hide
Free keywords:
Transcription
Glucocorticoid Receptor
Transcriptional Memory
Androgen Receptor
Abstract:
The glucocorticoid receptor (GR) is a transcription factor which becomes activated upon binding to glucocorticoids, a class of steroid hormones. Upon activation, GR binds to various genomic locations and induces large-scale changes in transcription and chromatin structure. Clinically, GR is an important therapeutic target, since glucocorticoids are widely applied to treat autoimmune and inflammatory conditions. However, long-term treatment with glucocorticoids is associated with glucocorticoid-resistance and severe side-effects. At the molecular level, the effects of prolonged GR activation on a cell’s transcriptional responses are not fully understood. Here, I investigated if exposure to glucocorticoids results in long-term changes in chromatin and transcription. In addition, I studied GR’s genomic binding preferences by investigating mechanisms that shape DNA binding specificities between GR and its paralog, the androgen receptor (AR). In the first part of the thesis, I investigated the immediate and the long-term effects of GR activation on chromatin and transcription. By examining GR binding as well as glucocorticoid-induced changes in chromatin accessibility and transcription, I found that genomic regions that lose chromatin accessibility were enriched near downregulated genes. Interestingly, these ‘closing’ regions were largely not bound by GR, indicating that repression, in part, does not depend on nearby GR binding and might occur through indirect effects of GR activation. To study the long-term effects of GR activation, I investigated changes in chromatin accessibility and transcription after washout of glucocorticoids. GR-induced changes in chromatin accessibility were found to be reversible following a 24-hour washout period. Similarly, transcriptional activity reverted to basal levels after washout. Moreover, I tested if a prior exposure to hormone changes the response to a subsequent treatment. Most genes showed similar transcriptional responses upon hormone-re-stimulation compared to the first stimulation. However, the GR-target gene ZBTB16 showed enhanced upregulation upon reinduction, suggesting that prior glucocorticoid exposure results in priming of this gene. Single-cell analysis showed that enhanced expression of ZBTB16 upon reinduction was a consequence of an increased probability of cells transcribing the gene as well as individual cells showing increased ZBTB16 transcription. In the second part of the thesis, I assayed the role of chromatin and DNA sequence in generating divergent genomic binding patterns of transcription factors with nearly identical DNA-binding preferences, specifically, GR and its paralog AR. Investigating binding of GR and AR in the same cell type revealed that both transcription factors occupy overlapping as well as unique sites. Examining the chromatin landscape at receptor binding regions showed that many GR-specific sites were situated within relatively inaccessible chromatin, suggesting that binding specificity is, in part, achieved through GR’s ability to bind to inaccessible chromatin. Furthermore, motif enrichment analysis at GR- and AR-specific regions provided further evidence that the receptors exhibit subtle differences in the recognition sequences they preferentially bind to. Lastly, analysis of GC-content revealed that receptor-specific binding is also driven by GC-content at the binding sites and the larger surrounding area, as mean GC-content was found to to be higher at GR- compared to AR-specific sites. In summary, these results provide evidence that GR is capable of inducing gene-specific transcriptional memory, even though GR-induced chromatin structural and transcriptional changes are largely reversible. Given GR’s biological role as an effector to fluctuating levels of glucocorticoids, the reversibility of GR-induced chromatin and transcriptional changes is to be expected. However, future experiments involving longer, or more frequently repeated, hormone exposures might yield insights into the underlying mechanisms of glucocorticoid resistance and side-effects associated with long-term glucocorticoid treatment. Furthermore, the chromatin landscape as well as DNA sequence composition contribute to driving receptor-specific genomic occupancy of GR and AR. These findings might represent a general mechanism that shapes differential binding among paralogous transcription factors and could contribute to our understanding of how genomic binding specificities are established for other related transcription factors.
