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HUMAN ENHANCERS OF FILAMENTOUS GROWTH IN YEAST

Over the last forty years, genetic and biochemical studies of the simple eukaryote Saccharomyces cerevisiae have established paradigms for cell growth control, gene expression, and metabolism. These paradigms were found to apply to more complicated eukaryotes up through and including humans, and have been used to identify and characterize key regulators of cell division control, greatly advancing our general understanding of the process of cancer. We have postulated that if parts of a genetic pathway are conserved between humans and yeast, it might be possible to identify novel important human genes by virtue of their ability to alter the normal functioning of the pathway in yeast. The primary focus of my laboratory is to characterize a number of human genes that, based on their ability to enhance formation of pseudohyphae in yeast, are potential regulators of cell growth control during carcinogenesis or during stress response. A second focus in my laboratory is the continuation of work on a protein-protein interaction system, called the Interaction Trap, to improve the utility of this yeast-based technology for identification and characterization of novel proteins.

THE INTERACTION TRAP. GOLEMIS, KHAZAK, ESTOJAK

In the Interaction Trap/two hybrid system, protein-protein interaction is assayed using a genetic strategy in which a protein (P1), or the "bait," is expressed as a fusion protein with a DNA-binding domain (LexA); and a second protein (P2) is expressed as a fusion protein with a transcriptional activation domain. Coexpression of the two protein fusions in yeast in which the binding site for the DNA binding domain is located upstream of a reporter gene will result in transcriptional activation of the reporter gene by the P2-fused activation domain if P1 and P2 are able to associate. This two-hybrid approach has gained considerable popularity because it provides a rapid and convenient assay for a large number of interactions without the need to purify proteins. In addition, we have shown that by substituting a cDNA library for a defined P2, the two-hybrid approach can be readily converted into a screening system for novel interacting proteins. We are currently conducting experiments to characterize and improve the basic functioning of the Interaction Trap. As one approach, we are attempting to introduce an additional "decoy" reporter system to eliminate the tedious tasks of isolating and eliminating false positive cDNA s obtained in library screens. In a continuing collaboration with Roger Brent, we also have performed a series of calibration experiments to determine how the affinity of two proteins as measured by two hybrid system correlates with in vitro biochemical measures of affinity.

RNA POLYMERASE SUBUNITS THAT MAY REGULATE CELL MORPHOLOGY AND STRESS RESPONSE. KHAZAK, ESTOJAK, in collaboration with SADHALE, WOYCHIK

Diploid S. cerevisiae undergo a conversion to pseudohyphal (filamentous) budding when grown on severely nitrogen-limited media. In this conversion, the budding pattern changes from bipolar to unipolar with a concomitant alteration in cell morphology from rounded to extremely elongated. Colonies of pseudo-hyphal cells have a stellate appearance that is easily distinguishable from vegetatively growing cells. The yeast budding pathway contains functional homologs of a number of human oncogenes and antioncogenes, suggesting that human genes that can specifically affect budding control might function as regulators of cell transformation and metastasis. In addition, because pseudohyphal conversion is induced by nutritional stress, human genes that enhance stress response might also enhance formation of pseudohyphae. We transformed yeast with a human cDNA expression library, then screened the transformed yeast and isolated several human genes that enhanced pseudohyphal formation.

One human gene identified in the pseudohyphal enhancement screen possesses significant sequence homology (21% to 53% identity) to a family of RNA polymerase subunits of which the most well characterized member is the yeast RPB7 subunit (McKune et al., Yeast 9: 295, 1993), leading us to designate it hsRPB7. Deletion of RPB7 in yeast is normally lethal. HsRPB7 can functionally replace its yeast counterpart and allow growth at moderate temperatures; we have used antibody directed against the largest subunit of pol II in co-immunoprecipitation experiments to demonstrate that hsRPB7 is capable of assembling into complete RNA pol II. HsRPB7-containing yeast are strikingly deficient for survival relative to wild-type yeast during culture at high and low temperatures, after heat shock, or when maintained at stationary phase. We postulated that this reduced ability to resist stress might be due to reduced ability of hsRPB7 to associate with another RNA pol II subunit, RPB4. Work from other laboratories had previously shown that an RPB4/RPB7 subcomplex was specifically required for effective stress response (Choder and Young, Mol. Cell. Biol. 13: 6984, 1993). Using the Interaction Trap, we have determined that hsRPB7 is able to interact with RPB4 in vivo, but does so with a lower affinity than the yeast RPB4-RPB7 subunit pair. This property provides a possible mechanism for the defective stress response of hsRPB7-dependent yeast.

The above data and other experiments indicated that the RPB7 subunit in yeast plays a role in both stress response and cell morphology control, and that the human gene preserves at least some of these functions. This observation suggests a parallel mechanism to some prokaryotic systems in which specific sigma factors associate with RNA polymerase during stress response and function, in part, by reprogramming cell shape to one better suited for survival (reviewed in Hengge-Aronis, Cell 72: 165, 1993). Our main interest is to determine whether hsRPB7 similarly functions to control stress response and morphology in human cells. We have begun a series of experiments to address these issues.

To date, we have found that the RNA encoding hsRPB7 is expressed at dramatically different levels (80-fold variation) in different tissues; the highest levels are in heart, muscle, kidney, and liver. We developed an antibody to hsRPB7 and used it to assay for protein expression in mammalian cells. In a Western blot of kidney cell proteins, the antibody detects one band of ~18 kD, which corresponds to the size of hsRPB7 predicted from its 176 amino acid coding sequence, and a second band of ~24 kD, which may correspond to a phosphorylated or otherwise modified form of the protein. Using hsRPB7 in Interaction Trap library screening, we obtained multiple isolates of two cDNAs encoding proteins that interacted strongly and specifically with LexA-hsRPB7. The first of these is a human homolog of the RPB4 subunit, which we have designated hsRPB4. Preliminary Northern analysis indicates that expression of this gene is also tissue specific. The highest levels of RNA were detected in the heart and muscle tissue, which parallels the expression pattern of hsRPB7. The second cDNA encodes a novel member of a family that includes the Arabidopsis (plant) EMB30 gene. The EMB30 gene was isolated because plants that have this gene deleted cannot form roots as a result of deficiencies in cell division and cell expansion (Shevell et al., Cell 77: 1051, 1994)­morphological phenotypes reminiscent of those involved in pseudohyphal transition. Over the next year, our goal is to explore in mammalian cells the increasingly intriguing relationship between hsRPB7 expression and modification, and the transcription of genes required for stress response and cell growth control.

HEF1. LAW, ESTOJAK, BILYAK, WANG

HEF1 (Human Enhancer of Filamentous growth 1) is the sole cDNA isolated from the screen that induces constitutive pseudohyphal growth under all nutrient conditions. The original HEF1 clone is 900 bp long and contains an open reading frame (ORF) that encodes a putative protein of 186 amino acids. We have recently isolated a murine genomic clone for HEF1 (MEF1). Analysis of MEF1 sequence indicates it is likely that the HEF1 clone represents one exon of a longer gene. The MEF1 and HEF1 amino acid sequences are 96 percent identical. The high degree of conservation suggests that these sequences encode an important function. Using a Northern blot of RNAs from several tissues, a HEF1-complementary probe identified a number of cross-hybridizing bands, including an ~3.4 kb and a 5 kb RNA species that were present in all tissues, but most abundant in kidney, lung, and placenta; and two other weakly cross-reacting species, one of ~8 kb that was detected only in kidney and lung, and one of ~1.2 kb that was detected only in liver.

Using a HEF1-specific probe, we have isolated a 3.7 kb cDNA sequence with an ORF coding for ~830 amino acids that contain an SH3 domain and multiple SH2-binding motifs. We found very recently that HEF1 is closely related to the gene for p130Cas, which was cloned based on its association with the oncogene v-crk (Sakai et al, EMBO J. 13: 3748, 1994). P130Cas appears to associate with and act as a substrate for multiple tyrosine-kinase containing oncogenes. The current model for the function of this protein suggests that it may act as a central assembly point for signals relevant to growth control and carcinogenesis. Preliminary immunohistochemical experiments with an antibody generated using a HEF1- specific peptide suggest that HEF1 associates with the actin cytoskeleton. Cumulatively, these data suggest that HEF1 may directly modulate cell growth function in response to oncogenic signals. The short ORF originally isolated in the pseudohyphal screen may correspond to an unregulated effector domain. Work on this gene remains a high priority.

PUBLICATIONS

DATTA, K., T.F. FRANKE, T.O. CHAN, A. MAKRIS, S.-L. YANG, D. KAPLAN, D.K. MORRISON, E.A. GOLEMIS, and P.N TSICHLIS. AH/PH domain mediated dimerization of Akt and its potential role in Akt regulation. Mol. Cell. Biol. (in press).

GOLEMIS, E., J. GYURIS, and R. BRENT. Interaction trap/Two-hybrid system to identify interacting proteins. Unit 13.14.1­13.14.17. In Current Protocols in Molecular Biology, edited by F.M. Ausubel, et al. John Wiley & Sons, NY, 1994.

KLICKSTEIN, L.B., R.L. NEVE, E. GOLEMIS, and J. GYURIS. Conversion of mRNA into double-stranded cDNA. Unit 5.5.1­5.5.13. In Current Protocols in Molecular Biology, edited by F.M. Ausubel, et al. John Wiley & Sons, NY, 1995.

SATO, T., M. HANADA, S. BODRUG, S. IRIE, N. IWAMA, L, BOISE, C. THOMPSON, E. GOLEMIS, L. FONG, H.-G. WANG, and J.C REED. Interactions among members of the BCL-2 protein family analyzed with a yeast two-hybrid system. Proc. Natl. Acad. Sci. USA 91: 9238­9242, 1994.

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