Changes in nascent transcripts upon glucocorticoid stimulation in macrophages

Glucocorticoids are widely prescribed anti-inflammatory drugs with tissue-specific effects. Beneficial anti-inflammatory effects are caused in cells of the immune system whereas metabolic adverse effects of glucocorticoid therapy are seen in metabolic tissues such as liver. The glucocorticoid receptor (GR), a nuclear receptor targeted by glucocorticoids, bind its DNA response elements upon ligand exposure in a tissue-specific manner. Tissue-specific GR binding patterns depend on the access of its binding sites, which determines the tissue-specific glucocorticoid response. Here, we investigated this response by nascent RNAseq in murine bone marrow-derived macrophages (BMDMs) after stimulation with lipopolysaccharide (LPS) and dexamethasone (Dex). After labelling newly synthesized transcripts with 4-thiouracile (4sU), we identified macrophage-specific non-coding transcripts expressed at intergenic GR binding sites. Those transcripts are regulated by the GR and correlate with its anti-inflammatory function in macrophages. Those findings add another layer to the mechanisms underpinning GR's tissue-specific gene regulation and represent potential drug targets in anti-inflammatory therapy and/or management of adverse effects in glucocorticoid therapy. Overall design: We performed 4sU-seq in murine bone marrow-derived macrophages upon LPS (2h 100 ng/ml) and LPS plus Dex (2.5 h 1 mM Dex, 2h 100ng/ml LPS) stimulation. One hour before cell lysis nascent transcripts were labelled with 200 mM 4-thiouridine (4sU, Carbosynth, Cat.No: 13957-31-8). Three biological replicates were analyzed per treatment condition. For each sample, we performed total (Illumina Stranded Total RNA Library Prep Kit) and PolyA-specific (Illumina TrueSeq Stranded mRNA Library Prep Kit) RNAseq library preparation.