TGF-β–Mediated Foxp3 Gene Expression Is Cooperatively Regulated by Stat5, Creb, and AP-1 through CNS2

Abstract

Foxp3 plays an important role in the development and the function of regulatory T cells (Treg). Both the induction and maintenance of Foxp3 gene expression are controlled by several regulatory regions including two enhancers in the conserved noncoding sequences (CNS). The functions of Enhancer 1 in CNS1 are well established, whereas those of Enhancer 2 in CNS2 remain unclear. Although CNS2 contains enhancer activity, methylated CpG sequences in this region prevent Foxp3 gene expression in Foxp3− T cells. These sequences are however demethylated in Foxp3+ Treg by mechanisms as yet unknown. To investigate the role of CNS2, we have determined the Enhancer 2 core sequence by luciferase reporter assays in the absence of methylation to exclude the inhibitory effect, and shown that transcription factors AP-1, Stat5 and Creb cooperate in regulating Enhancer 2 activity. We have then determined the methylation sensitivity of each of the transcription factors. AP-1 was found to be methylation sensitive as has previously been described for Creb. However, Stat5 was active even when its binding site in CNS2 was methylated. Stat5 binding to Enhancer 2 occurred early, and preceded that of AP-1 and Creb during Treg induction. In addition, Stat5 activation is itself dependent on TGF-β signaling through Smad3 mediated blockade of Socs3 expression. These findings suggest that Stat5 is a key regulator for opening up the CNS2 region during iTreg induction, while AP-1 and Creb maintain Enhancer 2 activity.

An increased CD25-positive intestinal regulatory T lymphocyte population is dependent upon Cox-2 activity in the Apcmin/+ model

“…However, an alternative tumour-specific signal to LP T reg generation during the process of tumorigenesis (which was not the purpose of this study) cannot be excluded. With reference to our observations, it has been proposed that, in different circumstances, TGF-b can generate T regs via diverse molecular pathways [dependent upon or independent of CD25 signalling] [32]. Furthermore, a previous study showed conversion of CD25 -T regs to CD25 1 T regs by TGF-b to mediate immunosuppression in tumours [33].…”

Control of the Inheritance of Regulatory T Cell Identity by a cis Element in the Foxp3 Locus

“…Thus, IL-2 could be a factor that “positively” acts via CNS2 and whose deprivation leads to a pronounced loss of Foxp3 expression in the absence of CNS2 . Indeed, CNS2 contains multiple STAT5 binding motifs and can be bound by STAT5 (Figure S1A) (Ogawa et al, 2013; Yao et al, 2007). …”

FOXP3+ regulatory T cells and their functional regulation

“…14 DNA methylation experiments have revealed that the Foxp3 promoter and conserved non-coding DNA sequence 2 (CNS2) region are highly demethylated in tTregs, which facilitate mRNA transcription of Foxp3 and contribute to lineage stability through FOXP3 expression, while in vitro induced Treg cells are substantially methylated at the CNS2 region. 15,16 TGF-b treatment decreases the methylation status of CNS2 and promotes FOXP3 expression. Knockdown of the DNA methyltransferase Dnmt1 can also induce FOXP3 expression.…”