1/7/2024 0 Comments Vicinity definition![]() Protein-DNA complexes were resolved on 6% native polyacrylamide gels. Briefly, 2 μg of recombinant purified GST-SATB1 (lanes 2 to 10 in panel E and lanes 3 to 7 in panel F) or GST alone (lane 1 in panel E and lane 2 in panel F) was incubated with radiolabeled, PCR-amplified DNA probes in the presence of 1 μg of poly(dI-dC) competitor DNA. The specificity of binding of SATB1 to HIV-1 in vivo integration clones BH609471 (E) and BH609700 (F) was demonstrated by competition EMSA. ChIP analysis of two representative in vivo integration clones, BH609797 and BH609646 (C), and two representative in vitro integration clones, BH610076 and BH609954 (D), is depicted. Distal promoter region P1 of IL- 2 was used as a positive control for SATB1 binding, whereas proximal promoter region P2 of IL- 2 and region P6 of IL- 2Rα were used as negative controls. In vivo binding of SATB1 was demonstrated by PCR amplification of DNA isolated by ChIP (C and D) with anti-SATB1 (lane 1) or rabbit IgG (R-IgG lane 2). Protein-DNA complexes were resolved on native polyacrylamide gels. Briefly, 10 to 100 ng of recombinant purified GST-SATB1, GST-PARP, or GST was incubated as indicated with radiolabeled, PCR-amplified DNA probes in the presence of 1 μg of competitor DNA. In vitro binding of SATB1 to HIV-1 integration clones BH609797 (A) and BH609646 (B) was demonstrated by EMSA as described in Materials and Methods. SATB1 specifically binds to HIV-1 integration sequences in vitro and in vivo. We propose that definitive sequence features such as the Alu-like motifs and SATB1-binding sites provide a unique chromatin context in vivo which is preferentially targeted by the HIV-1 integration machinery. Strikingly, many of these regions were disfavored for integration when SATB1 was silenced, providing unequivocal evidence for its role in HIV-1 integration site selection. Moreover, many of these sequences were preferentially partitioned in the DNA associated tightly with the nuclear matrix and not in the chromatin loops. ![]() The cloned sequences flanking HIV-1 integration sites were specifically immunoprecipitated and amplified from the pool of anti-SATB1-immunoprecipitated genomic DNA fragments isolated from HIV-1 NL4.3-infected Jurkat T-cell chromatin. ![]() To specifically isolate sequences flanking the viral integration sites and also harboring both Alu-like repeats and SATB1-binding sites, we combined chromatin immunoprecipitation with sequential PCRs. Alu repeats make up nearly 10% of the human genome and have been implicated in the regulation of transcription. Additionally, these sequences were preferentially bound by SATB1, the T lineage-restricted chromatin organizer, in vitro and in vivo. We show here the occurrence of Alu-like motifs in the sequences flanking the reported viral integration sites that are significantly different from those obtained from the randomly picked sequences from the human genome, suggesting that unique primary sequence features exist in the genomic regions targeted by human immunodeficiency virus type 1 (HIV-1). However, the mechanism for integration site selection by retroviruses is not clear. Retroviral integration has recently been shown to be nonrandom, favoring transcriptionally active regions of chromatin.
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