(259a) Synthetic Chromatin-Based Transcriptional Logic, Spatial Regulation, and Memory

Biomedical and bioindustrial processes often depend upon robust and accurate transcription of genomically-integrated expression constructs. In eukaryotes, chromatin plays a central role in regulating gene transcription and vastly extends the information potential of the eukaryotic genome beyond the underlying DNA sequence. Hundreds of chromatin regulator (CR) proteins modulate access to genomic information by biochemically modifying DNA-bound histones as well as catalyzing the physical movement and recruitment of histones and other regulatory proteins. Yet, despite being the subject of extensive studies, it remains unclear what types of quantitative and qualitative regulatory behaviors CRs may enable, nor has chromatin been exploited for engineering applications. Synthetic biology and site-specific DNA-targeting technologies offer a unique approach to decipher the complexity of chromatin and to engineer novel transcriptional behaviors through combinatorial, spatial, and temporal CR recruitment.

We individually recruited 223 yeast zinc-finger-CR fusions to a GFP reporter locus and identified activators and repressors. To provide design rules in selecting CRs for future applications, CRs were clustered by gene ontology (GO) function terms to reveal proteins functions associated with activation and repression. This library was also co-recruited with a transcriptional activator, VP16, enabling engineering of a range of two-input transcriptional logic. This included CRs that fully repressed transcription even when co-recruited with VP16, and CRs that additively or synergistically activated transcription upon co-recruitment with VP16. We discovered that synergistic activation occurred with CRs that have distinct molecular mechanisms to VP16, while those with similar mechanisms to VP16 activated transcription additively. Next, we engineered CRs capable of simultaneously activating a GFP reporter gene when recruited 5’ of the gene while repressing a mCherry reporter gene when recruited 3’ of that gene, among other unique asymmetric spatial regulatory behaviors. Finally, we identified CRs capable of long-range repression, repressing three tandem genes when targeted upstream of only the first gene, as well as CRs that stably repressed reporter expression after only a transient pulse of CR expression. These novel chromatin-based regulatory behaviors, gene ontology-derived design principles, and synthetic biology tools could be useful in engineering cellular devices and biosensors and may expand our understanding of chromatin’s role in disease and development.

Reference:  Keung et. al., Using Targeted Chromatin Factors to Engineer Combinatorial and Spatial Transcriptional Regulation, In Press, Cell, 2014.