FIG 3.

NF-κB and IRF1 interact with the ISREs in the ERVK 5′ LTR and synergize to enhance ERVK gene transcription. (A) In silico-predicted IRF1 and NF-κB binding sites, including two ISREs in the ERVK 5′ LTR consensus sequence. The data are from reference 1. (B) IRF1 and NF-κB p65 significantly enhance ERVK pol transcription in 293T cells. 293T cells were transfected with 1 μg of pCMVBL or pCMV2 empty vector (negative controls) or with plasmids encoding wild-type or phosphomimetic forms (5D, 4D, and 6D) of IRF and NF-κB for 48 h. The modulation of ERVK pol RNA levels was measured by using SYBR green detection and Q-PCR, and data were normalized to the negative control (**, P < 0.01; ****, P < 0.0001; n = 3). 18S rRNA was used as the endogenous control. Only IRF1 and NF-κB p65 significantly induced ERVK pol transcription. (C) IRF1 and NF-κB p65 and p50 synergize to significantly enhance ERVK pol transcription in astrocytes. SVGA cells were transfected with 1 μg of empty vector (negative control) or with plasmids encoding IRF1 and NF-κB isoforms, individually and in combinations, for 48 h. The modulation of ERVK pol RNA levels was measured by using SYBR green detection and Q-PCR, and data were normalized to the negative control (*, P < 0.05; ****, P < 0.0001; n = 3). 18S rRNA was used as the endogenous control. Although IRF1 or NF-κB p65 alone was sufficient to significantly induce ERVK pol transcription, IRF1 and NF-κB p65 and p50 synergized to further enhance ERVK pol RNA levels (up to 68-fold). (D) TNF-α and LIGHT markedly enhanced the binding of IRF1 and NF-κB p65 and p50 to both ISREs in the ERVK 5′ LTR, in a cell-type-dependent manner. Chromatin was extracted from SVGA cells and ReNcell CX cell-derived neurons treated with TNF-α (10 ng/ml) or LIGHT (10 ng/ml) for 8 h. Chromatin immunoprecipitation (ChIP) was performed with anti-human IRF1 or NF-κB p65, p50, or p52 antibody. Q-PCR was used to amplify immunoprecipitated ISRE sequences within the ERVK 5′ LTR, and products were detected using SYBR green detection. The fold enrichment of transcription factors at each ISRE was first normalized to the input control and then to the IgG negative control. All transcription factors were bound to the ISREs at basal levels. However, the binding of NF-κB p65 and p50 was significantly enhanced by LIGHT treatment, but not TNF-α treatment, in astrocytes (top panels) (n = 3; *, P < 0.05; ****, P < 0.0001). In contrast, the binding of NF-κB p65 and p50 was significantly enhanced by TNF-α treatment, but not LIGHT treatment, in neurons (bottom panels) (n = 2; *, P < 0.05; ***, P < 0.001).