEDGF/p75 in Hela cells and interact in vitro in the presence of DNA. Finally, single round VSV-G pseudotyped HIV-1 but not MLV infection is inhibited in cells overexpressing these two PIRs. The observed inhibition of infection can be attributed to a defect in the integration step. Our data suggest that a regulation of LEDGF interaction with chromatin by cellular partners of its PWWP domain could be involved in several processes linked to LEDGF tethering properties, such as lentiviral integration, DNA repair or transcriptional regulation.
    Full-text · Article · Nov 2013 · PLoS ONE
Show more