Molecular Regulation of SATB1 in Regulatory T-cells

Abstract

In this study we have identified SATB1, a nuclear protein that recruits chromatin-remodeling factors and regulates numerous genes, as a novel effector molecule in T_reg cells. Our interest in SATB1 resulted from a genome wide expression profile of T_reg cells and conventional T-cells (T_conv cells). SATB1 was a prominent candidate gene that constantly repressed in T_reg cells and highly expressed in T_conv cells. The dominant repression of SATB1 expression in T_reg cells could be confirmed at mRNA, protein, and single cell level under resting and different stimulation conditions in humans and mice. In contrast, SATB1 is expressed at high levels in T_conv cells and is further enhanced following physiological stimulation.<br /> The inverse expression pattern of FOXP3, the main transcription factor in shaping and maintaining T_reg cell identity, in relation to SATB1 led us to hypothesize its active involvement in regulation of SATB1. On the one hand, induction of FOXP3 was associated with inhibition of SATB1. This could be demonstrated by induction of FOXP3 in naïve CD4+ T-cells converted to induced T_reg cells (iT_reg) or in CD4⁺ T-cells ectopically overexpressing FOXP3 after lentiviral transduction. On the other hand, using different genetic approaches loss of FOXP3 expression in T_reg cells results in relieving the FOXP3-mediated repression and leads to an upregulation of SATB1. Furthermore, confocal microscopy on lymphocytes form <em>scurfy</em> and normal mice interestingly showed mutually excluding staining patterns. While the SATB1 signal is low in normal FOXP3-expressing thymocytes, it is high in thymocytes expressing a mutated non-functional FOXP3 from <em>scurfy</em> animals. <br /> FOXP3 as a transcription factor has been linked to direct binding to DNA, thereby regulating gene expression. To investigate whether FOXP3 can directly bind to the SATB1 genomic locus FOXP3-ChIP tiling arrays were performed. The analysis of tiling array data provided us with several putative FOXP3 binding sites in the promoter and intronic regions of the SATB1 locus which were confirmed by ChIP qRT-PCR. Furthermore, we were able to demonstrate high specificity of the binding and determine the binding coefficients of FOXP3 to several motifs in the SATB1 locus by filter retention assays. To assess whether this binding has functional relevance, we performed reporter assays and showed that FOXP3 reduces lucifierase activity for several binding regions clearly supporting that FOXP3 regulates SATB1 transcription by direct binding to the genomic locus. Interstingly, we showed that FOXP3 also controls SATB1 gene expression indirectly at post-transcriptional level via miRNAs. Indeed we identified several FOXP3 dependent miRNA that have been linked to post-transcriptional regulation of gene expression. FOXP3-ChIP tiling arrays showed FOXP3 peaks within these miRNAs loci. Furthermore, silencing of FOXP3 reversed this enrichment, whereas over-expression of FOXP3 induced their expression. Binding of FOXP3-dependent miRNAs to the 3´UTR of SATB1 in reporter assays confirms the suppressive effect of these miRNAs on SATB1 expression. An additional level of regulation of gene expression is exerted by epigenetic modifactions of the respective genomic locus. Epigenetic changes control the accessibility of a genomic locus by permissive or inhibitory histone modifications as well as methylation of CpG islands. Although, we did not observe differences in the methylation pattern of the CpG islands at the SATB1 locus between T_reg cells and T_conv cells, we observed more permissive and less repressive histone marks at the SATB1 genomic locus in T_conv cells and the opposite in T_reg cells which is in line with the expression data and aforementioned described regulatory mechanism of SATB1 expression in T_reg cells. <br /> Besides the molecular mechanism regulating SATB1 expression in T_reg cells, we further delineated the functional consequences of induction of SATB1 in T_reg cells. Lentiviral over-expression of SATB1 in human and murine T_reg cells resulted in the edition of gene expression and function of T_reg cells. The striking observation was the abrogation of the capacity of T_reg cells to suppress the proliferation of responder cells <em>in vitro</em>, in addition to the production of proinflammatory cytokines like IL-4 and IFN-γ. These findings suggested that T_reg cells acquire an effector phenotype a finding which is further corroborated on a genome wide level. Gene expression profiles of SATB1 overexpressing T_reg cells showed that many proinflammatory genes have been switched on upon induction of SATB1 expression in T_reg cells which promotes skewing of regulatory towards effector programs. To further prove the antagonistic effect of SATB1 on the regulatory function of T_reg cells <em>in vivo</em>, we adoptively transferred T_reg cells overexpressing SATB1 with naïve CD4+ cells into RAG2-/- mice. In this experimental setting T_reg cells failed to suppress inflammation in vivo and subsequently the mice developed colitis. <br /> In conclusion, SATB1 is an important effector molecule whose expression is tightly regulated in T_reg cells. SATB1 upregulation in T_reg cells results in aquisition of proinflammatory properties and attenuated suppressive function <em>in vitro </em>and<em> in vivo</em>. Therefore, FOXP3-mediated repression of SATB1 expression in T_reg cells seems to be an important regulatory circuit crucial to maintain suppressive function of these cells.