Periodic Reporting for period 4 - SILENCE (Mechanisms of Gene Silencing by the Glucocorticoid Receptor)
Berichtszeitraum: 2020-02-01 bis 2021-07-31
Glucocorticoids are steroid hormones secreted by the adrenal gland that bind to their cognate receptor, the Glucocorticoid Receptor (GR), which then enters the nucleus to regulate gene expression as a transcription factor. Synthetic glucocorticoids (such as Dexamethasone, Cortisone) are some of the most potent anti-inflammatory drugs in clinical use today, and some of the most powerful metabolic regulators. Unfortunately, their unique ability to efficiently shut off inflammatory gene expression is accompanied by serious side effects. These undesired effects are attributed to the transcriptional activation of GR's metabolic target genes and limit its therapeutic use.
With SILENCE, we proposed to decipher the unresolved molecular paradox of positive versus negative gene regulation by the Glucocorticoid Receptor. Inflammation is known to contribute to the pathogenesis of numerous human illnesses, including cancer, autoimmune disorders, diabetes and cardiovascular disease. Understanding the specific mechanisms involved in the silencing of inflammatory gene expression carries transformative potential for novel therapies and safer drugs. In addition the clinical relevance of this project is underscored by the fact that currently, the gold standard of care for Covid-19 patients hospitalized with respiratory support, mainly consists of these anti-inflammatory steroids.
The ERC Starting Grant ‘SILENCE’ was focused on the identification of DNA-encoded factors (proteins, noncoding RNAs or cis-regulatory sequences) mediating transcriptional silencing of inflammatory genes by the Glucocorticoid Receptor (GR).The molecular mechanisms of how GR turns off inflammatory gene expression are far from understood, which is what this project was addressing.
With SILENCE, we proposed to decipher the unresolved molecular paradox of positive versus negative gene regulation by the Glucocorticoid Receptor. Inflammation is known to contribute to the pathogenesis of numerous human illnesses, including cancer, autoimmune disorders, diabetes and cardiovascular disease. Understanding the specific mechanisms involved in the silencing of inflammatory gene expression carries transformative potential for novel therapies and safer drugs. In addition the clinical relevance of this project is underscored by the fact that currently, the gold standard of care for Covid-19 patients hospitalized with respiratory support, mainly consists of these anti-inflammatory steroids.
The ERC Starting Grant ‘SILENCE’ was focused on the identification of DNA-encoded factors (proteins, noncoding RNAs or cis-regulatory sequences) mediating transcriptional silencing of inflammatory genes by the Glucocorticoid Receptor (GR).The molecular mechanisms of how GR turns off inflammatory gene expression are far from understood, which is what this project was addressing.
In the course of the project, we successfully developed genetic screening tools to perform genome-wide screening for loss-of-function of novel proteins (coding genes) involved in the anti-inflammatory action of the glucocorticoid receptor. In addition, we adapted and established the new technique called ChIP-Nexus for our applications and were able to map GR binding sites together with inflammatory co transcription factors at unprecedented resolution. Most importantly we were able to identify novel mechanistic insights into GRs mode of action by the dissection of tethered GR binding sites versus those that require direct DNA binding. In contrast to the generally accepted view that protein-protein interactions with transcription factors such as AP-1 or NFkB are responsible for the suppression of inflammatory responses by GR, we have shown for the first time that DNA binding is essential for both activation and suppression of transcription. (Escoter et al 2020)
Furthermore we have successfully identified additional modulators of GR target locus binding proteins belonging to the STAT family (Quagliarini et al 2019).
By using state-of-the-art techniques such as siRNA, Crispr/Cas9 and proteomics, we were also able to identify and characterize important co-regulators of the anti-inflammatory GR transcriptional complex that belong to the chromatin landscape, such as the COMPASS-H3K4 methyltransferase complex and components of the SWI/SNF complex. Finally, we found several noncoding RNAs, such as miRNAs, eRNAs, uORFs, lncRNAs, which are differentially regulated in activated macrophages in response to GR ligand (Greulich et al 2021). In summary, we have performed the experiments described in the grant proposal, with minor technical modifications, as methods were evolving and improving.
Furthermore we have successfully identified additional modulators of GR target locus binding proteins belonging to the STAT family (Quagliarini et al 2019).
By using state-of-the-art techniques such as siRNA, Crispr/Cas9 and proteomics, we were also able to identify and characterize important co-regulators of the anti-inflammatory GR transcriptional complex that belong to the chromatin landscape, such as the COMPASS-H3K4 methyltransferase complex and components of the SWI/SNF complex. Finally, we found several noncoding RNAs, such as miRNAs, eRNAs, uORFs, lncRNAs, which are differentially regulated in activated macrophages in response to GR ligand (Greulich et al 2021). In summary, we have performed the experiments described in the grant proposal, with minor technical modifications, as methods were evolving and improving.
When looking at the bigger picture painted by my combined publications over the past several years, it is emerging that transcription factor binding sites are not only cell type, time point and signal specific, but that these cistromes display surprising complexity. Transcription factor binding profiles are not only highly dynamic and exert plasticity in response to external stimuli, but within one cell type, under the same conditions, in response to identical signals, distinct subsets of binding sites exist. That means that the Glucocorticoid Receptor exerts locus specific modes of gene regulation, and that different fractions of target genes are regulated by a combinatorial code consisting of varying co-regulators, cofactors, and co-bound transcription factors. For example, in macrophages, there are some inflammatory enhancers which depend on the COMPASS complex, some that depend on the BRG1 SWI/SNF complex, some that express eRNAs, some that are co-bound by AP-1, by NF-kappaB or by STAT3/5, and some that have yet unknown features. So not all binding sites are the same, which is probably true for all transcription factors, and which is an important contribution to the field.
Overall, our discovery has important implications for the development of novel glucocorticoid receptor agonists or modulators with lower side-effect profiles and for our understanding of transcriptional regulation by GR.
Overall, our discovery has important implications for the development of novel glucocorticoid receptor agonists or modulators with lower side-effect profiles and for our understanding of transcriptional regulation by GR.