Ction with cellular cofactors. We, thus, focused our work on the
Ction with cellular cofactors. We, thus, focused our work on the better determination of this final local association between IN and nucleosomes.Benleulmi et al. Retrovirology (2015) 12:Page 3 ofThe functional interaction between retroviral intasomes and nucleosomes in cells remains elusive. There are no preferential positions for nucleosomes across the majority of the human genome [40]. Consequently, the dynamic nature of chromatin makes it difficult to accurately predict the positions of nucleosomes in the cell. For this reason, in vitro approaches based on the use of chromatinized templates, in which nucleosome positioning is exclusively sequence-driven, are valuable for understanding the specific influence of the chromatin structure on retroviral integration site selectivity. Early results showed that DNA distorsion and nucleosome could favor retroviral integration in vitro [41-46]. More recently, in vitro study using chromatinized template has shown that HIV-1 integration into nucleosomes could be modulated by chromatin structure and dynamics [36]. Additionally, partial one end and physiological two ends concerted HIV-1 integration reactions were shown to be affected differently by chromatin in vitro suggesting that the structure of the different intasomes catalyzing these various integration reactions could impact their sensitivity to nucleosomal DNA [36]. In order to determine how integration could be modulated both by chromatin structure and intasome architecture, we compared the ability of various retroviral intasomes to accommodate different chromatin structures both in vitro and in vivo. We selected retroviral integrases that vary in their integration site preference in infected cells and in the size of the duplicated target site sequence governed by the distance separating the two active sites of the intasome and, thus, the architecture of the IN catalytic pocket. To determine the intrinsic sensitivity of the selected integrases toward chromatin compactness was determined by analyzing their integration efficiency, selectivity and fidelity in naked and nucleosomal DNA in vitro using specific biochemical conditions allowing efficient full site integration reactions in the lack of cofactors. Our results indicate that the impact of the chromatin structure at the integration site is mainly driven by intrinsic physical constraints within the retroviral intasomes. These constraints could thus dictate their capability to bind nucleosomes functionally in specific chromatin contexts after targeting into specific regions of the host genome via the interaction with cellular cofactors.A-836339 cancer ResultsIn vitro integration catalyzed on chromatin by integrases from various retroviral generaHIV-1, PFV, MLV and ASV INs were compared using their specific donor DNAs (described in Additional file 1: Figure S1) and p5S acceptor plasmids derived from the previously described pBSK-Zeo-S5G5E4 receptor vector (Figure 1A and PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28667899 Additional file 2: Figure S2A) containing a 5S-G5E4 fragment carrying two times fiverepeats of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/26780312 5S sequences surrounding a central sequence containing five gal4 DNA binding sites and the adenovirus 2 E4 minimal promoter. This construct allows the in vitro association of nucleosomes in stable, regularly spaced and defined positions of the DNA template leading to dense polynucleosome (PN) [36]. A nucleosome positioning prediction algorithm performed on the acceptor plasmid sequence (Figure 1B) shows that it contains two regions allowing the f.