Chromatin-Modifying Elements Overview

Eukaryotic chassis vary significantly from prokaryotic chassis in certain elements pertaining to regulation of gene expression. Eukaryotes have a complex epigenetic code that involves histone modifications and DNA methylation, as well as innate defense mechanisms that target exogenous DNA inserts (Matzke et al., 2000). These factors contribute to the gradual reduction in expression of an integrated gene over time, commonly known as gene silencing. Furthermore, integrated genetic constructs often contain DNA regulatory elements such as enhancers and promoters that may interact bidirectionally with those endogenous to the genome (Recillas-Targa et al., 2004). Referred to as neighbourhood effects, these interactions can lead to highly variable and unpredictable expression levels of all genes involved.

In order to mitigate the aforementioned issues, our team investigated eukaryotic chromatin-modifying elements (CMEs). Within this broad category of DNA elements, insulators and ubiquitously-acting chromatin opening elements (UCOEs) were selected for further exploration due to their unique properties. We were interested in creating a collection of parts in the registry that would help to increase the utility and efficiency of eukaryotic synthetic biology.

Insulators

The function of insulator sequences has been the focus of extensive investigation and as such, the mechanisms involved in this function have been previously described in literature. When placed in between enhancer and promoter, insulator sequences are capable of interfering with interactions between these regulatory elements (Recillas-Targa et al., 2004). By designing a system in which our gene inserts would be flanked by a well-characterized 1.2kb insulator sequence from the 5' end of the chicken β-globin gene, we aimed to create an isolated and protected pocket virtually free from such neighbourhood effects (Pikaart et al., 2018).

In addition to their enhancer-blocking function, insulators can act as a barrier to prevent the spread of closed, unexpressed DNA known as heterochromatin which ultimately contributes to gene silencing. This is accomplished by recruiting or sequestering histone-modifying enzymes, as well as by blocking DNA methylation mechanisms (Giles et al., 2010).

Ubiquitously-Acting Chromatin Opening Elements (UCOEs)

UCOEs can be defined as methylation-free, CpG-rich sequences found upstream of housekeeping genes within eukaryotic genomes (Romanova et al., 2017). They function not only to prevent gene silencing, but can actively re-open closed chromatin by promoting demethylation of promoter DNA (Romanova et al., 2017). We selected A2UCOE for use in our system, as it is the most commonly researched sequence that falls into the UCOE category (Harraghy et al., 2015). The sustained increase in gene expression levels observed when using such a sequence in eukaryotic gene integration schemes has been well documented, though the mechanism utilized by these elements is not well understood (Kunkiel et al., 2017).

Our Combinatorial Approach

The function of insulator sequences has been the focus of extensive investigation and as such, the mechanisms involved in this function have been previously described in literature. When placed in between enhancer and promoter, insulator sequences are capable of interfering with interactions between these regulatory elements (Recillas-Targa et al., 2004). By designing a system in which our gene inserts would be flanked by a well-characterized 1.2kb insulator sequence from the 5' end of the chicken β-globin gene, we aimed to create an isolated and protected pocket virtually free from such neighbourhood effects (Pikaart et al., 2018).