ChIP-nexus of mineralocorticoid receptors and glucocorticoid receptors in neuroblastoma cells

Purpose: To compare the binding profiles of mineralocorticoid and glucocorticoid receptors after corticosterone treatment in neuroblastoma cells. Methods: Tagged glucocorticoid receptors (eGFP-rat GR) and mineralocorticoid receptors (mCherry-rat MR) were transiently transfected into Neuro2A mouse neuroblastoma cells and treated with (i) vehicle, (ii) corticosterone 100 nM for 20 mins and (iii) corticosterone 100 nM for 20 mins followed by media repacement twice and 40 mins further incubation in charcoal-stripped serum media. ChIP-nexus samples were prepared in triplicate for deep sequencing using Illumina NextSeq, as well as control samples taken from cell lysates before immunoprecipitation. Sequence data was analyzed and processed for the identification of enriched binding sites, using MACS2, and for the characteristic staggered ChIP-nexus borders, using the MACE pipeline (Wang et al., 2014). Results: Corticosterone-sensitive glucocorticoid receptor (GR) binding sites and mineralocorticoid receptor (MR) binding sites showed considerable overlap, by MACS2 analysis. GR and MR binding sites were highly similar showing recruitment to the same DNA sites, by MACE analysis (Wang et al., 2014). Further analyses showed GR could tether MR to the DNA by GR in a functional manner such that gene expression could be altered by the presence of tethered MR. Conclusions: MR can be recruited to DNA-binding sites by GR, even in the absence of MR DNA-binding. MR can interact in multiple modes at genomic DNA binding sites. ChIP-nexus samples were prepared in triplicate from expressed tagged glucocorticoid receptors (eGFP-mouseGR) and mineralocorticoid receptors (mCherry-mouseMR) transiently transfected into Neuro2A mouse neuroblastoma cells for deep sequencing using Illumina NextSeq, for each of 3 treatment groups: (i) vehicle, (ii) corticocsterone 100 nM 20 mins, (iii) corticosterone 100 nM 20 mins followed by 2 washed and incubation without steroid for a further 40 mins. Additional gene expression and ChIP testing was performed using DNA-binding domain mutants to confirm findings.