ABSTRACT
The pharmacokinetic properties of three pyrrole-imidazole (Py-Im) polyamides of similar size and Py-Im content but different shape were studied in the mouse. Remarkably, hairpin and cyclic oligomers programmed for the same DNA sequence 5'-WGGWWW-3' displayed distinct pharmacokinetic properties. Furthermore, the hairpin 1 and cycle 2 exhibited vastly different animal toxicities. These data provide a foundation for design of DNA binding Py-Im polyamides to be tested in vivo.
Subject(s)
Imidazoles/chemistry , Imidazoles/pharmacokinetics , Nylons/chemistry , Nylons/pharmacokinetics , Pyrroles/chemistry , Pyrroles/pharmacokinetics , Animals , Body Weight/drug effects , Imidazoles/adverse effects , Mice , Mice, Inbred C57BL , Nylons/adverse effects , Pyrroles/adverse effectsABSTRACT
Glucocorticoids rapidly and robustly induce cell fate decisions in various multipotent cells, although the precise mechanisms of these important cellular events are not understood. Here we showed that glucocorticoids repressed Per3 expression and that this repression was critical for advancing mesenchymal stem cells to the adipocyte fate. Exogenous expression of Per3 inhibited adipogenesis, whereas knocking out Per3 enhanced that fate. Moreover, we found that PER3 formed a complex with PPARγ and inhibited PPARγ-mediated transcriptional activation via Pparγ response elements. Consistent with these findings, Per3 knock-out mice displayed alterations in body composition, with both increased adipose and decreased muscle tissue compared with wild-type mice. Our findings identify Per3 as potent mediator of cell fate that functions by altering the transcriptional activity of PPARγ.
Subject(s)
Adipocytes/metabolism , Adipogenesis/physiology , PPAR gamma/biosynthesis , Period Circadian Proteins/metabolism , Response Elements/physiology , 3T3-L1 Cells , Adipocytes/cytology , Animals , COS Cells , Chlorocebus aethiops , Gene Expression Regulation/physiology , Gene Knockdown Techniques , Mice , PPAR gamma/genetics , Period Circadian Proteins/geneticsABSTRACT
Circadian clock genes are regulated by glucocorticoids; however, whether this regulation is a direct or secondary effect and the physiological consequences of this regulation were unknown. Here, we identified glucocorticoid response elements (GREs) at multiple clock genes and showed that 3 were directly regulated by the glucocorticoid receptor. We determined that a GRE within the core clock gene Per2 was continuously occupied during rhythmic expression and essential for glucocorticoid regulation of that gene in vivo. We further demonstrated that mice with a genomic deletion spanning this GRE expressed elevated leptin levels and were protected from glucose intolerance and insulin resistance on glucocorticoid treatment but not from muscle wasting. We conclude that Per2 is an integral component of a particular glucocorticoid regulatory pathway and that glucocorticoid regulation of the peripheral clock is selectively required for some actions of glucocorticoids.
Subject(s)
Circadian Rhythm/genetics , Glucocorticoids/physiology , Glucose/metabolism , Animals , Cell Cycle Proteins/genetics , Circadian Rhythm/drug effects , Gene Expression Regulation , Glucocorticoids/pharmacology , Homeostasis , Leptin/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/physiology , Mice , Nuclear Proteins/genetics , Period Circadian Proteins , Polymerase Chain Reaction , Transcription Factors/genetics , Transcription, GeneticABSTRACT
Genes are not simply turned on or off, but instead their expression is fine-tuned to meet the needs of a cell. How genes are modulated so precisely is not well understood. The glucocorticoid receptor (GR) regulates target genes by associating with specific DNA binding sites, the sequences of which differ between genes. Traditionally, these binding sites have been viewed only as docking sites. Using structural, biochemical, and cell-based assays, we show that GR binding sequences, differing by as little as a single base pair, differentially affect GR conformation and regulatory activity. We therefore propose that DNA is a sequence-specific allosteric ligand of GR that tailors the activity of the receptor toward specific target genes.
Subject(s)
DNA/chemistry , DNA/metabolism , Receptors, Glucocorticoid/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Humans , Ligands , Models, Molecular , Mutation , Protein Conformation , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , Receptors, Glucocorticoid/chemistry , Receptors, Glucocorticoid/genetics , Transcriptional ActivationABSTRACT
The androgen receptor (AR) mediates the physiologic and pathophysiologic effects of androgens including sexual differentiation, prostate development, and cancer progression by binding to genomic androgen response elements (AREs), which influence transcription of AR target genes. The composition and context of AREs differ between genes, thus enabling AR to confer multiple regulatory functions within a single nucleus. We used expression profiling of an immortalized human prostate epithelial cell line to identify 205 androgen-responsive genes (ARGs), most of them novel. In addition, we performed chromatin immunoprecipitation to identify 524 AR binding regions and validated in reporter assays the ARE activities of several such regions. Interestingly, 67% of our AREs resided within approximately 50 kb of the transcription start sites of 84% of our ARGs. Indeed, most ARGs were associated with two or more AREs, and ARGs were sometimes themselves linked in gene clusters containing up to 13 AREs and 12 ARGs. AREs appeared typically to be composite elements, containing AR binding sequences adjacent to binding motifs for other transcriptional regulators. Functionally, ARGs were commonly involved in prostate cell proliferation, communication, differentiation, and possibly cancer progression. Our results provide new insights into cell- and gene-specific mechanisms of transcriptional regulation of androgen-responsive gene networks.
