We showed here that continuous presence of TGF-β was required fol

We showed here that continuous presence of TGF-β was required following restimulation to maintain the inducible binding activity of the PcG protein Mel-18 at the Il17a promoter. In its absence, the binding of Mel-18 18 h following restimulation was comparable to that in resting cells. However, TGF-β

was not sufficient to induce the binding activity of Mel-18 at the Il17a promoter in the absence of TCR stimulation. Therefore, signaling pathways downstream to the TCR and polarizing cytokines synergize to induce and maintain, LBH589 respectively, the binding activity of Mel-18 at the Il17a promoter, and consequently to promote its expression. Eighteen hours following restimulation, the downregulation in the expression of the Th17 cytokines and transcription factors was IL-12-independent. IL-12 was more important for the upregulation of the expression of the Th1 key genes Tbx21 and Ifng. In accordance with that, IL-12 only modestly increased the detachment of Mel-18 from the Il17a promoter. It was previously shown that the differentiation of Mel-18-deficient RXDX-106 Th2 cells is impaired 73. Our recently published results demonstrated that PcG proteins positively regulate the expression

of the signature cytokine genes in Th1 and Th2 cells 74. The knockdown experiments here showed that Mel-18 positively regulates the expression of Il17a in restimulated Th17 cells. Considering that: (i) Mel-18 was associated with Il17a in correlation with gene expression and (ii) its binding was regulated synergistically by signaling pathways crucial for Il17a expression – our results support the idea that Mel-18 functions directly to increase Il17a expression, but indirect effects cannot be excluded. The binding activity of Ezh2 at the Il17a promoter was dependent on signaling pathways downstream to the Dichloromethane dehalogenase TCR, but in 18 h-restimulated Th17

cells the binding was TGF-β independent. Yet, knockdown of Ezh2 resulted in the downregulation of Il17a. Since Ezh2 is associated with Il17a promoter, a direct regulation of Il17a expression is suggested. However, as with Mel-18, it is also possible that Ezh2 indirectly regulates the expression of Il17a, for example by modulating the TCR signaling pathway; Ezh2 interacts with Vav 75 and is involved in actin polymerization 76. Ezh2 may also have a context-dependent functional role at the Il17a gene; it can function as transcriptional activator in the presence of Mel-18 but following its removal in the absence of TGF-β, Ezh2 may turn into a conventional PcG repressor. It was shown indeed that H3K27me3 is increased at the Il17a promoter in the presence of IL-12 and absence of TGF-β 42. However, this change requires a longer kinetics of 48 h, and therefore it was suggested by the authors that this is probably not the earliest event that initiates the repression of Il17a.

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