RICHARD S. ZITOMER
Professor, Department of Biological Sciences
University of Pennsylvania · B.S. May 1968
University of Pennsylvania · Ph.D. May 1972
Present Research Support
National Institutes of Health Research Grant: Regulation of Yeast Cytochrome c Genes.
Link to NIH Crisp Index for abstract of NIH grant.
Selected, Recent Publications
Link to abstracts of publications.
CURRENT RESEARCH INTERESTS
Regulation of Gene Expression by Heme
There are two classes of genes in bakers' yeast whose expression responds to heme levels as a sensor for molecular oxygen. Heme biosynthesis requires molecular oxygen as a substrate at two steps, thus it is well suited to a regulatory role. The first set of genes encodes components of the respiratory apparatus and the oxidative stress response, and the transcription of the genes in this class is activated by heme. The second class is comprised of the hypoxic genes. A subset of these encode functions that allow more efficient utilization of the limiting substrate oxygen in respiration and in heme, sterol, and fatty acid biosyntheses. The transcription of these genes is repressed by heme. Our long term goal is to determine the molecular mechanisms involved in regulating the expression of these latter genes.
We have studied the expression of three heme regulated genes. CYC1 encodes the major cytochrome c protein in aerobically-grown cells and is our prototype heme activated gene. ANB1 which encodes the translation initiation factor eIF-5A, is a heme-repressed gene. (There is a heme-activated homologue, and the role of these two oppositely regulated translation factors is currently under study.) The third gene CYC7 encodes the minor species of cytochrome c which comprises only 5% of the cytochrome c in aerobically grown cells, but the greater percentage in cells grown in limiting oxygen. Its usefulness lies in the fact that it contains cis-acting regulatory elements common to both heme induced and repressed genes making it weakly inducible in the presence and absence of heme. In addition, CYC7 is a member of the family of global stress-response genes of yeast, and our recent studies have uncovered some members of the signal transduction pathway induced by a variety of stresses.
Heme repressed genes- The regulatory region of the ANB1 gene contains two operator sites, one 200 bp and the other 290 bp 5' to the coding region. Each site consists of two copies of an 12 bp sequence also repeated in other heme-repressed genes. Activation of ANB1 transcription is mediated through a 120 bp region further upstream that contains extensive runs of A residues in the coding strand. This region is responsible for the constitutive expression of ANB1 in the absence of the repression site.
Repression at the operator sites is mediated through a protein Rox1; a mutant carrying a deletion of the rox1 gene is constitutive for expression of the heme repressed genes. The ROX1 gene itself falls into the heme-activated regulatory class. Thus heme repression occurs as a result of synthesis of the Rox1 protein when heme concentrations are high, while in the absence of heme, Rox1 is not made and heme repressed genes can be expressed. The actual function of Rox1 is heme-independent. In addition, ROX1 is auto-repressed which serves to regulate Rox1 protein levels tightly. The Rox1 protein sequence places it in the HMG class of DNA binding proteins. We have purified the Rox1 protein through over expression in bacterial cells and demonstrated that it binds specifically to the hypoxic consensus sequence. Rox1 bends DNA when it binds, and this bending may be required for repression. Using two different selection schemes, we have isolated several dozen point mutations in ROX1, and are using them to characterize the nature of the DNA-protein interaction and the mechanism of repression.
The heme-dependent expression of the ROX1 gene is mediated by the Hap1 protein which both activates its transcription aerobically and represses transcription anaerobically. In addition, Rox1 represses its own transcription, ensuring low levels of expression. Rapid induction of the hypoxic genes results from both the rapid repression of ROX1 transcription at the onset of anaerobiosis, and a rapid degradation of the protein.
Other trans-acting regulatory mutations- We have conducted a number of mutant hunts designed to isolate trans-acting mutations that cause constitutive expression of either CYC1 or ANB1 or increased CYC7 expression. We identified five unlinked genes, designated ROX1 (described above) and ROX3-6.
ROX4 and 5 are identical to a the TUP1 and SSN6 genes, respectively. The two proteins interact to form a general repression complex required for repression of a number of yeast regulons. This Tup1/Ssn6 complex functions in conjunction with regulon-specific repressors including: the repressor 2 and the 2-a1 complex for the repression of the a-mating type and haploid specific genes, respectively; the Mig1 repressor for the repression of catabolite repressed genes; and, of course, Rox1 for the hypoxic genes. Other regulon affected by this complex control genes involved in flocculence, DNA repair, and plasmid stability. The current hypothesis for the activity of this general repressor is that it is anchored to specific genes through specific DNA binding proteins, then interacts with the general transcriptional machinery. We have purified recombinant Tup1, Ssn6, and Rox1 to study their interactions and have obtained evidence that Rox1 interacts with Ssn6. We are also carrying out a genetic analysis of the TUP1 and SSN6 genes.
The role of Rox3 and Rox6 in the stress-induced pathway-A fourth ROX gene, ROX3, was identified as effecting the stress-response of CYC7 expression. CYC7 is transcriptionally activated by heat shock, glucose starvation, and osmotic shock, all acting through a set of STRE (stress-responsive-elements) sequences. ROX3 encodes a 220 amino acid long, basic protein localized in the nucleus. It is an essential protein; a deletion of ROX3 is lethal. We isolated extra-genic suppressors of rox3 mutants to identify genes encoding proteins that interact with Rox3. One, RTS1, encodes a novel regulatory subunit of phosphatase 2A. In collaboration with Ana DePaoli-Roach at the Indiana University Medical School, we have found that the cDNA encoding the rabbit skeletal homologue complements the temperature sensitive/osmotic sensitive phenotype of a yeast rts1 deletion.
We have found that the expression of ROX3 is itself induced by stress through a novel regulatory element, GA10GG. Recently, Roger Kornberg has demonstrated that Rox3 is part of the RNA polymerase II mediator complex.
Rox6 is Srb10, a cyclin in the mediator complex. A deletion of the gene results in constitutive expression of the stress induced genes.
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