Standardization of DNA amount for bisulfite conversion for analyzing the methylation status of LINE-1 in lung cancer.

Highly methylated Long Interspersed Nucleotide Elements 1 (LINE-1) constitute approximately 20% of the human genome, thus serving as a surrogate marker of global genomic DNA methylation. To date, there is still lacking a consensus about the precise location in LINE-1 promoter and its methylation threshold value, making challenging the use of LINE-1 methylation as a diagnostic, prognostic markers in cancer. This study reports on a technical standardization of bisulfite-based DNA methylation analysis, which ensures the complete bisulfite conversion of repeated LINE-1 sequences, thus allowing accurate LINE-1 methylation value. In addition, the study also indicated the precise location in LINE-1 promoter of which significant variance in methylation level makes LINE-1 methylation as a potential diagnostic biomarker for lung cancer. A serial concentration of 5-50-500 ng of DNA from 275 formalin-fixed paraffin-embedded lung tissues were converted by bisulfite; methylation level of two local regions (at nucleotide position 300-368 as LINE-1.1 and 368-460 as LINE-1.2) in LINE-1 promoter was measured by real time PCR. The use of 5 ng of genomic DNA but no more allowed to detect LINE-1 hypomethylation in lung cancer tissue (14.34% versus 16.69% in non-cancerous lung diseases for LINE-1.1, p < 0.0001, and 30.28% versus 32.35% for LINE-1.2, p < 0.05). Our study thus highlighted the optimal and primordial concentration less than 5 ng of genomic DNA guarantees the complete LINE-1 bisulfite conversion, and significant variance in methylation level of the LINE-1 sequence position from 300 to 368 allowed to discriminate lung cancer from non-cancer samples.

Induction of Short NFATc1/αA Isoform Interferes with Peripheral B Cell Differentiation.

In lymphocytes, immune receptor signals induce the rapid nuclear translocation of preformed cytosolic NFAT proteins. Along with co-stimulatory signals, persistent immune receptor signals lead to high levels of NFATc1/αA, a short NFATc1 isoform, in effector lymphocytes. Whereas NFATc1 is not expressed in plasma cells, in germinal centers numerous centrocytic B cells express nuclear NFATc1/αA. When overexpressed in chicken DT40 B cells or murine WEHI 231 B cells, NFATc1/αA suppressed their cell death induced by B cell receptor signals and affected the expression of genes controlling the germinal center reaction and plasma cell formation. Among those is the Prdm1 gene encoding Blimp-1, a key factor of plasma cell formation. By binding to a regulatory DNA element within exon 1 of the Prdm1 gene, NFATc1/αA suppresses Blimp-1 expression. Since expression of a constitutive active version of NFATc1/αA interfered with Prdm1 RNA expression, LPS-mediated differentiation of splenic B cells to plasmablasts in vitro and reduced immunoglobulin production in vivo, one may conclude that NFATc1/αA plays an important role in controlling plasmablast/plasma cell formation.

NFATc1 supports imiquimod-induced skin inflammation by suppressing IL-10 synthesis in B cells.

Epicutaneous application of Aldara cream containing the TLR7 agonist imiquimod (IMQ) to mice induces skin inflammation that exhibits many aspects of psoriasis, an inflammatory human skin disease. Here we show that mice depleted of B cells or bearing interleukin (IL)-10-deficient B cells show a fulminant inflammation upon IMQ exposure, whereas ablation of NFATc1 in B cells results in a suppression of Aldara-induced inflammation. In vitro, IMQ induces the proliferation and IL-10 expression by B cells that is blocked by BCR signals inducing NFATc1. By binding to HDAC1, a transcriptional repressor, and to an intronic site of the Il10 gene, NFATc1 suppresses IL-10 expression that dampens the production of tumour necrosis factor-α and IL-17 by T cells. These data indicate a close link between NFATc1 and IL-10 expression in B cells and suggest NFATc1 and, in particular, its inducible short isoform, NFATc1/αA, as a potential target to treat human psoriasis.

NF-κB factors control the induction of NFATc1 in B lymphocytes.

In peripheral lymphocytes, the transcription factors (TFs) NF-κB, NFAT, and AP-1 are the prime targets of signals that emerge from immune receptors. Upon activation, these TFs induce gene networks that orchestrate the growth, expansion, and effector function of peripheral lymphocytes. NFAT and NF-κB factors share several properties, such as a similar mode of induction and architecture in their DNA-binding domain, and there is a subgroup of κB-like DNA promoter motifs that are bound by both types of TFs. However, unlike NFAT and AP-1 factors that interact and collaborate in binding to DNA, NFAT, and NF-κB seem neither to interact nor to collaborate. We show here that NF-κB1/p50 and c-Rel, the most prominent NF-κB proteins in BCR-induced splenic B cells, control the induction of NFATc1/αA, a prominent short NFATc1 isoform. In part, this is mediated through two composite κB/NFAT-binding sites in the inducible Nfatc1 P1 promoter that directs the induction of NFATc1/αA by BCR signals. In concert with coreceptor signals that induce NF-κB factors, BCR signaling induces a persistent generation of NFATc1/αA. These data suggest a tight connection between NFATc1 and NF-κB induction in B lymphocytes contributing to the effector function of peripheral B cells.

NFATc1 induction in peripheral T and B lymphocytes.

NFAT transcription factors control the proliferation and survival of peripheral lymphocytes. We have reported previously that the short isoform NFATc1/αA whose generation is induced by immune receptor stimulation supports the proliferation and inhibits the activation-induced cell death of peripheral T and B cells. We will show in this study that in novel bacterial artificial chromosome transgenic mice that express EGFP under the control of entire Nfatc1 locus the Nfatc1/Egfp transgene is expressed as early as in double-negative thymocytes and in nonstimulated peripheral T and B cells. Upon immune receptor stimulation, Nfatc1/Egfp expression is elevated in B, Th1, and Th2 cells, but only weakly in T regulatory, Th9, and Th17 cells in vitro whose generation is affected by TGFß. In naive lymphocytes, persistent immune receptor signals led to a 3-5 increase in NFATc1/αA RNA levels during primary and secondary stimulation, but a much stronger induction was observed at the protein level. Whereas anti-CD3(+)CD28 stimulation of primary T cells induces both NFATc1/αA and their proliferation and survival, anti-IgM stimulation of B cells induces NFATc1/αA and proliferation, but activation-induced cell death after 3-d incubation in vitro. The anti-IgM-mediated activation-induced cell death induction of B cells in vitro is suppressed by anti-CD40-, LPS-, and CpG-mediated signals. In addition to inducing NF-κB factors, together with anti-IgM, these signals also support the generation of NFATc1/αA. According to these data and the architecture of its promoter region, the Nfatc1 gene resembles a primary response gene whose induction is affected at the posttranscriptional level.