Genome binding/occupancy profiling by high throughput sequencing Expression profiling by high throughput sequencing
Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we identify the protein network around chromatin-bound GR by using selective isolation of chromatin-associated proteins and show that the network is affected by receptor SUMOylation, with several nuclear receptor coregulators and chromatin modifiers preferring interaction with SUMOylation-deficient GR and proteins implicated in transcriptional repression preferring interaction with SUMOylation-competent GR. This difference is reflected in our chromatin binding, chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in binding and opening chromatin at glucocorticoid-regulated enhancers and inducing expression of target loci. Blockage of SUMOylation by a SUMO-activating enzyme inhibitor (ML-792) phenocopied to a large extent the consequences of GR SUMOylation deficiency on chromatin binding and target gene expression. Our results thus show that SUMOylation modulates the specificity of GR by regulating its chromatin protein network and accessibility at GR-bound enhancers. We speculate that many other SUMOylated TFs utilize a similar regulatory mechanism.
Examination of NCOA1, NCOR1, and H3K4me2 binding by ChIP-seq from isogenic HEK293 cells stably expressing GRwt (HEK293flpGR) or SUMOylation deficient GR (GR3KR; HEK293flpGR3KR). Examination of GR binding after SAE inhibitor ML-792 treatment by ChIP-seq from HEK293flpGR and HEK293flpGR3KR cells. In addition, examination of SUMO2/3, PIAS1+2, H3K4me2, and Pol2 binding by ChIP-seq from HEK293flpGR and isogenic HEK293 cells expressing in a tetracycline (tet)-inducible manner PIAS1 (HEK239flpPIAS1). Sequencing with Illumina HiSeq 2000, or Illumina NextSeq 500. IgG immunoprecipitated sample from HEK293flpFRT cells was used as control. Chromatin accessibility examined by ATAC-seq from HEK293flpGR and HEK293flpGR3KR cells, in biological duplicates, using Illumina NextSeq 500. Gene expression examined by RNA-seq after SAE inhibitor ML-792 treatment from HEK293flpGR or HEK293flpGR3KR cells, in biological duplicates, using Illumina NextSeq 500.