The Regulatory Mechanism Of Lymphocyte-activation Gene3 (Lag3) Expression

Lymphocyte-activation gene 3 (lag3), also known as CD223, was originally identified as a CD4-like transmembrane molecule that locates upstream of CD4, belonging to the immunoglobulin superfamily (IgSF). Early studies showed that LAG3 was restrictedly expressed on a few subpopulations of the PBMCs (peripheral blood mononuclear cells), such as activated T lymphocytes and plasmacytoid dendritic cells. Extensive following studies suggest that LAG3 has diverse biological effects on regulating CD8+ T cell functions and is involved in immune dysfunctions under various disease conditions. However, mechanisms underlying lag3 gene regulation remain incompletely understood. In this study, we utilized a model consisted of three T cell lines (CEM, Jurkat E6-1, Jurkat ZM cells), which express differential LAG3 mRNA and protein levels, to partially investigate the regulatory mechanism of lag3 gene expression in T cells, and revealed that the DNA methylation occurred on the CpG island shore locates in a transcriptionally regulatory region would inhibit lag3 gene transcription.First, we detected LAG3 mRNA and protein expression levels in three T cell lines (CEM, Jurkat E6-1, Jurkat ZM cells) and found that LAG3 expression was regulated at the transcriptional level in these cell lines. Through sequence alignments between human and mouse genomes, we identified two potentially regulatory regions CRA and CRB (conserved region A&B), locating upstream of lag3 transcription start site (TSS). Then, we performed DNase 1 hypersensitive site assays on LAG3-/+ subsets of Jurkat E6-1 cells and found that CRA and CRB were both sensitive to DNase I treatment in the LAG3+ subset. Cloning of a 1446-bp genomic DNA fragment contains CRA and CRB and gene reporter assays showed that the cloned region had robust promoter activity in Jurkat E6-1 cells. Further, CRB was functionally characterized as the core region of lag3 promoter by progressively truncating the 1446-bp fragment. Surprisingly, all these transcription-competent fragments exhibited similar or even higher promoter activities in LAG3 low-expressed CEM and Jurkat ZM cells, suggesting that the endogenous LAG3 expression might be epigenetically regulated. Next, using the Methprimer program, we predicted a single putative CpG island (IS, GC content=68%, Observed/Expected CpG value=0.77) lies across the third exon and a CpG island shore presents in CRB with lower CpG density (GC content=66%, Observed/Expected CpG value=0.45) lies lkb upstream of IS. Sequencing of bisulfite treated genomic DNA from the three T cell lines suggested that IS was steadily hypermethylated among these cell lines, while the methylation status of the CpG island shore inversely correlated with LAG3 expression levels in these cell lines. In vitro methylation of CRB could significantly reduce its promoter activity, implying that CRB methylation lead to lag3 transcriptional repression. In addition, treatment of LAG3 low-expressed CEM and Jurkat ZM cells with DNA methyltransferase inhibitor,5-Aza-dc, could restore LAG3 expression at both mRNA and protein levels and demethylated CRB. In comparison to B cell counterpart, CRB was hypomethylated in all CD8+ T cell memory differentiation subpopulations.Taken together, our data suggest that CpG island shore methylation is one of the key epigenetic regulators for lag3 gene transcription in human T cell lines, and hypomethylated CRB might poise primary CD8+ T cells for rapid LAG3 expression in order to counter activation signal and terminate CD8+ T cell immune response promptly.