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Strategy

In order to create a bistable in S. pombe based on epigenetic regulation, we have mimic an architecture flanked by silencer sequences and designed a gene expression reporter system (promoter:GFP) which is flanked upstream and downstream by binding sites for engineering silencing proteins (SP). This reporter system will be integrated in S. pombe genome. The silencing proteins consist of fusion proteins or kymeras, composed by tetracycline repressor (tetR) and one silencing protein, or a domain of it. TetR will cause binding to tetR operator sites (tetO) and silencing proteins will be responsible for the recruitment of silencing proteins that will compact and inactivate the promoter. We expect that the recruitment and spreading of silencing proteins at flanking region of promoters will have a drastic effect on gene expression, due to the interference of promoter activity. When silencing proteins are expressed, compaction of DNA sequence will lead to gene repression, resulting in the OFF state. The ON state will be attained by repressing the expression of silencing proteins that will result in decompaction of nucleated structure, just like a zip. The result of this experimental design will be an epigenetic bistable.

ON state: To reach this state it is necessary the addition of three different chemicals to the cell culture: thiamine, doxicycline and uracil. Thiamine is used to repress the promoter of silencing genes. Thiamine regulates expression of Pnmt1 promoter (no message in thiamine promoter, 41X). Doxicycline functions as a decompaction compound, because of it ability to dissolve the structure nucleated around operator sites of reporter module, allowing the expression of GFP. Uracil is the activator of inducible P urg promoter, that regulates the expression of marker protein GFP. Addition of this thee chemical leads to inhibition of silencing proteins expression, decompaction of nucleated structure and finally synthesis of the marker protein, in a short interval of time, less than half an hour, due to chemicals and promoter activities (Bähler et al, 1998; Watt et al, 2008).

OFF state: To reach this state it is necessary the removal of the three chemicals in the medium: doxicycline, thiamine and uracil. For that reason chemical must be removed from the medium by sequential washing in cell culture. After washing out these compounds it will begin the expression of silencing genes and the repression of GFP expression. Synthesized proteins get back to the nucleus and stochastically find tetR operator sites and form a nucleation point. Spreading of SP produce a total repression of reporter module. Due to promoter characteristics, around 10-12 hours are needed to reach this state (Bähler et al, 1998; Watt et al, 2008).