John M. Lopes
Professor of Microbiology, University of Massachusetts
Ph.D.: University of South Carolina Postdoctoral Training: Carnegie Mellon University
The goal of the research in my lab is to understand the transcriptional regulation of phospholipid biosynthesis in yeast, and how phospholipid biosynthesis is coordinated with other biological processes. To address this goal, my lab is carrying on two inter-related projects. One project is designed to determine how phospholipid biosynthesis is coordinated with other biological processes via a set of transcription factors that belong to the basic helix-loop-helix (bHLH) family. Another project is focused on the regulationof the only essential phospholipid biosynthetic gene (PIS1) required for the synthesis of phosphatidylinositol (PI). This project is driven by the fact that virtually nothing is known about the transcriptional regulation of the PIS1 gene except that it is not coordinated with expression of the other phospholipid biosynthetic genes. We have been very successful with both projects publishing 38 papers with 2 more accepted for publication. My contributions in these areas are perhaps best evidenced by 4 solicited reviews. To carry out these projects, I have maintained a steady stream of extramural funding from the American Cancer Society and the National Science Foundation. More recently, we have begun to examine the evolutionary conservation of the regulatory pathways that control phospholipid biosynthesis in the Saccharomyces genus. This project is in its infancy and I plan to apply for funding in the near future. Each of these three projects is summarized below. I. Combinatorial regulation of gene expression by basic helix-loop-helix proteins. Regulation of yeast phospholipid biosynthesis requires the Ino2p:Ino4p activators, which belong to the bHLH family of transcription factors. Initially, we were focused on the regulation of the INO2 and INO4 genes and found that INO2 expression is auto-regulated in a pattern that is identical to the regulation of the Ino2p target genes. More recently we also carried out genetic analyses of these genes to define their roles in transcriptional regulation. Currently, we have turned our attention to how phospholipid biosynthesis is coordinated with other biological processes via the bHLH proteins. Yeast has nine bHLH proteins that regulate several different processes including:phosphate utilization, retrograde response (control of nuclear genes in response to mitochondrial damage), and glycolysis (Fig.1). Since bHLH proteins are known to form multiple dimer combinations in other organisms, we reasoned that dimer partner selection could be a mechanism for coordination of the biological processes regulated by yeast bHLH proteins. Thus, we defined the yeast bHLH protein interaction map using the yeast two-hybrid system and co-purification of epitope-tagged recombinant proteins. As we predicted, this map identifies several novel bHLH dimer combinations (Fig. 1). In addition, since specific growth conditions affect expression ofspecific bHLH genes, then different growth conditions would be expected to change the bHLH protein interaction map. We are presently testing this prediction. We are also determining the role of the yeast bHLH proteins in coordination of different biological processes using well-characterized candidate target genes for each biological process. To complement the candidate gene approach, microarray analysis is being used to define the target genes for each bHLH protein under conditions that affect the expression of the different bHLH encoding genes. Lastly, to define the mechanism(s)whereby bHLH dimers coordinate gene expression, we are determining the autoregulatory circuits that control expression of each bHLH-encoding gene. II. Genomic analysis of PI synthesis in yeast. Recently, we have also focused our attention on the regulation of PIS1 expression that is required for the synthesis of PI. PI and its metabolites (inositol polyphosphates, phosphoinositides, and sphingolipids) play important roles in a myriad of processes, including: chromatin remodeling, export of mRNA from the nucleus, vesicle trafficking, and signal transduction. In spite of the importance of PI to the cell, relatively little is known about how its levels are regulated, particularly at the level of transcription of the PIS1 gene. Promoter deletion analysis reveals that the PIS1 promoter includes five regulatory elements: three upstream activation sequences (UAS) and two upstream repression sequences (URS). Our published studies reveal the carbon source regulates PIS1 expression through the URS GLY element and probably via a novel regulatory cascade. We have also determined that oxygen regulates PIS1 expression through the Rox1p repressor protein and the URS ROX element. Screening a yeast gene deletion set, we identified 120 genes that affect expression of a PIS1 reporter. These genes suggest that processes such as peroxisomal function and DNA repair are coordinated with PI synthesis. We are determining the role of a select setof these genes in PI biosynthesis and determining which of the five PIS1 promoterelements are required for their action. Using several strategies, including the above screen and mining of a ChIP-chipdatabase, we identified transcription factors and promoter elements that regulate PIS1 expression. These results suggest regulatory networks that we are testing for effects on PI biosynthesis. The knowledge that PI metabolites have roles that affect gene expression suggests that regulation of PI synthesis may affect several cellular processes. We are testing this directly by determining the phenotype of strains with altered PI synthesis and by using microarray hybridization to identify genes that are regulated in response to PI levels. III. Evolution of membrane phospholipid composition in the Saccharomyces genus. Comparative genome analysis of related species can provide a glimpse into the evolution of biochemical pathways and their regulation. There is a major ongoing effort to use comparative genome analysis to identify the regulatory sequences in the yeast genome. To better understand the evolution of cellular physiology, we compared the phospholipid compositions of several species of Saccharomyces to that of Saccharomycescerevisiae . We found that phospholipid composition varies dramatically between related species of Saccharomyces, specifically in the synthesis of PI and phosphatidylcholine (PC), two major phospholipids, and their common precursor, CDP-diacylglycerol (CDP-DAG). However, these differences cannot be explained by simply comparing the promoter sequences of relevant structural genes. Furthermore, examination of transcript levels of PI biosynthetic genes in the various Saccharomyces species reveals that the major transcriptional regulatory response, repression in response to inositol, is not conserved. These results strongly suggest that studies comparing regulation of membrane synthesis will be fertile ground for understanding evolution of cellular physiology. We have coined this area of investigation “evo-physio.” We have initiated experiments to characterize the transcriptional regulation of the phospholipid biosynthetic genes in the different species of Saccharomyces. Currently, we are developing the necessary tools to swap transcriptional regulatory factors and promoter elements between different members of the Saccharomyces. These experiments will enable to us determine how different levels of the regulatory cascades that control phospholipid biosynthesis have evolved.