Molecular Genetics of the Adenovirus E1A Oncoproteins

The laboratories

This summary describes a collaborative project ongoing in the Engel laboratory and the Smith laboratory . The two labs share a common interest in gene expression and cellular regulation, and they bring together a unique combination of techniques in yeast and mammalian cell molecular biology and genetics. The research summarized here was carried out by Mary Miller for her Ph.D. thesis, working in the two labs.

[Photograph of Dan Engel, Mitch Smith, and Mary Miller]

Figure 1 Dan Engel, Mitch Smith, and Mary Miller plotting experiments at the Department of Microbiology Retreat, Graves Mountain Lodge, November 1994.

Introduction

The adenovirus E1A gene encodes potent oncoproteins that modify the normal transcriptional regulation of key cellular genes and thus alter cell cycle control. Over the last 20 years, the E1A gene has been subjected to extensive genetic analysis and its functional domains are known in considerable detail. However, little is understood about the cellular pathways affected by the E1A oncoproteins, in large part because of the technical difficulties of genetic analysis in mammalian tissue culture cells. Thus, this project began with the goal of applying the powerful molecular genetic tools available for Saccharomyces cerevisiae to the question of E1A function in the cell. The first results of the project are published in a recent paper and this link will take you to the full text of Miller, Engel, and Smith, Oncogene 11, 1623-1630 (1995).

E1A expression in yeast inhibits cell growth

To begin the project, Mary first constructed a series of plasmids in which derivatives of the E1A gene were expressed from a yeast expression vector under control of the GAL1 promoter. As shown in Figure 2, the expression of E1A protein strongly inhibits the growth of yeast cells. This inhibition is characterized by an accumulation of cells in the G₁ phase of the cell division cycle.

[Plate assays of cell growth]

Figure 2 Growth inhibition phenotype of cells expressing E1A. (A) wild type cells; (B) cells expressing E1A.

E1A function in yeast requires protein domains also needed in mammalian cells

As shown in Figure 3, distinct domains of E1A interact with mammalian cell proteins. Using a series of E1A deletion mutants, Mary showed that the growth inhibition in yeast cells required at least three of these same protein domains: the N-terminal region, CR1, and CR3. Further experiments showed that the N-terminal region and CR1 were sufficient to cause severe growth inhibition.

[Diagram of E1A protein domains]

Figure 3 Functional domains of E1A. A map of the predicted 2D structure of E1A₂₈₉ is shown with four functional domains highlighted: the N-terminal region, and conserved regions 1, 2, and 3 (CR1, CR2, and CR3). Predicted hydrophilic regions are represented by the cyan boxes, while hydrophobic regions are shown in red. The domains necessary for protein binding (p300, pRb, TBP) and functional activities (transformation, c-fos activation) are indicated by the dark blue bars. The domains necessary for function in yeast (S. cerevisiae) correspond to domains also required for cellular transformation and gene activation in mammalian cells.

E1A function in yeast requires the cAMP pathway

Since the Engel laboratory had previously shown that the activation of the mammalian c-fos gene by E1A requires the cAMP signaling pathway, we tested the role of this pathway for E1A function in yeast. Adenylate cyclase is encoded by the CDC35 gene in S. cerevisiae and at semipermissive temperatures cdc35 Ts^- mutants have reduced levels of cAMP. As seen in Figure 4, cdc35 cells expressing either E1A₂₈₉ or E1A₂₄₃ are unable to grow at permissive temperature (24C), but readily form colonies at semipermissive temperature (28C).

[Plate assays of E1A function in cdc35 mutants]

Figure 4 E1A function requires the normal cAMP pathway. Strains transformed with E1A plasmids were streaked for growth on galactose plates to induce expression. For each plate shown, the cells expressed either control plasmid vector (top section), E1A₂₄₃ (lower right section) or E1A₂₈₉ (lower left section). (B) LRA85 (cdc35) at 24C; (E) LRA85 (cdc35) at 28C;

E1A and the SWI/SNF transcription activation complex

Taking advantage of the growth inhibition caused by E1A expression, Mary conducted a large-scale genetic screen for yeast mutants insensitive to E1A expression. This screen yielded 5 mutants in 4 complementation groups. The first of these genes has been cloned, and together with Randy Levinson of the Yamamoto laboratory (UCSF) and Brad Cairns of the Kornberg laboratory (Stanford) it was identified as encoding a subunit of the SWI/SNF transcription activation complex. A manuscript describing these results is currently submitted. The model derived from these studies is shown in Figure 5.

[Diagram of E1A interaction with SWI/SNF]

Figure 5 E1A and SWI/SNF. A variety of genes depend on the action of the SWI/SNF complex for transcription activation. For these genes, chromatin proteins such as histones are believed to negatively regulate the function of transcription factors by blocking their access to the DNA. The role of the SWI/SNF complex is to negatively regulate the inhibitory function of chromatin. Our results suggest that E1A acts to repress the expression of SWI/SNF-dependent genes by directly inhibiting the ability of the SWI/SNF complex to relieve the repression by chromatin proteins.

The identification of the yeast SWI/SNF complex as a functional target of E1A has important implications for the activity of E1A in mammalian cells. There are at least two types of SWI/SNF transcription activation complexes conserved in mammalian cells. In yeast, E1A blocks SWI/SNF-dependent transcription activation through its N-terminal region and CR1, two domains required for activation and repression in mammalian cells. We propose that some of these effects are likely due to the interaction of E1A proteins with one or more of the mammalian SWI/SNF complexes.

Future directions

These results clearly raise many exciting questions. In yeast, exactly how does E1A block SWI/SNF function? Are there direct physical interactions between E1A and SWI/SNF subunits? What specific SWI/SNF-dependent genes are critical for viability? In mammalian cells, can we obtain evidence for our proposal that E1A modifies the function of the SWI/SNF complex? Do the known E1A binding proteins play a role in this function?

Currently, Mary Miller is cloning and analyzing the genes for the other yeast mutants that came out of her screen. We anticipate that these will shed additional light on the mechanism of E1A action in yeast and mammalian cells. Caroline Kuleza is conducting a completely different genetic screen of E1A function in yeast that should identify other key players and pathways. Daniel Murphy is working on the mammalian side of the project, seeking evidence for the functional interaction of cellular E1A binding proteins with the SWI/SNF complex pathway.

Acknowledgements

We thank all the members of the Engel and Smith labs for helpful discussions during the course of these experiments, Randy Levinson and Brad Cairns for sharing results and materials prior to publication, and Stockton Press for permission to place the full-text version of our Oncogene paper on line. Mary Miller was supported in part by training grant 5T3 CA09109 from the National Cancer Institute. The work described in this summary was supported by a grant from the Life and Health Insurance Medical Research Foundation awarded to Dan Engel and National Institutes of Health grant GM28920 awarded to Mitch Smith.

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