[14, 36] A small set of seemingly FOXP3-activated, Treg-cell-specific enhancers existed, but even these were recapitulated in FOXP3-negative cells upon activation and were enriched for motifs of the TCR activated transcription factors, AP-1 and NFAT.[14] Therefore, as with GATA3, TBET and RORγt, FOXP3 has a minimal role in the de novo activation of enhancers during differentiation, and instead functions subsequently, binding to previously active regulatory elements to augment or tune activity. The study FDA approved Drug Library in vitro by Rudensky and colleagues also reveals an extensive collection of regulatory DNA elements in ex vivo isolated, mature,

unstimulated CD4 T-cells. Almost 6000 uniquely accessible chromatin sites were present in mature naive CD4 T-cells, compared with B cells. This array of DNase I hypersensitive sites probably STAT inhibitor represents poised or active regulatory elements and may reflect the differentiation potential of these cells (almost all of these were shared with Treg cell DNase I hypersensitive sites).[14] Certainly, in the context of T-cell activation, AP-1, NFAT, IRF4 and other TCR-activated or induced transcription

factors have essential roles in de novo accessibility and activation of regulatory elements. However, while these recent studies expose the activity of several transcription factors in the activation of Th-cell-specific enhancers (previously inactive or poised in naive CD4 T-cells), the factors responsible for poising the enhancer landscape that exists in naive CD4 T-cells during thymocyte differentiation are largely unknown. Although a number of transcription factors are critical for thymocyte development (PU.1, NOTCH, GATA3, E2A, TCF-1, LEF-1, RUNX1, etc),[33] those responsible for the de novo accessibility and heritable maintenance of poised or active enhancer states are not well understood. Such factors could function analogously to PU.1 and C/EBP in myeloid cells and PU.1, EBF and E2A

in early B-cell differentiation – binding co-operatively to lineage-specific enhancers to mediate de novo chromatin remodelling and acquisition of H3K4me1 on enhancer-flanking nucleosomes.[37-39] Notably these studies found AP-1 motif enrichment at a portion of lineage-specific Teicoplanin enhancers, and AP-1 and NFAT motifs were also enriched among enhancers activated during Th cell polarization without Th1 or Th2 bias.[13] Furthermore, activation of a subset of MYOD enhancers appears to be dependent on AP-1; knockdown of c-Jun resulted in reduced H3K4me1 and H3K27ac at AP-1 and MYOD co-bound enhancers.[9] It is intriguing to consider then that both MRFs (MYOD) and ERFs (IRFs and STATs) could engage AP-1 as a common factor involved in de novo enhancer activation. Given its broad expression, what determines the activity of AP-1 in a given cell type? Several recent studies have characterized co-operative binding of AP-1 with IRF4 and IRF8.