Regulation of CCL2 expression by an upstream TALE homeodomain protein-binding site that synergizes with the site created by the A-2578G SNP

Abstract

CC Chemokine Ligand 2 (CCL2) is a potent chemoattractant produced by macrophages and activated astrocytes during periods of inflammation within the central nervous system. Increased CCL2 expression is correlated with disease progression and severity, as observed in pulmonary tuberculosis, HCV-related liver disease, and HIV-associated dementia. The CCL2 distal promoter contains an A/G polymorphism at position -2578 and the homozygous -2578 G/G genotype is associated with increased CCL2 production and inflammation. However, the mechanisms that contribute to the phenotypic differences in CCL2 expression are poorly understood. We previously demonstrated that the -2578 G polymorphism creates a TALE homeodomain protein binding site (TALE binding site) for PREP1/PBX2 transcription factors. In this study, we identified the presence of an additional TALE binding site 22 bp upstream of the site created by the -2578 G polymorphism and demonstrated the synergistic effects of the two sites on the activation of the CCL2 promoter. Using chromatin immunoprecipitation (ChIP) assays, we demonstrated increased binding of the TALE proteins PREP1 and PBX2 to the -2578 G allele, and binding of IRF1 to both the A and G alleles. The presence of TALE binding sites that form inverted repeats within the -2578 G allele results in increased transcriptional activation of the CCL2 distal promoter while the presence of only the upstream TALE binding site within the -2578 A allele exerts repression of promoter activity.

Figure 1. Distinct TALE site binding preferences for PREP1, PBX2, HoxA9 and IRF1 in the CCL2 promoter.

ChIP results from U87-MG cells transfected with appropriate plasmids (as described above) and activated with IL-1β for 3 hours. A. Western blot results from U87-MG cells transfected with indicated siRNAs. GAPDH represents loading control. B,C. ChIP results from U87-MG cells co-transfected with the -2578 G pGL4.11 plasmid and indicated siRNA. Graphics show percent of total DNA immunoprecipitated by each indicated antibody. IgG represents the negative control. Results are expressed as the mean ± S.E. of three independent experiments carried out in duplicate. Differences between multiple groups were tested using ANOVA (Figure 1B, P = 0.0115 and Figure 1C, P = 0.0014). Subsequent pair wise comparisons of groups with each treatment against the control group utilized the Bonferroni correction.

Figure 2. Distinct TALE site binding preferences for PREP1, PBX2, HoxA9 and IRF1 in the CCL2 promoter with and without TALE site repeats.

A. Sequence of double stranded CCL2 promoter DNA representing the distal regulatory region containing the two TALE binding sites (in red) with the A-2578G polymorphism within parentheses and the incorporated mutation within the upstream TALE site (denoted by *m). The CCL2 ISRE sequence is within the black box. Graphics show the percent of total DNA immunoprecipitated from U87-MG cells by each indicated antibody. B-I. U87-MG cells were transfected with either the -2578 A pGL4.11 plasmid or the -2578 Am pGL4.11 plasmid. F-I. U87-MG cells were transfected with either the -2578 G pGL4.11 plasmid or the -2578 Gm pGL4.11 plasmid. Percent input was calculated by 100×2∧(Ct_input – Ct_enriched). Input was determined from 1% of the cell lysate and results are expressed as the mean ± S.E. of three independent experiments carried out in duplicate.

Figure 3. siRNA silencing of PREP1 and PBX2 in transfected U87-MG cells differentially affects promoter activity of the -2578 A and G alleles.

A,B. Luciferase expression in U87-MG cells transfected with either the -2578 A pGL4.11 plasmid or the -2578 G pGL4.11 plasmid along with indicated siRNAs. C. Luciferase expression in U87-MG cells transfected with either the -2578 A pGL4.11 plasmid or the -2578 Am pGL4.11 plasmid. D. Luciferase expression in U87-MG cells transfected with either the -2578 G pGL4.11 plasmid or the -2578 Gm pGL4.11 plasmid. All results were normalized to Renilla luciferase values. Results are expressed as the mean ± S.E. of three independent experiments carried out in duplicate. Differences between multiple groups were tested using ANOVA (Figure 3A, P = 0.0495 and Figure 3B, P<.0001). Subsequent pair wise comparisons of groups with each treatment against the control group utilized the Bonferroni correction. Student's two-sided t tests were used for 3C and 3D.