Inhibitors of Maf1-dependent transcriptional repression

Project Summary Maf1 is a conserved nutrient- and stress-sensitive repressor of gene transcription, best known for its role as a global regulator of RNA polymerase III transcription in yeast and humans and more recently understood to affect the expression of a subset of genes transcribed by RNA polymerase II in mammalian cells. The major targets of MAF1-mediated repression, 5S rRNA and tRNA genes, encode highly abundant non-coding RNAs that are also major consumers of nucleotides in gene transcription. Thus, the universal requirement for MAF1 in the repression of these genes has led to the view that MAF1 function is important for metabolic economy. This function is now dramatically demonstrated by the profound resistance of mice with a whole body knockout of Maf1 to diet-induced obesity and non-alcoholic fatty liver disease. Maf1 KO mice represent a new model of obesity resistance. Phenotypic studies reveal that reduced food intake and increased energy expenditure due to metabolic inefficiencies underlie the lean phenotype and the resistance of Maf1 KO mice to weight gain on a high fat diet. More specifically, the inefficient metabolism of Maf1 KO mice is demonstrated by their increased synthesis and turnover (futile cycling) of tRNAs and hepatic lipids and elevated levels of autophagy among other metabolic changes. Coupled with the apparent good health (extended lifespan) of the mice, MAF1 has emerged as an appealing therapeutic target for the treatment of obesity and related diseases. This grant application represents the first step to test the idea that MAF1 inhibitors will be anti-obesity drugs. Accordingly, the long term goals of this research are to identify and characterize biologic inhibitors of human MAF1 as mechanistic probes of MAF1-dependent transcriptional repression and as potential anti-obesity therapeutics and drug discovery tools. In aim I we will use phage display to isolate peptides that bind with high affinity to human Maf1. Subsequent experiments will characterize peptide-MAF1 interactions and evaluate the activity of the peptides in vitro and in cell culture systems for readouts relevant to Maf1 KO mice. In aim II we will employ photocrosslinking methods to define the sites on MAF1 that are important for interactions with its natural ligands. In aim III we will isolate synthetic antibodies against functionally important regions of MAF1 for use as crystallographic chaperones to obtain high resolution structures of full length human Maf1 in its active state.