Systems for induction of gene expression and protein depletion in yeast
A system allows for rapid and specific induction of individual genes in eukaryotic cells using a chimeric transcriptional activator that is responsive to hormone inducer. Upon addition of the hormone, cytoplasmic transcriptional activator localizes to the nucleus and subsequently binds to promoters containing sequences that bind to its DNA-binding domain. Genetic modifications allow for rapid and specific degradation of a targeted protein upon addition of hormone by means of a regulated degron method that utilizes a protease variant. This system is useful for discovering new compounds by high throughput screening when introducing compound libraries to these protein-depleted cells.
1. A method of targeted graded protein depletion in a eukaryotic cell comprising:
a. providing a eukaryotic cell comprising:
i. a target gene having a modified degron sequence at the initiator codon of the open reading frame, the target gene being capable of expressing a target protein;
ii. a protease-encoding gene that expresses a protease that is capable of hydrolyzing the target protein to yield a protein having an N-end rule destabilizing amino acid at the amino terminus;
iii. a transcriptional activator comprising a DNA-binding domain and a hormone-binding domain, wherein, when bound by a hormone, the transcriptional activator induces expression of the protease-encoding gene; and
vi. a transcriptional activator gene comprising a constitutive promoter that expresses the transcriptional activator at a level that is sufficient to achieve a graded response of activation to hormone induction; and
b. exposing the eukaryotic cell to an amount of a hormone that is effective to induce the transcriptional activator in a graded response to the hormone; wherein the hormone binds to the hormone-binding domain of the transcriptional activator, the hormone-bound transcriptional activator induces expression of the protease by the protease-encoding gene, the protease cleaves the target protein to expose the N-end rule destabilizing amino acid and the target protein is degraded.
2. The method of claim 1 wherein the DNA-binding domain is a GAL4 DNA binding domain, a zinc finger domain, or a DNA-binding domain of a transcriptional activator that is not from yeast.
3. The method of claim 1 wherein the hormone-binding domain is from the human estradiol receptor, the human glucocorticoid receptor or the human mineralocorticoid receptor.
4. The method of claim 1 wherein the transcriptional activator is GEV, the hormone is β-estradiol, and the protease-encoding gene encodes the tobacco etch virus (TEV) protease.
5. The method of claim 1 wherein the protease-encoding gene is driven by a synthetic promoter operably linked to a modified TEV protease-coding region, a spliceosome subunit p14-peptide coding region, a terminator, and a selectable marker.
6. The method of claim 1 wherein the protease is TEV, HRV 2A or HRV 3C.
7. A eukaryotic cell comprising:
a. a target gene having a modified degron sequence at the initiator codon of the open reading frame, the target gene being capable of expressing a target protein;
b. a protease-encoding gene that expresses a protease that is capable of hydrolyzing the target protein to yield a cleaved target protein having an N-end rule destabilizing amino acid at the amino terminus;
c. transcriptional activator comprising a hormone-binding domain, wherein, when bound by a hormone, the transcriptional activator induces transcription of the protease-encoding gene;
d. a transcriptional activator gene comprising a constitutive promoter that expresses the transcriptional activator at a level that is sufficient to achieve a graded response of activation to hormone induction.
8. The cell of claim 7 wherein the transcriptional activator is GEV, ZEV or 4ZEV, and the hormone is β-estradiol.