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Identifying chromosomal regions associated with glucocorticoid-regulated gene transcription


Glucocorticoid (GC) response elements (GREs) are genomic segments that confer GC-regulated transcription in by recruiting hormone-bound glucocorticoid receptor (GR) and nucleating assembly of transcriptional regulatory complexes (TRCs). The locations of GR binding, the functionality of those GR occupied regions (GORs) as GREs, and the molecular features and spatial organization that characterize active GREs are gene-, cell- and physiological-context specific, and poorly understood. Moreover, identification of the gene(s) targeted for regulation by a given GRE has been inferred by proximity, or examined outside the normal chromosomal context, rather than rigorously validated. We approached these two issues in two human cell lines with distinct tissue origins, treated or not with a hormonal ligand that activates GR. First, we took a systems approach to examine the GC response, cataloging GORs by ChIP-seq, comparing RNA-seq defined transcriptome datasets from three different laboratories, mapping short bidirectional transcripts by Pro-seq, and assessing higher order genome structure by in situ Hi-C. To identify a functional GRE, we focused on a single 1.4 Mb topological domain bearing a GC-regulated gene and multiple GORs, and used Cas9 mutagenesis for in-genome GOR editing, coupled with transcriptional analysis to assess GRE activity and identify target gene(s). Our work established an experimental and analytic workflow for identification of robust sets of GC-regulated genes, and for unequivocal determination and validation of GRE activity. We found some but not all of the GORs dispersed across the topological domain contributed to GRE activity, the GRE directly regulated only one or two of the seven genes within the domain, and that features such as bidirectional transcripts or chromosome looping were seen at some but not all functional GORs. These results are consistent with context-specific combinatorial assembly of TRCs into functional GREs, which together enable GCs to orchestrate organismal developmental and physiological actions comprised of gene- and cell-specific transcriptional regulatory events.

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