derived hematopoietic precursors and generated bone marrow chimeras that contained transduced GFP+ and non-transduced GFP2 T cells. Although statistically not significant, we observed a trend pointing to a higher frequency of IL-17 producing cells among the CD4+ Th and CD42 cells in chimeras from bone marrow transduced with either miR-133b or miR-206 but not the empty vector (Fig. 7B, C). Discussion In this work, we revealed a tight co-regulation of the expression of the miR-206/133b cluster with the potential to produce the pro-inflammatory cytokine IL-17A in lymphocytes. This finding was equally valid for cells that had acquired the capacity to produce IL-17 cytokines in vivo and for in vitro polarized CD4+ Th17 cells. Furthermore, this co-regulation was similarly observed in those subsets of cd T cells that were reported to be genuine IL-17A producers, i.e. characterized by the expression of CCR6 [14,15] and the IL-23R [36]. However, at present it is not clear whether expression of miR-206/133b plays a functional role in Th17 lineage differentiation by targeting specific mRNAs or whether its expression may just be involved in epigenetic regulation of the Il17a/f locus. In fact, experiments, in which miR-206 or miR-133b or both were over- expressed in T cells showed no significant changes in the capacity of naive ab T cells to become polarized towards Th17 or other Th lineages as well as iTreg. Similarly, over-expression of miR-206 or miR-133b in bone marrow chimeras did not reveal striking changes in the frequency of IL-17 producing CD4+ Th or CD42 cells. Synteny of the miR-206/133b and Il17a/f loci is conserved among mammals and chicken, suggesting that the transcriptional co-regulation is physiologically relevant. Along this line, genome- wide gene expression studies suggest that neighboring co-expressed genes in eukaryotes are often functionally connected, implying some operational similarity to prokaryotic operons [16]. However, despite the high expression levels of miR-206/133b we found no detectable expression of Il17 in muscle tissue (Fig. S3) suggesting that the co-regulation of the miR-206/133b cluster and the Il17a/f locus is specific for T lymphocytes. Recently, Akimzhanov et al. characterized the size of the locus control region of the Il17a/f genes, which extends beyond the highly conserved non-coding sequences upstream of the Il17a/f locus, where miR-206/133b is located [37]. Furthermore, miR- 206/133b are close to a conserved noncoding sequence (CNS-1 or -60 region), which was recently reported to undergo chromatin remodeling in Th17 cell differentiation and therefore possibly provides accessibility to regulatory elements [38]. In addition to classical roles of miRNAs, namely gene-regulation by targeting 39- UTRs of mRNA, it is thus conceivable that expression of the miRNAs miR-133b and miR-206 is involved in the regulation of Il17a and Il17f locus accessibility comparable to the involvement of miRNA in DNA methylation in plants [39]. Transcriptional activity of the miR-206/133b cluster may promote opening of the whole region including the Il17a/f locus. Likewise, the miR-181- a1/181-b1 cluster is located upstream of the CD45 gene locus and is known to be involved in the T versus B lineage decision in CD45+ lymphocytes [35]. Using quantitative Real-Time PCR (qRT-PCR), we found considerable differences of miR-206/133b expression levels between IL-17 producing and non-producing cells suggesting high specificity of the assay. Indeed, a very recent comprehensive resource study based on deep-sequencing reproduced our findings that miR-206/133b are specifically upregulated in Co-Regulation of miR-133b and miR-206 in the Il17a/f Locus Th17 cells compared to other T cell subsets [40]. However, overall expression levels of miR-133b in lymphocytes were much lower when compared to samples of skin and muscle tissue. Notably, miR-206 was expressed to considerable extents in Th17 cells with more than 1000 sequence tags per million. Further- more, expression of this miRNA was only 14-fold and 19-fold lower in Th17 cells when compared to skin and muscle [40]. Accordingly, miR-206/133b were initially described to be specifically expressed in skeletal muscle as well as osteoblasts suggesting that the Il17a/f locus might be accessible for gene transcription in these tissues as well. So far, expression of IL-17A and/or IL-17F has not been reported for these cell types. Whereas IL-17A expression is thought to be restricted to lymphocytes, IL-17F expression has also been demonstrated in colonic epithelial cells [41]. Several targets of miR-133b and miR-206 such as DNA pol a, connexin43, histone deacetylase 4 (HDAC4) and the transcription factor Pitx3 have been reported to play a role in muscle development, regeneration of neuro- muscular synapses as well as osteoblast function [22,42,43,44]. Therefore, it will be interesting to see whether such targets are also functionally regulated in IL-17A producing lymphocytes. In addition, we performed in silico analysis of potential target genes for both miRNAs with the target prediction tools miRANDA, TargetScan and PicTar in order to identify novel target genes of potential immunological interest. Interestingly, the gene coding for the transcription factor Ets1 was predicted to be a target of both miR-133b (one predicted recognition site) and miR-206 (four predicted recognition sites). Ets1 was previously suggested to regulate Th17 cell differentiation as a direct target of miR-326 [12]. However, we could not confirm an effect of miR-133b or miR-206 overexpression on constructs containing the 39-UTR of the Ets1 gene in the context of the BW5147a-b- thymoma cell line (Fig. S4). Co-regulation of neighboring genes may be controlled by multiple non-mutually exclusive factors. Thus, changes in chromatin status may occur over extended distances. Location of miR-206/133b within the reported locus control region of the Il17a/f gene is consistent with this hypothesis. Alternatively, neighboring genes may directly share promoter or enhancer elements. In this study we found that both miR-206/133b and IL- 17A can be induced by IL-23 signaling. IL-23/p19 induced activation of STAT3 leads to direct binding of phosphorylated STAT3 to IL-17A and IL-17F promoters suggesting that, in addition to chromatin remodeling, sharing of regulatory elements may as well contribute to co-regulation of miR-206/133b and IL- 17A [45]. Taken together, we discovered co-regulation of miR- 133b and miR-206 with the Il17a/f locus as a novel feature of T cell differentiation that is shared between mouse ab and cd T cells and also extended to human Th17 cells. This qualifies transcrip- tion of miR-133b and miR-206 as a novel marker for T cells of an IL-17-producing phenotype. Materials and Methods Mice Six- to ten-wk-old C57BL/6N and B6.129P2(Cg)-Rorctm2Litt/J [25], BALB/c DO11.10 TCR-transgenic mice were purchased from either Charles River (Germany) or from The Jackson Laboratory. C57BL/6-Tcrd-H2BeGFP mice have been described before [27]. IL-23Rgfp.KI mice have been described previously [30,46]. Mice were bred and housed under specific pathogen free (SPF) conditions in individually ventilated cages (IVC) either at the Hannover Medical School animal facility or at the SPF facility at the Technical University Munich and all animal experiments were PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e20171