Allele-specific repression of lymphotoxin-α by activated B cell factor-1

Abstract

Genetic variation at the human LTA locus, encoding lymphotoxin-α, is associated with susceptibility to myocardial infarction, asthma and other diseases. By detailed haplotypic analysis of the locus, we identified a single-nucleotide polymorphism (SNP) at LTA+80 as a main predictor of LTA protein production by human B cells. We found that activated B-cell factor-1 (ABF-1) binds to this site in vitro and suppresses reporter gene expression, but only in the presence of the LTA+80A allele. Using haplotype-specific chromatin immunoprecipitation, we confirmed that ABF-1 is preferentially recruited to the low-producer allele in vivo. These findings provide a molecular model of how LTA expression may be genetically regulated by allele-specific recruitment of the transcriptional repressor ABF-1.Identifying human DNA polymorphisms that regulate gene expression is essential for understanding the genetic basis of common diseases but is technically challenging. Valuable insights into molecular mechanisms can be derived by in vitro analysis of protein-DNA binding and reporter gene expression, but we have limited tools to interrogate putative regulatory polymorphisms in vivo. Such analysis needs to consider natural chromatin structure and natural haplotypic combinations of polymorphisms. It also must be able to detect small differences in gene expression, which could be crucial for disease susceptibility if a physiologically important mediator is involved.We attempted to address these issues for LTA, an important early mediator of immune and inflammatory responses 1 . Regulation of LTA is finely calibrated to resist dangerous pathogens but not to cause inflammatory damage to the host, as seems to occur in bacterial sepsis 2 and a b Figure 1 LTA protein production is associated with LTA haplotype. (a) Haplotype structure at the LTA locus. SNPs present at an allele frequency >0.1 in the panel of 26 LCLs are shown (minor allele frequencies given in brackets) together with haplotypes (haplotype frequencies shown at the head of the columns). Designations of SNPs refer to the nucleotide position relative to transcriptional start site. Clade A is uniquely defined by LTA+10A, LTA+252G and LTA+723A; clade B is uniquely defined by LTA+80A and LTA+368C; and clade C is uniquely defined by LTA+438(10) and LTA+495C. The frequencies of these main haplotype clades were 0.27, 0.46 and 0.27, respectively. (b) Analysis of LTA production by haplotype pair. A scatter plot of LTA concentration in cell culture supernatant from the panel of LCLs is shown per 10 6 cells, plotted as a symbol representing each cell line together with the geometric mean (horizontal lines). A total of six replicate experiments were done. The cell lines heterozygous with respect to haplotypes A, B and C are shown categorized at a given time point according to the pair of haplotypes present in that cell line (A+B, A+C or B+C). At 24 h after stimulation, cells bearing haplotypes B+C or B+A were associated with significantly lower LTA concentrations than those bearing haploty...

Genetic variation at the TNF locus and the risk of severe sequelae of ocular Chlamydia trachomatis infection in Gambians

“…The LTA þ 77 SNP has been reported to affect LTA protein production in human EBV-transformed B cells. 31 The functionality of the risk þ 77T allele is thought to reside in the fact that the activated B cell Factor-1 (ABF-1) only binds in the presence of this low producing þ 77T allele in vivo to suppress gene expression. 31 Conversely, the LTA þ 252G allele, present in HAP2 and HAP3 but absent in HAP1, has been linked to higher LTA transcription levels in vitro.…”

“…31 The functionality of the risk þ 77T allele is thought to reside in the fact that the activated B cell Factor-1 (ABF-1) only binds in the presence of this low producing þ 77T allele in vivo to suppress gene expression. 31 Conversely, the LTA þ 252G allele, present in HAP2 and HAP3 but absent in HAP1, has been linked to higher LTA transcription levels in vitro. 13,14 Our data support the suggestion from in vitro functional studies of independent and opposite functional effects for LTA þ 77 T and þ 252G.…”

Haplotype-Based Association of Four Lymphotoxin-Alpha Gene Polymorphisms with the Risk of Coronary Artery Disease in Han Chinese

“…Therefore, we agree that replication/validation of those LTA SNPs in larger, well-designed studies within genetic and functional framework is critical. Knight et al (2004) had identified the A allele of A+80C polymorphism as a main predictive variable of LTA protein production by a detailed haplotype analysis of TNF/ LTA locus. Contrarily, we did not find a significant singlelocus association between CAD and A+80C polymorphism, which was one of the SNPs harboring the protective haplotype G-C-T-C.…”

Resolving the Variable Genome and Epigenome in Human Disease

“…For genes without transcribed genetic markers, relative allelic expression can be assessed by haplotype-specific chromatin immunoprecipitation (haploChIP) for RNA polymerase II using antibodies specific for the phosphorylated serine residues in the C-terminal domain characteristic of actively transcribing RNA polymerase II [135]. This approach can also be used to resolve allelespecific recruitment of specific transcription factors such as activated B cell factor-1 which we demonstrated was recruited to the LTA (encoding lymphotoxin alpha) gene [136] in the presence of a genetic variant subsequently associated with susceptibility to leprosy [137].…”