Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer

Abstract

The transcription factor Foxp3 is involved in the differentiation, function and survival of CD4+CD25+ regulatory T (T(reg)) cells. Details of the mechanism underlying the induction of Foxp3 expression remain unknown, because studies of the transcriptional regulation of the Foxp3 gene are limited by the small number of T(reg) cells in mononuclear cell populations. Here we have generated a model system for analyzing Foxp3 induction and, by using this system with primary T cells, we have identified an enhancer element in this gene. The transcription factors Smad3 and NFAT are required for activity of this Foxp3 enhancer, and both factors are essential for histone acetylation in the enhancer region and induction of Foxp3. These biochemical properties that define Foxp3 expression explain many of the effects of transforming growth factor-beta on the function of Foxp3+ T(reg) cells.

Unravelling the molecular basis for regulatory T‐cell plasticity and loss of function in disease

“…CNS2 contains the TSDR and is responsible for maintenance of FOXP3 in all dividing Treg and CNS3 is the pioneer site for FOXP3 expression in thymic origin nTreg 63. Occupancy by specific transcription factors at each of these loci is now characterised, and this includes AP1 and NFAT at CNS1,59 Runx1 and CBFβ at CNS264 and cREl at CNS3 63. The observation that naïve human CD4+ T cells induce FOXP3 upon activation,65, 66, 67 and that in the presence of TGFβ and all‐trans retinoic acid (ATRA)63 the expression of FOXP3 is stabilised to some degree, was defined transcriptionally by the finding that the activation‐induced expression of FOXP3 results from partial but not complete demethylation of the FOXP3 locus in iTreg 37.…”

Regulation of the Immune Response by TGF-β: From Conception to Autoimmunity and Infection

“…The TGF-b signaling pathway plays a role in the early development of tTreg cells, and 3-to 5-day old mice lacking the TGF-b type I receptor (TbRI) show a dramatic reduction in the frequency of Foxp3 þ thymocytes (Liu et al 2008). Although a conserved DNA sequence for Smad3 binding is present in Foxp3 gene regulatory sequences (Tone et al 2008), TGF-b signaling is dispensable for the induction of Foxp3 expression in tTreg cells (Zheng et al 2010;Schlenner et al 2012), showing that TGF-b does not promote tTreg-cell development by directly regulating Foxp3 expression. Instead, TGF-b signaling promotes tTreg-cell development by antagonizing thymic-negative selection, and therefore promoting the survival of tTreg-cell precursors (Ouyang et al 2010).…”

“…The Foxp3 gene contains an enhancer element that allows for direct binding of Smad3 and nuclear factor of activated T (NFAT) cells (Tone et al 2008) to a conserved DNA sequence (Xu et al 2010;Zheng et al 2010). TGF-b also promotes locus activation by opposing the recruitment of a DNA methyltranferase to the Foxp3 locus (Josefowicz et al 2009).…”

Imprinting of Skin/Inflammation Homing in CD4+ T Cells Is Controlled by DNA Methylation within the Fucosyltransferase 7 Gene

“…For methylated oligonucleotides, all cytosines in CpG motifs were substituted by 5-methyl-2-deoxycytidin during synthesis. After annealing, 200 pmol of oligonucleotides were coupled to 25 ml of prewashed streptavidin agarose beads (Sigma-Aldrich) for 1 h in 400 ml of binding buffer (60 mM KCl, 12 mM HEPES [pH 7.9], 4 mM TrisHCl [pH 8], 0.5 mM EDTA, 5% glycerol) (23). Fifty micrograms of nuclear extract (nuclear extract kit; Active Motif) of 5 h PMA/ionomycin-stimulated RLM-11-1 cells was preincubated for 1 h at 4˚C in 400 ml of binding buffer supplemented with 5 mg of salmon sperm DNA (Invitrogen), 1 mM DTT, and cOmplete protease inhibitors (Roche).…”