Genome-Wide Binding Studies of Acetyl-STAT3 Demonstrates a Novel Regulatory Pathway in Dendritic Cells

Dendritic cells play a vital role in the induction of either activation or tolerance of an immune response.  However the molecular pathways regulating these opposite responses to immune stimuli remain poorly understood. STAT3 is a master transcriptional regulator of immune response including those mediated by DCs. STAT3 can either positively or negatively regulate DC responses, but the mechanisms are unknown. STAT3 is post-translationally modified by either acetylation of phosphorylation. While much is understood about transcriptional targets of phosphorylated STAT3, the gene targets and the functional impact of acetylated STAT3 remain unclear. We aimed to answer  the gene targets of acetylated-STAT3 and test the hypothesis that acetylation of STAT3 plays a key role on negative regulation of DCs.

To determine the transcriptional targets of acetyl-STAT3, we first performed genome-wide binding analysis of acetyl-STAT3 by ChIP-Seq. We additionally performed gene expression microarrays on these samples and coupled gene expression to the acetyl-STAT3 binding datasets then analyzed with Gene Set Enrichment Analysis (GSEA). Inhibition of histone deacetylases (HDACi) acetylates STAT3, therefore, we treated DC with either HDACi or diluent and performed ChIP-Seq and analyzed with Genomic Regions Enrichment Annotation Tool (GREAT). The analysis revealed 3598 binding sites in 4605 gene loci as potential targets of acety-STAT3. Theses binding sites were mostly proximal but some were also distal up to over 100 kb from transcription start site. Gene expression array showed 1701 genes up-regulated and 1668 genes down-regulated. Proximal binding of acety-STAT-3 showed more effective transcription function than distal binding.  Detailed analysis was performed utilizing TRANSFAC to analyze canonical GAS motif (TTCnnnGAA) and the non-canonical motif (single-nucleotide variants) of binding for acetyl-STAT3. In top 500 binding peaks, canonical motif sites bound by acetyl-STAT3 were 349 versus 87 in control (p<0.00001). Analysis of acetyl-STAT3 binding to all motifs, including the one canonical motif and 18 non-canonical motifs revealed that although all 19 motifs come in up-regulation and 16 motifs come in down- regulation, the canonical motif bound by acetyl-STAT-3 was associated with the highest percent of most significantly regulated target genes (92% and 91% for up- or down-regulation respectively), demonstrating the best transcription potential for canonical motif.

We next validated the expression of some of the up-regulated (IL-10Ra, Cdk6, E2F2, Rb1, Mef2c, and E2F2) and down-regulated genes (Map3k, Fcgr3, Slfn2, and Prdm1) that are not known to be direct targets of phospo-STAT3 and further confirmed that the acetyl-STAT-3 binding peaks at these gene loci were associated with H3K4me3 marks

We next performed analysis for functional pathways utilizing 2 different bioinformatics tools: IPA (Ingenuity Pathway Analysis) and MSigDB conjugated with and displayed by GREAT. IPA predicted that the most up-regulated pathways were those involved in Th2 differentiation and negative regulation of cytokine production and the most down-regulated pathways involved in antigen processing and presentation. MSigDB predicted that IL-10 signaling was among the most significantly altered pathway in the acetylated STAT-3 group. We next validated with ChIP-qPCR the genes involved in IL-10 and its signaling. We found the acetyl-STAT3 binding peaks at IL-10Ra and Ido1 gene loci were associated with H3K4me3 marks, demonstrating open configuration and increased transcription.  We next determined its biological relevance. We found that DCs with increased acetylated-STAT3 showed enhanced response to IL-10 and showed reduced responses to TLR (LPS) stimulation and reduction in activation of T cells. Further functional studies showed that IL-10 treatment of DCs with acetyl-STAT3 caused greater expression  in total STAT3 and IL-10 demonstrating a positive feed forward mechanism for sustaining tolerogeneic functions of DCs. These data thus collectively demonstrate (a) acetylation of STAT3 targets different genes than phosphorylation of STAT3 and (b) acetylated STAT3 acts in a feedforward mechanism to enhance DC tolerance by increasing IL-10Ra and thus enhancing sensitivity to IL-10. Thus, we identified a novel acetylated-STAT3 dependent regulatory pathway in DCs.