rs13900 heterozygous individuals exhibit AEI in CCL2.

(A) Schematic depicting distal, proximal regulatory elements extending 3 kb on either side of CCL2 gene and the LD between regulatory polymorphism rs1024611 and the transcribed polymorphism rs13900. rs1024611 is located 2578 base pairs upstream of the CCL2 translation start site and rs13900 located in the CCL2 3′ UTR. (B) Allelic expression imbalance (AEI) in heterozygous donors is measured as a ratio of alternative allele (ALT) to reference allele (REF) in a transcribed polymorphism. (C) Representative chromatograms obtained following Sanger sequencing of PCR products obtained from genomic DNA (gDNA) and reverse transcription-PCR of mRNA (cDNA) from three individuals heterozygous for rs13900. gDNA and mRNA were obtained from PBMC treated with LPS for 3 h as previously described. The allelic ratios shown were determined by PeakPicker analysis. Peakpicker calculates allelic ratios by dividing the peak height of the alternate allele (rs13900 T allele) by that of reference allele (rs13900 C allele). The gDNA peaks were used for normalization. (D) Allelic ratio for cDNA and gDNA in six individuals heterozygous for rs13900 after treatment with LPS for 3 h. Statistical significance for the difference in the level of expression between the alleles was determined using Student’s t-test (P < 0.003).

rs13900T confers greater stability to CCL2 mRNA.

(A) CCL2 mRNA expression in peripheral monocytes of heterozygous individuals (n = 4) after treatment with LPS for 3 h and then incubated with 5 µg Act D for indicated times. mRNA was detected by RT-PCR. Results, normalized to 18S rRNA levels, are expressed as fold increase over unstimulated cells (CNT). Levels shown in bar graph represent mean ± SEM of result at time 0. (*P = 0.019) versus unstimulated cells (N = 4). (B) CCL2 mRNA half-life, calculated for each condition as the time (in hours) required for the transcript to decrease to 50% of its initial abundance [t ½ = Ln (0.5)/slope]. (C) Nascent RNA was isolated from treated monocytes from three individuals in the presence and absence of ActD. Allelic ratio was determined after 4 h of incubation with or without ActD. Expression of rs13900 T allele was much higher in ActD-treated samples. The difference between the groups were assessed by ANOVA with Fisher LSD method (*P < 0.05, ** P < 0.005).

Bioinformatic analysis of the rs13900.

(A) Validation RBP binding sites and polymorphism located on the 3′ UTR of CCL2 transcript from the Atlas of UTR Regulatory Activity and analysis of ENCODE genome-wide data sets detected specific enrichment of HuR (ELAVL1) at the region that contains the rs13900. (B) Predicted changes in the secondary structure using Vienna RNA package 2.0; changes in secondary structure are indicated by arrows. (C) Sequence logo of HuR binding site as determined by HOMER. (D) Relative structural preference of HuR to different structural contexts, the letters P, L, U, M indicate that the nucleotide is paired (P), in a hairpin loop (L), in an unstructured (or external) region (U), and Miscellaneous (M) region.

The rs13900 T allele shows increased in vitro binding of HuR.

(A) REMSA with labelled oligoribonucleotide containing either rs13900 C or T allele and whole cell extracts from 293T cells. § denotes free probe; black arrow, bound probe; red arrow, supershift. (B) Representative quantitative densitometric analysis of the antibody shifted complexes suggested increased HuR binding to the oligoribonucleotide bearing rs13900 T allele. The signals in the bound fraction(s) were normalized using the free probe (N = 4). The top panel represents the data from four independent experiments (mean ± SEM). Statistical analyses were performed using Student’s t test (*P < 0.001). The bottom panel shows the relative fold enrichment of the bound protein complexes to the oligoribonucleotide containing the rs13900 T allele relative to that containing the rs13900 C allele. Statistical significance was calculated using Student t test (*P < 0.001) (C) REMSA with labelled oligoribonucleotides containing either rs13900 T or C allele and purified HuR protein. (D) Plot showing the fraction of bound rs13900 C or rs13900 T oligoribonucleotides with increasing HuR concentration.

rs13900 C and T alleles are associated with differential binding to HuR ex vivo.

(A) HuR enrichment in immunoprecipitated material from macrophages stimulated with LPS. (B) CCL2 3′ UTR was detected at significant levels in the samples precipitated by α-HuR antibody when compared to the control IgG. (C) CCL2 mRNA expression in anti-HuR antibody enriched immunoprecipitated material analyzed by RT-qPCR (N = 4). Statistical significance was calculated using Student t-test (*P < 0.005). The error bars represent SEM. (D) Relative expression levels for rs13900 C and T alleles in macrophages stimulated with LPS (N = 4). Statistical significance was calculated using a Student t-test (*P < 0.005).

Differential effects of rs13900 alleles in reporter assays and role of HuR.

(A) Schematic representation of the luciferase reporter vectors containing CCL2 3’ UTR with either rs13900 C or T allele. (B) HEK-293 cells were transfected with the equal quantities of CCL2 3′ UTR reporter vectors and luciferase activity was measured 48 hrs later. The relative luciferase activities of the 3′ UTR reporter plasmids were expressed as percent reduction in the luminescence when compared to the control vector that was set to 100% after normalizing for the protein content of the lysates. The error bars indicate the standard error of mean and statistical significance was calculated using two-tailed Student’s t test (*P < 0.05). (C) HEK-293T cells were transfected with either pCMV6-HuR (0.5 μg) or pCMV-Entry (0.5 μg) and after 72 hours they were co-transfected with the two plasmid constructs (0.5 μg). Twenty-four hours after transfection the relative change in luciferase activity was determined. (D) Cells were co-transfected with 125 pmol HuR siRNA or control siRNA and with the two plasmid constructs (0.5 μg). Twenty-four hours after transfection the relative change in luciferase activity was determined (normalized to total protein concentration, data from four independent experiments (mean ± SEM; N = 4). Statistical analyses were performed using Fisher LSD method (*P < 0.05).

Influence of rs13900 C and T alleles on CCL2 translatability.

(A) HEK-293 cells were transfected by nucleofection with control and CCL2 3′ UTR reporter constructs with rs13900 C and T alleles. The nucleofected cells were plated separately and harvested for total RNA isolation or lysed for mRNA level or protein level expression of luciferase respectively after 24 h. The reporter mRNA levels from the transfected 293 T cells were quantified by qRT-PCR, and 18S rRNA was used for normalization. (B) The relative luciferase activities of the 3′ UTR reporter plasmids were expressed as percent reduction in the luminescence when compared to the control vector that was set to 100% after normalizing for the protein content of the lysates. (C) mRNA translatability was calculated as luciferase activity normalized by the reporter luciferase mRNA level. The error bars indicate the standard error of mean from six independent experiments (N = 6) and statistical significance was calculated using ANOVA and post hoc contrast with Fisher LSD method. (*P < 0.05, **P < 0.005).

HuR differentially regulates CCL2 haplotypes.

(A) HuR expression in primary human macrophages following lentiviral transduction. Macrophages obtained from four individuals who are homozygous for either rs13900 C or rs13900 T allele were transduced with either CMV-null or HuR expressing lentiviral particles (pCMV6-HuR) for 72 hours followed by LPS stimulation for 3 h. (B) CCL2 expression determined by RT-qPCR. Error bars represent SEM. Statistical analyses were performed using Student’s t test (* P < 0.05).

Allele carriages and allele frequencies of rs13900 in healthy volunteers

Differential loading of rs13900 C>T onto polysome isolated from macrophages of heterozygous donors. C>T ratio < 1 indicates increased levels of the minor T allele relative to C allele. Result shows enrichment of rs13900T allele on polysomes.