Probing the mechanisms of synergy control motifs in the glucocorticoid receptor: A role for posttranslational modification by the small ubiquitin -like modifier (SUMO).

The recruitment of a given factor to multiple response elements within the promoter of a gene often results in a more than additive, or synergistic, transcriptional response compared to that seen from a single element. Our group has identified a novel class of peptide sequences, termed synergy control, or SC motifs, that function in multiple regulators by selectively limiting the activity emanting from multiple, but not single, response elements. This dissertation examines the mechanisms of action of SC motifs using the Glucocorticoid Receptor (GR) as a paradigm. Through in vivo and in vitro approaches, we demonstrate that SC motifs serve as sites for post-translational modification by members of the small ubiquitin-like modifier (SUMO) protein family. This modification is directly responsible for the inhibitory effects of SC motifs. The basis for selective action of SC motifs is not due to targeting of synergy per se, but, rather, is dependent on binding of SUMO-modified factors to multiple sites on DNA. A comprehensive mutational analysis to probe the structural determinants of SUMO required for its inhibitory properties, identified a cluster of basic and hydrophobic residues that define a critical surface essential for transcriptional inhibition. An analysis of receptor-specific requirements for SC motif function revealed that the inhibitory actions of SUMO are dependent on stable binding of GR to DNA. The systematic alteration of the number and arrangement of response elements revealed that the repressive effects of SUMO extend over a relatively short distance (∼80bps). We propose that the stable binding of SC motif-bearing regulators to multiple sites on DNA allows for the recruitment of specific co-repressors through their interaction with a critical functional surface of SUMO.Ph.D.Biological SciencesHealth and Environmental SciencesMolecular biologyPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125665/2/3208469.pd

LRH-1 and PTF1-L coregulate an exocrine pancreas-specific transcriptional network for digestive function

ChIP-seq and RNA-seq technologies were combined with analysis of LRH-1-null mice to describe the first complete analysis of the cistrome and transcriptome for the nuclear receptor LRH-1 in an adult animal. The authors found that LRH-1 interaction with the pancreas pioneering factor PTF1-L is required for the expression and secretion of exocrine pancreas proteins that aid in digestion

Experimental microdissection enables functional harmonisation of pancreatic cancer subtypes

© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ. Objective Pancreatic ductal adenocarcinoma (PDA) has among the highest stromal fractions of any cancer and this has complicated attempts at expression-based molecular classification. The goal of this work is to profile purified samples of human PDA epithelium and stroma and examine their respective contributions to gene expression in bulk PDA samples. Design We used laser capture microdissection (LCM) and RNA sequencing to profile the expression of 60 matched pairs of human PDA malignant epithelium and stroma samples. We then used these data to train a computational model that allowed us to infer tissue composition and generate virtual compartment-specific expression profiles from bulk gene expression cohorts. Results Our analysis found significant variation in the tissue composition of pancreatic tumours from different public cohorts. Computational removal of stromal gene expression resulted in the reclassification of some tumours, reconciling functional differences between different cohorts. Furthermore, we established a novel classification signature from a total of 110 purified human PDA stroma samples, finding two groups that differ in the extracellular matrix-associated and immune-associated processes. Lastly, a systematic evaluation of cross-compartment subtypes spanning four patient cohorts indicated partial dependence between epithelial and stromal molecular subtypes. Conclusion Our findings add clarity to the nature and number of molecular subtypes in PDA, expand our understanding of global transcriptional programmes in the stroma and harmonise the results of molecular subtyping efforts across independent cohorts

The G Protein-Coupled Bile Acid Receptor, TGR5, Stimulates Gallbladder Filling

A novel physiological role for the bile acid responsive GPCR, TGR5, is described that involves relaxation of the gallbladder in response to bile acids

SUMO-Mediated Inhibition of Glucocorticoid Receptor Synergistic Activity Depends on Stable Assembly at the Promoter But Not on DAXX

Multiple transcription factors, including members of the nuclear receptor family, harbor one or more copies of a short regulatory motif that limits synergistic transactivation in a context-dependent manner. These synergy control (SC) motifs exert their effects by serving as sites for posttranslational modification by small ubiquitin-like modifier (SUMO) proteins. By analyzing the requirements for both synergy control and SUMOylation in the glucocorticoid receptor (GR), we find that an intact ligand-binding domain and an engaged DNA- binding domain dimerization interface are necessary for effective synergy control. However, these features, which promote stable assembly of GR-DNA complexes, are required downstream of SUMOylation because their disruption or deletion does not interfere with SUMO modification. Remarkably, in the absence of these features, sensitivity to the effects of SUMOylation can be restored simply by stabilization of DNA interactions through a heterologous DNA binding domain. The data indicate that stable interaction with DNA is an important prerequisite for SUMO-dependent transcriptional inhibition. Analysis of genomic regions occupied by GR indicates that the effects of SC motif SUMOylation are most evident at multiple, near-ideal GR binding sites and that SUMOylation selectively affects the induction of linked endogenous genes. Although the SUMO-binding protein DAXX has been proposed to mediate the inhibitory effects of GR SUMOylation, we find that inhibition by DAXX is independent of GR SUMOylation. Furthermore, neither expression nor knockdown of DAXX influences SUMO effects on GR. We therefore propose that stable binding of GR to multiple sites on DNA allows for the SUMO-dependent recruitment of inhibitory factors distinct from DAXX