Glucocorticoid-Mediated Transcriptional Regulation in the Human Genome

Abstract

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.