4C). Antibodies recognizing pS73 c-Jun were not sensitive enough to detect binding to the TNF proximal promoter/TSS in quiescent polarized T cells (Fig. 4C). No binding of NFATc2 or c-Jun was detected at the proximal promoter of the LTα gene (−148 −44); therefore, we considered

the corresponding amplicon PI3K inhibitor as a negative control (Fig. 4B and C). Overall, the level of c-Jun binding better correlated with the open conformation of TNF TSS than the level of NFATc2 binding. To investigate further the possible role of the TCR-activated transcription factors in the regulation of chromatin conformation at the TNF TSS, we performed Western blot analysis of the nuclear fractions from quiescent and activated T cells. In accordance with earlier reports [25-27, 49, 51], we detected an increase in NFATc2 concentration, including its active dephosphorylated form (lower band of approximately 130 kDa), in the nucleus already 15 min after activation of cells with anti-CD3 and anti-CD28 antibodies, while phosphorylation

of c-Jun (pSer63 and pSer73) became prominent only 1 h after stimulation and increased further at 3 h (Fig. 5). Such kinetics correlated with binding of NFATc2 and c-Jun with the TNF proximal promoter/TSS (Fig. 4B and C). Extended analysis of nuclear concentrations buy RAD001 of AP-1, NFAT, and NF-κB family members (Supporting Information, Results and Fig. 5) demonstrated that both NFATc2 and c-Jun transcription factors are required for chromatin remodeling at the TNF

TSS in T cells upon activation. We next compared chromatin status of the TNF TSS and the nuclear concentrations of NFATc2 and c-Jun transcription factors in mouse CD4+ T-cell subsets (Fig. 6A). In quiescent polarized T cells, we observed higher levels of expression and phosphoryl-ation of transcription factor c-Jun in Th1 and Adenosine Th17 cells regardless of the polarization method (either with soluble or immobilized anti-CD3 antibodies), while NFATc2 in quiescent polarized T cells remained at comparable levels except Th17 cells, where it was higher (Fig. 6A). We also detected similar or comparable levels of RelA/p65 and c-Rel transcription factors in the nuclei of quiescent polarized T cells (Fig. 6A), while c-Fos member of AP-1 family was not detected (data not shown). The level of JunB transcription factor was higher in Th2 and Th17 cells polarized in the presence of soluble anti-CD3 antibodies (Fig. 6A). Importantly, c-Jun appeared to be critical for the maintenance of open chromatin conformation at the TNF TSS in quiescent T cells polarized under Th1 and Th17 conditions. Incubation of these cells with c-Jun N-terminal kinase (JNK) inhibitor SP600125, blocking c-Jun phosphorylation (Supporting Information Fig. 5C), but not with cyclosporine A (CsA), blocking NFATc2 migration to the nucleus (Supporting Information Fig. 5C), facilitated the restoration of closed chromatin configuration at the TNF TSS (Fig. 6B and Supporting Information Fig. 6).