Endy:Notebook/DNA Methyl Bit/Initial Discussion

Why bi-stability

Bi-stable cells, which can stably exist in two or more expression expression states, have advantage over mono-stable cells in a variable environment. Thus, a population is hedged in an uncertain world by stably existing in two or more expression states.

Engineered genetic switches take advantage of mechanisms

Engineered memory switches can take advantage of natural mechanisms that confer bi-stability, which include:

• Feedback regulation
• DNA methylation

Critical aspects of engineered memory switch

• Two stable states
• Controllable switching

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Feedback regulation

Collins toggle switch (2000)

Parameter space for bi-stability

Switch controlled by proteins it creates. Proteins bind to promoter regulatory sites to repress or activate expression. This results in two stable states: proteins (repressor) repress their own repressor, so they will remain stably expressed. But, switching is controllable switching: dominant repressor (state) can be de-activated with addition of inducer (IPTG, thermal shock, etc). This allows expression of the other repressor, and enables controllable state change of the system Collins, 2000.

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Feedback regulation versus Methylation

Feedback regulation

• State : determined by the proteins they generate
• Bi-stability : relative expression rate of repressors
• Control : inducer
• Metabolically costly

Epigenetic

• State : determined by methylation of DNA
• Bi-stability : intermediate states biased to resist occupation
• Control : not clear
• Not metabolically costly

The below section discusses methylation, based upon : Hasty review and Multi-step epigenetic switch

Methylation switch in E. Coli

Epigenetic ON and OFF

On and Off state

• Context
  • Cis-regulatory region downstream of promoter can be methylated for a gene, Agn45, in E. Coli that codes for outer membrane protein involved in bio-film formation

• Switching on
  • Methylase enzyme methylates cis region, resulting in ON expression state

• Switching off
  • Only way to leave state is via DNA replication, which adds de-methylated DNA

• When de-methylated, OxyR competes with DAM for binding
  • When OxyR binds, the system is stable in the OFF state

Transition between states

Epigenetic switching mechanism

Bi-stable gene expression involves transitions through several rarely occupied intermediate states. Cells that leave on or off enter one of these immediate and states and experience pressure to revert to their initial state. These intermediate states are strongly biased to resist occupation, and quickly distribute cells towards the two extremes.

The puzzle

Simulation shows few cells leave the off state when started in the off state

So, the question is: how to switch?

With feedback regulation, protein concentration determines the state and state change is driven by altering protein expression levels. Bi-stability is therefore observed only within a particular regime of relative repressor protein expression levels. Here, bi-stability is observed independent of the expression level of the gene that is being controlled. However, the repressor switch can be controlled by addition of inducer, which interacts with and turns off the repressor currently expressed. This drives the system into the other expression state. Here, there appears to be no inducer analog. How can switching between on and off methylation states be controlled? Moving from ON to OFF requires dilution of methylated DNA via replication. Moving from OFF to ON requires dissociation of OxyR.