Browsing by Subject "Glucocorticoids"

Glucocorticoids (GCs) are a class of steroid hormones released from adrenal gland to mediate multiple physiological processes including the immune responses, cognitive functions and development. Glucocorticoids exert their gene regulatory effects through a ligand-activated transcription factor, glucocorticoid receptor (GR). Upon GC activation, GRs are particularly recruited to promoter-distal regulatory elements enriched with other transcription factors (TFs) and co-regulators including active protein 1 (AP-1). AP-1 is a heterodimeric TF potentially composed of subunits belongs to JUN, FOS and activating protein TF family. While the genomic function of AP-1 in response to GCs is well studied, the effect of specific configurations of AP-1 subunits on GR-mediated transcription remains unknown. In chapter 1, I introduce various regulatory components for transcriptional regulation. In chapter 2, I demonstrate that AP-1 subunits may not form preferential dimers between specific subunits, but rather bind each other promiscuously. I further show that the convergence of AP-1 subunits to enhancers is a key determinant for GR-mediated transcription and, by extension, cell-type specific environmental responses. GR binds DNA both directly and indirectly. While genome wide binding activity of GR can be effectively characterized by ChIP-seq, the binding mode (i.e. direct vs. indirect) at a specific site can’t be directly inferred. In chapter 3, I describe a machine learning approach to predict direct and indirect GR-DNA interactions using Protein Binding Microarray data. I demonstrate that motif-directed GR binding remains to be persistent after stimulus whereas indirect GR binding is likely transient. I further illustrate that robust transcriptional activation requires persistent GR binding and direct GR binding have the higher regulatory potential than indirect GR binding. GR activation represses certain genes. Along with the regulatory actions of GR cofactors, histone deacetylation is thought to regulate gene expression, especially gene repression. Therefore, I hypothesize that limiting histone deacetylases (HDAC) activity promotes robust gene activation. To test this hypothesis, in chapter 4, I delve into the GC-mediated transcriptional change after inhibiting the activity of histone deacetylases by HDACi to determine HDAC effect on GC-meditated transcriptional outputs. I demonstrate that the inhibition of HDAC activity reduces the magnitude of GC-mediated repression as well as activation in transcription. I also show that HDACi x GR-mediated intronic changes quantified from RNA-seq are minimally confounded by mRNA half-life linked to exonic changes, thereby accurately capturing transcriptional activity. Throughout the dissertation, I investigate GR-mediated transcriptional regulation by integrative analyses for numerous functional genomic datasets and by a predictive modeling for differential GR-DNA binding modes. In particular, the dissertation demonstrates that TF cooperativity, especially from subunits of AP-1 TF family, is a key determinant that drives the control of transcriptional output in cell-type specific manner. The dissertation further shows the potential for the generality of this regulatory mechanism beyond glucocorticoid stimulus and human cell types, suggesting that the TF convergence to a site, especially from the same TF family, may determine their functional specificity in a given cellular context.

As humans continue to introduce stressors into the marine environment, we are obligated to understand how our behaviors impact wildlife. For many cetacean populations, anthropogenic noise poses a significant conservation threat, but the ability to monitor these animals is constrained by their often-remote habitats and limited time at the surface. Researchers have developed innovative solutions to overcome these challenges, including the development of techniques that enable physiological sampling with minimal disturbance. As frontiers, these methods require careful development and validation before they can be used reliably in experimental studies. In this dissertation, I employ one of these innovative techniques, measuring steroid hormones in remote blubber biopsy samples, in short-finned pilot whales (Globicephala macrorhynchus). As modulators of reproduction and stress, steroid hormones provide information that is advantageous for wildlife monitoring. Because a validated method for measuring these compounds in short-finned pilot whale blubber did not yet exist, I adapted a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify 11 steroid hormones of interest. Before proceeding with its application, I tested its analytical and biological validity with blubber from three stranded short-finned pilot whales. Next, I applied the validated LC-MS/MS method to an archive of blubber biopsies, collected from free-ranging short-finned pilot whales in the Western North Atlantic. Leveraging the comprehensive nature of LC-MS/MS profiling, I investigated relationships between hormones and characterized steroid hormone profiles across demographic groups and seasons. In the fourth chapter, I conducted an acoustic response study, using the previously established methods to collect and quantify steroid hormones after exposure to simulated mid-frequency active sonar (MFAS). I modeled the responses of cortisol and cortisone over time to gain insight into steroid perfusion rates in cetacean blubber and asked whether demography contributed to these responses. Together, my results demonstrate the reliability of blubber for measuring steroid hormones and reflecting relevant physiological states, like stress and pregnancy. While LC-MS/MS enables extensive steroid measurement, it struggled to detect some steroids of interest in this matrix. This dissertation shows the relevance of multi-steroid profiling and offers reference points for baseline steroid concentrations in analytes relevant to behavior, reproduction, and stress. The observation of post-exposure stress responses confirms a relationship between noise and physiology in short-finned pilot whales and illustrates the ability of blubber sampling to be applied in this context.