No abstract

The glucocorticoid receptor (GR) regulates transcription through binding to specific DNA motifs, particularly at enhancers. While the motif to which it binds is constant across cell types, GR has cell type-specific binding at genomic loci, resulting in regulation of different genes. The presence of other bound transcription factors (TFs) is hypothesized to strongly influence where GR binds. Here, we addressed the roles of other TFs in the glucocorticoid response by comparing changes in GR binding and nascent transcription at promoters and distal candidate cis-regulatory elements (CCREs) in two distinct human cancer cell types. We found that after glucocorticoid treatment, GR binds to thousands of genomic loci that are primarily outside of promoter regions and are potentially enhancers. The majority of these GR binding sites are cell-type specific, and they are associated with pioneer factor binding. A small fraction of GR occupied regions (GORs) displayed increased bidirectional nascent transcription, which is a characteristic of many active enhancers, after glucocorticoid treatment. Non-promoter GORs with increased transcription were specifically enriched for AP-1 binding prior to glucocorticoid treatment. These results support a model of transcriptional regulation in which multiple classes of TFs are required. The pioneer factors increase chromatin accessibility, facilitating the binding of GR and additional factors. AP-1 binding poises a fraction of accessible sites to be rapidly transcribed upon glucocorticoid-induced GR binding. The coordinated activity of multiple TFs then results in cell type-specific changes in gene expression. We anticipate that many models of inducible gene expression also require multiple distinct TFs that act at multiple steps of transcriptional regulation.

No abstract

Voltage sensitive fluorescent dyes (VSDs) are important tools for probing signal transduction in neurons and other excitable cells. These sensors, rendered highly lipophilic to anchor the conjugated p-wire molecular framework in the membrane, offer several favorable functional parameters including fast response kinetics and high sensitivity to membrane potential changes. The impact of VSDs has however been limited due to the lack of cell-specific targeting methods in brain tissue or living animals. We address this key challenge by introducing a non-genetic molecular platform for cell and molecule specific targeting of synthetic voltage sensitive dyes in the brain. We employ a dextran polymer particle to overcome the inherent lipophilicity of voltage sensitive dyes by dynamic encapsulation and target the construct to specific axonal extensions using the monoamine transporter ligand dichloropane. VoLDeMo (<u>Vo</u>ltage Sensor-<u>L</u>igand-<u>De</u>xtran Targeted to <u>Mo</u>noaminergic Neurons) probes label dense dopaminergic axons in the mouse striatum and sparse noradrenergic axons in the mouse cortex in acute brain slices. We also demonstrate in whole adult <i>Drosophila</i> brains that VoLDeMo targeting is ligand dependent. VoLDeMo variants bearing either a classical electrochromic ANEP dye or state-of-the-art VoltageFluor dye respond to membrane potential changes in a similar manner to the parent dyes, as demonstrated by whole-cell patch recording. The VoLDeMo platform enables targeting of diffusible VSD probes to specific neuronal cells using endogenous expression levels of native components of neurotransmission machinery. We envision that modularity of our platform will enable its application to a variety of molecular targets (other receptors and covalent labeling-based tags) and sensors (including those in other imaging modalities), as well as lipophilic drugs and signaling modulators. This work demonstrates the feasibility of a chemical targeting approach and expands the possibilities of cell-specific imaging and pharmacology.