title: Response Element Composition Governs Correlations between Binding Site Affinity and Transcription in Glucocorticoid Receptor Feed-forward Loops.
abstract: Combinatorial gene regulation through feed-forward loops (FFLs) can bestow specificity and temporal control to client gene expression; however, characteristics of binding sites that mediate these effects are not established. We previously showed that the glucocorticoid receptor (GR) and KLF15 form coherent FFLs that cooperatively induce targets such as the amino acid-metabolizing enzymes AASS and PRODH and incoherent FFLs exemplified by repression of MT2A by KLF15. Here, we demonstrate that GR and KLF15 physically interact and identify low affinity GR binding sites within glucocorticoid response elements (GREs) for PRODH and AASS that contribute to combinatorial regulation with KLF15. We used deep sequencing and electrophoretic mobility shift assays to derive in vitro GR binding affinities across sequence space. We applied these data to show that AASS GRE activity correlated (r(2) = 0.73) with predicted GR binding affinities across a 50-fold affinity range in transfection assays; however, the slope of the linear relationship more than doubled when KLF15 was expressed. Whereas activity of the MT2A GRE was even more strongly (r(2) = 0.89) correlated with GR binding site affinity, the slope of the linear relationship was sharply reduced by KLF15, consistent with incoherent FFL logic. Thus, GRE architecture and co-regulator expression together determine the functional parameters that relate GR binding site affinity to hormone-induced transcriptional responses. Utilization of specific affinity response functions and GR binding sites by FFLs may contribute to the diversity of gene expression patterns within GR-regulated transcriptomes.
authors.affiliation: From the Department of Medicine, National Jewish Health, Denver, Colorado 80206.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Sasse
authors.affiliation: Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri 63108-8510.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zuo
authors.affiliation: From the Department of Medicine, National Jewish Health, Denver, Colorado 80206.
authors.forename: Vineela
authors.initials: V
authors.lastname: Kadiyala
authors.affiliation: Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Liyang
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Miles A
authors.initials: MA
authors.lastname: Pufall
authors.affiliation: Case Cardiovascular Research Institute and Harrington Heart and Vascular Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-7290, and.
authors.forename: Mukesh K
authors.initials: MK
authors.lastname: Jain
authors.affiliation: Department of Medicine, University of Colorado, Denver, Colorado 80045.
authors.forename: Tzu L
authors.initials: TL
authors.lastname: Phang
authors.affiliation: Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri 63108-8510.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Stormo
authors.affiliation: From the Department of Medicine, National Jewish Health, Denver, Colorado 80206, Department of Medicine, University of Colorado, Denver, Colorado 80045 gerbera@njhealth.org.
authors.forename: Anthony N
authors.initials: AN
authors.lastname: Gerber
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001980:Bronchi / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D003907:Dexamethasone / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011394:Proline Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D012443:Saccharopine Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014158:Transcription, Genetic
chemical_list: C414172:KLF15 protein, human; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; C043044:AASS protein, human; D012443:Saccharopine Dehydrogenases; D011394:Proline Oxidase; C582834:PRODH protein, human
keywords: DNA-protein interaction; KLF15; binding site affinity; feed-forward loop; gene regulation; glucocorticoid receptor; systems biology; transcription enhancer