BET proteins promote efficient murine leukemia virus integration at transcription start sites

Abstract

The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral murine leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy. (1-4). The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus studied (5-7). For example, the γ-retroviruses favor integration near transcription start sites, whereas lentiviruses favor integration within transcription units. These observations have suggested that different cellularbinding partners of retroviral integrases are likely to be responsible for integration target-site selection. However, to date, only one example has been reported: lens epithelium-derived growth factor (LEDGF/p75), which functions as a bimodal tether that engages HIV-1 intasomes and navigates them to active genes (8-14). Cellular cofactors of other retroviral genera are currently unknown.The molecular mechanisms of γ-retroviral murine leukemia virus (MLV) integration are of particular significance because MLV-based vectors are used for human gene therapy. In clinical trials, the use of γ-retroviral vectors to correct primary immunodeficiencies has been curative, but adverse events have occurred associated with insertion of MLV-based vectors near protooncogenes (reviewed in refs. 15-18). The identification of cellular factors for γ-retroviruses may provide mechanistic clues to facilitate the development of safer gene-therapy vectors.In this report, we have identified the bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as the cellularbinding partners of MLV IN and demonstrate their significance for stimulating and targeting MLV integration at transcription start sites. (Table 1, Table S1, and Fig. S1). Of these, Brd4 and Brd3 were the top hits in NIH 3T3 and Sup-T1 cells, respectively. Differential pull-down levels of these proteins (Table 1) could be attributable to the varying expression le...

Single-Nucleotide-Specific Targeting of the Tf1 Retrotransposon Promoted by the DNA-Binding Protein Sap1 of Schizosaccharomyces pombe

“…Retroviruses such as human immunodeficiency virus 1 (HIV-1) and murine leukemia virus (MLV), which share significant similarities with LTR retrotransposons in their genetic structures and mechanisms of propagation (Levin and Moran 2011), depend on host factors for targeting integration. HIV-1 insertion is directed to the body of RNA polemerase II-transcribed genes by host factor LEDGF, while MLV IN interacts with BET proteins to direct its integration into enhancer sequences of RNA polemerase II-transcribed genes (Ciuffi et al 2005;Llano et al 2006;Shun et al 2007;Gupta et al 2013;Sharma et al 2013). The mechanism by which Tf1 accomplishes targeting appears to be significantly different from the preceding examples except perhaps for MLV, which does integrate into promoter sequences.…”

Le rôle des protéines BET dans l’intégration des γ-rétrovirus

“…Pour comprendre comment IN de MLV sélectionne les sites d'intégration, trois équipes ont cherché les protéines cellulaires capables d'interagir avec elle. Les partenaires identifiés sont trois protéines apparentées, BRD2 (bromodomaincontaining protein 2), BRD3 et BRD4, qui présentent deux bromodomaines préférentiellement dans le corps des gènes transcrits ( Figure 1B) [2][3][4][6][7][8][9][10][11][12]. Les LV sont moins oncogéniques que les RV [10].…”

“…Leurs dérivés artificiels, défectifs et intégratifs à quelques exceptions près, seront appelés rétrovecteurs (dérivés des rétrovirus, quels qu'ils soient), -rétrovecteurs (RV, très souvent dérivés de gamma-rétrovirus murins) et lentivecteurs (LV, dérivés des lentivirus, généralement le VIH, virus de l'immunodéficience humaine). Les résultats présen-tés ici ont été principalement obtenus avec des RV défectifs dérivés du -rétrovirus murin MLV mais sont probablement valables pour tous les -rétrovirus et tous les RV ( Figure 1A) [3,8,11,12].…”

Multifunctional facets of retrovirus integrase

“…Besides LEDGF/p75, cellular Nup153 is indispensable for the integration of HIV DNA into the peripheral edge of the nuclear DNA suggesting that nuclear topography is also an important for HIV integration site selection [42] . For MLV IN, recent studies demonstrated that IN interacts with the chromatin BET (bromo-and extra-terminal domain) proteins (BRD2, BRD3 and BRD4) directing it to the promoter region of genes [39,[43][44][45] (Figure 4). To date, there are no reports that have identified cellular proteins involved in host site selection by avian retroviruses.…”