Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - SILENCE (Mechanisms of Gene Silencing by the Glucocorticoid Receptor)
Teaser
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. Glucocorticoids are some of the most potent...
Summary
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. Glucocorticoids 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. The molecular mechanisms of how GR turns off inflammatory gene expression are far from understood, which is where this project is needed. With this project, we decipher the unresolved molecular paradox of positive versus negative gene regulation by the Glucocorticoid Receptor.
SILENCE uses cutting-edge genome-wide approaches to identify the molecular mechanisms underlying the transcriptional repression, or silencing, of inflammatory genes by GR. The general, open question we address is how one transcription factor can simultaneously both activate and repress transcription. We postulate the existence of unknown coregulator proteins, cis-regulatory DNA sequences, noncoding RNAs, or combinations thereof. Therefore, we are performing a large scale genomic screen to identify those cofactors that specify repression versus activation, carrying out ChIP-exo experiments to map genomic GR binding sites at an unprecedented resolution, and profiling nascent RNAs to define the role of noncoding RNAs during the silencing of inflammatory genes.
Inflammation is known to contribute to the pathogenesis of numerous human illnesses, including cancer, autoimmune diseases, 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.
If we can understand how and why GR (or Glucocorticoids) can suppress inflammation so rapidly, we can hopefully develop safer immune suppressant drug with reduced side effect profiles in the future. This project (SILENCE) used cutting edge genome wide technologies such as next generation sequencing to address the unanswered question of how the silencing of inflammatory genes is achieved by the Glucocorticoid Receptor.
Work performed
We are currently establishing genetic screening tools to perform a genome wide loss of function screen for novel proteins (coding genes) involved in the anti-inflammatory actions of the Glucocorticoid Receptor. We are also applying a novel technique called ChIP-nexus to map GR binding sites together with inflammatory transcription factors such as AP-1 and NF-kB at unprecedented resolution. Finally, we have identified thousands of noncoding RNAs to be regulated by GR in immune cells called macrophages. We have functionally characterized miRNAs involved in inflammatory responses and are currently studying lncRNAs and eRNAs.
Final results
We expect to identify many novel candidates in our genome wide genetic screen. We also expect to have a high resolution map of the genomic landscape mediating inflammatory responses in macrophages, which will answer many open questions. We will have assigned immune modulatory function to many previously unknown noncoding RNAs. Most importantly, we will find out how GR turns inflammatory genes off while turning other genes on.
We have also performed proteomics experiments to purify the transcriptional complex assembled by GR on inflammatory enhancers and identified several putative novel coregulators.