This is a proposal by Li Zhang for five years of support to investigate heme signaling in yeast. The work focuses on the transcription factor HAP1, a master regulator controlling oxygen sensing and heme signaling in Saccharomyces cerevisiae. Target genes for HAP1 include cytochromes and the anaerobic repressor ROX1. The simple domain structure of HAP1 is well-characterized. There is a DNA binding and dimerization domain, belonging to the 6-cys cluster family of zinc finger proteins, a heme responsive domain with two types of repeated elements including heme binding sites, and a transcription activation domain. Key to this proposal DNA binding and activation are modulated by heme. DATA HAP1 association with a large complex (HMC) in cell extracts is also regulated by heme. Thus, Zhang's model for heme regulation proposes that in low heme HAP1 repressed by association with a large complex in the cell, possibly in a monomeric state. This HMCc has DNA binding activity. High heme levels lead to heme binding to HAP1 and dissociation of the complex. The de-repressed factor can now bind DNA as a dimer and activate transcription. Unresolved questions in this model include: what is the nature of the association of HAP1 with HMC and how does heme regulate this interaction?, which cellular factors, for example, components of HMC, are necessary for the regulation and how do these factors function? The current proposal consists of 4 specific aims.
Aim 1 determines the minimal domain of HAP1 necessary for heme regulation and performs physical characterizations + heme, including a NMR structural determination.
Aim 2 investigates the role of the high molecular weight complex by mutagenizing HAP1 elements proposed to be involved in HMC formation.
The third aim i nvestigates the mechanism by which HAP1 dimerization elements affect heme regulation and transcriptional activation.
This aim i ncludes both mutagenesis and tests for inter and intramolecular interactions as well as experiments designed to determine the subunit configuration of HAP1 in the HMC.
The final aim employs yeast genetics, a two-hybrid screen, and biochemical approaches to identify the cellular factors involved in heme regulation.
