Regulation of the lymphotoxin alpha gene : characterization of elements located between the transcription and translation start sites that impact expression
Yokley, Brian H.
Thesis (Ph.D.)--Georgetown University, 2010.; Includes bibliographical references.; Text (Electronic thesis) in PDF format. The lymphotoxin alpha (LTA) gene codes for an inflammatory cytokine whose expression is critical to numerous immunological processes. However, little research has investigated LTA gene regulation. In this study, we identify and further characterize functional elements within the LTA downstream segment (DSS; +1 to +459), which is bounded by the transcription and translation start sites. Luciferase assays demonstrate that the LTA DSS is required for full activity of the LTA promoter in T and B cells and that this region alone is capable of regulating LTA expression. DSS-mediated expression is dependent on an alternative promoter composed of a Sp1 and multiple TFII-I binding sites [initiator (Inr)-like element] as mutation of these sites greatly inhibits expression. ChIP assays confirm transcription factor binding in vivo and 5'RLM-RACE assays identify mRNA that initiate from the alternative promoter under specific stimulation conditions in both primary and immortalized T cells. Also identified are ten novel LTA mRNA transcripts, many of which utilize alternative splicing at position +19, whose 5'UTR lengths vary in a stimulation-dependent manner, presumably to influence translational efficiency. Functional analysis of single nucleotide polymorphisms (SNPs) located within the DSS indicates cell type-specific effects. Previously, the LTA +81C/A and +369G/C SNPs, which have been shown be functional and/or impact disease, had been analyzed only in B cells, yet T cells contribute most to LTA levels. Luciferase assays show that the LTA +81A variant significantly increases expression relative to +81C in T cells, opposite of its reported impact in B cells, and that the LTA +369G variant significantly decreases expression only in unstimulated T cells. EMSAs suggest that repression mediated by the +369G variant is likely due to an increased binding affinity to +369G by either a complex of Sp1 with an unidentified nuclear factor or the unidentified nuclear factor itself. ChIP assays confirmed Sp1 binding in vivo. Further, mutation of the putative Sp1 binding site associated with this SNP results in an expected change in expression. Taken together, these data indicate a complex cell type-specific and stimulation-dependent pattern of LTA gene regulation that utilizes both alternative promoters and functional SNPs.
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