Regulation of <i>NF-IL6</i> and HIV-1 expression
The research focused on the regulation of HIV-1 Long Terminal Repeat (LTR) and human Nuclear Factor for Interleukin 6 (NF-IL6) gene in activated U937 cells. The U937 cells represent human monocytes that differentiate to macrophages upon stimulation with Lipopolysaccharide (LPS) and Phorbol 12-Myristate-13-Acetate (PMA). It is thought that LPS might activate HIV-1 viral production in AIDS patients suffering from bacterial infection. DNA binding assays showed that stimulation of U937 cells with LPS+PMA induced interactions of NF-IL6 and NF-κB with the LTR. The assays further revealed that Sp1, Sp3, and Sp4 interacted with the Sp1 sites in the LTR. These interactions changed as a function of stimulation time while interactions of USF-1/USF-2 with the E-box remained relatively constant. The results of functional assays indicated that several individual factor-binding sites contributed to elevated levels of the LTR activity during cellular stimulation. However, multiple sites in the LTR were necessary for full-scale upregulation of transcription during LPS+PMA stimulation. The results of titration experiments, using a synthetic C/EBP-related peptide as decoy, further implied that in stimulated cells, both site-dependent and -independent pathways were involved in the regulation of HIV-1 gene expression by NF-IL6. To examine whether and how regulation of the NF-IL6 gene is correlated to the activation of LTR by LPS+PMA stimulation, the immediate 5′ end of the gene (1.2 kb) was sequenced and subsequently analyzed in biochemical and functional assays. Oligo-scanning analysis identified eleven regions that interacted with nuclear proteins including Sp1 and Sp3, NF-κB and EGR-1. The results of functional analysis further revealed that the NF-IL6 gene was subject to both positive and negative controls, both before and after cellular stimulation with LPS+PMA.
0307: Molecular biology