Transcriptional effects of a lupus-associated polymorphism in the 5' untranslated region (UTR) of human complement receptor 2 (CR2/CD21)

Abstract

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a strong genetic component that determines risk. A common three single-nucleotide polymorphism (SNP) haplotype of the complement receptor 2 (CR2) gene has been associated with increased risk of SLE (Wu et al., 2007; Douglas et al., 2009), and a less common haplotype consisting of the major allele at SNP1 and minor alleles at SNP2 and 3 confers protection (Douglas et al., 2009). SNP1 (rs3813946), which is located in the 5' untranslated region (UTR) of the CR2 gene, altered transcriptional activity of a CR2 promoter-luciferase reporter gene construct transiently transfected into a B cell line (Wu et al., 2007) and had an independent effect in the protective haplotype (Douglas et al., 2009). In this study, we show that this SNP alters transcriptional activity in a transiently transfected non B-cell line as well as in stably transfected cell lines, supporting its relevance in vivo. Furthermore, the allele at this SNP affects chromatin accessibility of the surrounding sequence and transcription factor binding. These data confirm the effects of rs3813946 on CR2 transcription, identifying the 5' UTR to be a novel regulatory element for the CR2 gene in which variation may alter gene function and modify the development of lupus.

Figure 1. Location of rs3813946 within the proximal promoter region of the human CR2 gene

The nucleotide sequence from the -133 upstream promoter region to the translation start site of the CR2 gene is shown. Previously identified functional elements, an AP2-like site and the Ebox1 and Ebox2 are shaded. Recently identified transcription factor binding sites for PAX5, C/EBP β and MAZ are shown as boxed regions. The transcription and translation start site is indicated with arrows. The putative TATA box binding site is in bold font. The single-nucleotide polymorphism studied herein (rs3813946) is indicated in the sequence at its nucleotide position (+21T/C).

Figure 2. The minor C allele of rs3813946 decreases proximal promoter activity

(A) Transcriptional activity of luciferase constructs containing the −315/+75 proximal promoter sequence with the major T (+21T) or minor C (+21C) allele of rs3813946. Constructs were transiently transfected into CR2-negative K562 cells. Results shown represent mean transcriptional activity +SEM (n=6–9) and are expressed as normalized transcriptional activity of each construct relative to the +21T construct. * p < 0.01 when comparing normalized transcriptional activity of the +21C to the +21T construct. (B) Representation of the +21 T/C-luciferase CR2 promoter reporter constructs. Both constructs were integrated into the K562 cell line using FLP recombination. (C) K562 cells containing the stably integrated CR2 promoter constructs were isolated and luciferase activity in cell lysates was assessed. Results show mean luciferase expression ±SEM (n=5).

Figure 3. Reduced chromatin accessibility in stably transfected reporter constructs containing the minor C allele at rs3813946 correlates with transcriptional activity

(A) Location of primers used to assess chromatin accessibility by CHART-PCR. (B) Pairs of “Uncut” and “Cut” samples from CHART-PCR assays were used as template in Q-PCR reactions with primer pairs targeting promoter regions of β-ACTIN (−239 to −62), PAX7 (−228 to −64), upstream CR2 (−1215 to −1052), and CR2-reporter transgene sequences (238 bp amplicon including −113 to +75 of the CR2 promoter sequence). MNase chromatin accessibility is expressed as the percentage cutting and plotted as the mean ± SEM from independent pairs of “Uncut” and “Cut” samples (n=6).

Figure 4. The T to C transition at rs3813946 alters transcription factor binding in vitro

EMSA analysis of the region surrounding rs3813946. Oligonucleotides utilized correspond to +10 to +37 of the proximal promoter sequence and contained either a T (+21T) or a C (+21C) at rs3813946. Nuclear extracts from Raji B cells were pre-incubated with poly(dI-dC) in binding buffer to reduce detection of non-specific DNA-protein interactions. Increasing fold molar excess of unlabeled selfcompetitor oligonucleotide demonstrate the presence of five specific protein-DNA complexes (A–E). Unbound oligonucleotides are represented as UB.

Figure 5. Absence of allele-specific binding of proteins to the 5`UTR of the CR2 mRNA

(A) The location of the +21T/C SNP within the transcribed region of the CR2 gene. Single-stranded 20 bp RNA oligonucleotides used for RNA-EMSA (REMSA) corresponded to +6 to +26 of the proximal promoter sequence (underlined). (B) REMSAs were performed using RNA oligonucleotides containing either a U (+21U) or a C (+21C) at position +21 and Raji cytoplasmic extracts (Raji CE). Increasing concentrations of tRNA were used for competition analysis. Unbound oligonucleotides are represented as UB. (C) UV crosslinking with Raji and K562 cytoplasmic (CE) and nuclear (NE) extracts identified two proteins bound to both CR2 transcripts in a sequence-specific manner (A and B). PSt denotes molecular weight protein standard.

Figure 6. Evidence that C/EBP β, PAX5 and MAZ interacts with the CR2 5` UTR in Raji cells

ChIP assays demonstrate specific enrichment of CR2 5` UTR sequences in Raji cells using antibodies targeting C/EBP β, Pax5 and MAZ compared to IgG and no antibody controls. (A) Gel analysis of QPCR products from a representative ChIP assay showing PCR marker (lane 1), IgG (lane 2), C/EBP β (lane 3), PAX5 (lane 4), MAZ (lane 5), no antibody (lane 6), serially diluted inputs (lanes 7–10) and no template control (lane 11). (B) Enrichment at specific promoters was quantified using a standard curve constructed with serially diluted input samples. ChIP was performed at least three times for each antibody and the enrichment (as a percentage of input samples) at target promoters was then normalized to the background level of enrichment generated by a non-specific IgG control.