Complement receptor 2 polymorphisms associated with systemic lupus erythematosus modulate alternative splicing.

Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971, rs17615, rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs 32.6% in controls, P=0.016, OR=0.90 (0.82-0.98)). Two of these SNPs are in exon 10, directly 5' of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon. Effects of these SNPs and a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact.

CR2/CD21 proximal promoter activity is critically dependent on a cell type-specific repressor.

Transcription of the human complement receptor type 2 (CR2/CD21) gene is controlled by both proximal promoter and intronic elements. CR2 is primarily expressed on B cells from the immature through mature cell stages. We have previously described the presence of an intronic element that is required for both cell- and stage-specific expression of CR2. In this study, we report the identification of a cell type-specific repressor element within the proximal promoter. This repressor sequence is shown by linker scanning mutagenesis to comprise an E box motif. By supershift analysis this element binds members of the basic helix-loop-helix family of proteins, in particular E2A gene products. Mutational analysis demonstrates that binding of E2A proteins is critical for functioning of this repressor. Thus, E2A activity is key not only for early B cell development, but also for controlling CR2 expression, a gene expressed only during later stages of ontogeny.

A site in the complement receptor 2 (CR2/CD21) silencer is necessary for lineage specific transcriptional regulation.

Expression of human complement receptor type 2 (CR2/CD21) is primarily restricted to mature B cells and follicular dendritic cells. We previously described an intronic transcriptional silencer that controls the appropriate B cell-specific and developmentally restricted expression of human CR2/CD21 in both stably transfected cell lines and transgenic mice. Here we report the identification of a nucleotide sequence within the 2.5 kb CR2 silencer (CRS) that is crucial to its silencer function. This site comprises a binding site for the transcriptional repressor CBF1 (RBP-J or RBP-Jkappa) as well as Sp1 and other as yet uncharacterized proteins. A 2-bp mutation which eliminates the binding of CBF1 and other protein(s) in vitro results in loss of silencer activity in vivo. These results demonstrate the importance of this site in regulating CR2 expression and suggest that CBF1, a component of the developmentally important Notch signaling pathway, may play a role in the control of human CR2 gene expression.

The role of Sp family members, basic Kruppel-like factor, and E box factors in the basal and IFN-gamma regulated expression of the human complement C4 promoter.

The fourth component of human complement (C4) is a serum protein that is expressed in the liver and other organs. The promoter region of the C4 gene has been analyzed in reporter gene assays in two cell lines that represent hepatic (HepG2) and monocytic (U937) lineages. Analysis indicated that regions important for basal transcription in HepG2 cells included Sp1 and E box sites within the first 100 bp upstream of the transcription initiation site but not the nuclear factor-1 site important in the control of the mouse C4 gene. Also, a region encompassing -468 to -310 was able to repress activity 2-fold. However, when a CACCC or GT box sequence at -140 was mutated the repressive activity of the upstream region resulted in almost no activity. The -140 region consists of a series of four closely positioned GT boxes that were shown to bind Sp1, Sp3, and basic Krupple-like factor in EMSA. This novel two-part regulatory element may be involved in the regulated expression of C4. However, IFN-gamma a major activator of C4 expression did not signal through this two-part regulatory element. We were able to map the position of an IFN-gamma responsive element in U937. IFN-gamma was able to increase transcription by up to 20-fold with mutations in the E box sequence at -78 to -73, thus completely abolishing induction. We conclude that the E box binding factors, which appear to be distinct from upstream stimulatory factors 1 and 2, are totally responsible for IFN-gamma induction of C4.

New Australian aboriginal complotypes characterised by increased C4 gene copy number.

Complement component C4 genes are located within the central region of the human MHC. The genomic arrangement of these genes is complex, with each chromosome usually encoding either one or two C4 genes. C4 allotyping of a group of Western Australian Aborigines demonstrated certain discrepancies in the densitometric ratios between the C4A4 and the C4A3 protein bands; however, the mechanism causing the increase in density of the C4A4 band was unknown. Our aim was to determine whether the increase in densitometry was due to an increase in the expression of the C4A4 isotype, or whether these individuals carried a new complotype characterised by an increased gene copy number. Using pulsed-field gel electrophoresis and Taq I RFLP analysis we will show that the apparent increase in C4A4 protein expression was due to the existence of new, previously uncharacterised Aboriginal complotypes defined by at least three C4 genes. Segregation analysis from an extensive family suggests that one of the new C4 complotypes is likely to contain the duplicated C4A4 isotype together with a C4B2 gene (C4A4, C4A4, C4B2) and is the first such chromosomal arrangement seen in this population group.

Extensive conservation of upstream C4 promoter sequences: a comparison between C4A and C4B.

Basal levels of expression of complement C4 vary in individuals but it is unknown whether this is due to gene copy number, protein clearance rates or differences in expression. To investigate whether differences in the promoter region may influence transcription of the C4 genes, we sequenced the promoter region of the C4B1 gene from the HLA-A1, B8, C4AQ0, C4B1, DR3 haplotype and compared this with an equivalent C4A3 region. The promoter regions of the C4 genes were highly conserved, indicating that transcriptional differences are unlikely. An unusual feature of the alignment was the higher level of polymorphism in the RP genes past the breakpoint of deletion/duplication in a region where the highest conservation would be expected.

Analysis of the major histocompatibility complex microsatellite CL1 in different human haplotypes.

Characterization of the region between HLA-B and the TNF loci in the human MHC revealed the presence of duplicated loci, named CL1 and CL2, that included repeat sequences. Development and use of a PCR typing methodology that amplified both CL microsatellites simultaneously indicated that PCR product patterns analysed on native agarose gels were allelic (Abraham et al., 1992). The purpose of the current study was to determine the molecular explanation for the unique patterns achieved. Sequence analysis of the CL1 locus from 32 chromosomes representing 10 ancestral haplotypes indicated that six alleles were present. The CL microsatellites also provided an opportunity to study the evolutionary relationships between MHC haplotypes from different racial groups. Sequence comparison of closely related ancestral haplotypes from different racial groups suggested that the CL1 microsatellite has not changed in the period since divergence.

Comparative analysis of the disease-associated complement C4 gene from the HLA-A1, B8, DR3 haplotype.

Complement component C4 is an important protein of the classical, or antibody-mediated pathway of complement activation. Human C4 is located within the central region of the major histocompatibility complex on chromosome 6. Partial C4 deficiency has been associated with an increased susceptibility to immune complex disease. The strongest association with partial C4 deficiency is with systemic lupus erythematosus (SLE) and has been shown in most racial groups studied. Interestingly, Caucasian population studies have demonstrated an increased prevalence of C4A null alleles in SLE patients, in particular in association with the haplotype HLA-A1, B8, BfS, C4AQ0, C4B1, DR3. To investigate whether the C4 gene on this haplotype had any structural irregularities which may explain disease association, we sequenced the entire C4B gene from this haplotype. The results revealed that the gene encoded on the disease-associated haplotype carried major structural differences (when compared to C4A3) at the exonic level only in the C4d region. A high degree of conservation in both the 5' and 3' untranslated regions imply that disease associations will not be due to differential C4 expression as a result of regulatory differences between C4 genes. It appears likely that protein clearance mechanisms may account for the altered levels of C4 seen between different isotypes.