The human genome consists of roughly 20,000 genes. Most of those genes contain instructions for making proteins, which work to build, repair, and regulate everything in our bodies. The genes are separated into distinct domains, and between those domains are boundary regions of DNA, which help to separate genes and ensure there isn’t crosstalk resulting in expression (genes turned on) or silencing (genes turned off) between the genes. Unfortunately, disruptions within boundary regions can still occur, leading to gene misexpression and disease in humans.

Some boundary regions contain barrier elements that can help block gene misexpression or shield against gene silencing, but the latter is far less studied due to the difficulty of identifying elements with such activity. In a recent study by the lab of Huimin Zhao (BSD leader/CABBI/MMG/), Steven L. Miller Chair of Chemical and Biomolecular Engineering at the University of Illinois Urbana-Champaign, the researchers describe a new technology, called SHIELD, which can effectively screen for barrier DNA elements that protect genes from being silenced. 

“Barrier DNA elements work to stop repressive heterochromatin spreading, which is one of the ways that genes become silenced,” explained Meng Zhang, a former graduate student in Zhao’s lab and first author on the study. “A gene that is flanked by these protective barrier DNA elements will still be able to express, even within highly repressed regions of the chromosome. These elements essentially function as a counter to the silencing effect.”   

Following this principle, the team decided to use a highly repressive area of the genome as the stage for their experiment – the Lamina-associated Domain. Often dubbed the “gene-silencing hub of mammalian genomes,” the LAD provides an ideal environment to screen for DNA elements that may contain barrier activity. Genes placed inside this region normally would be unable to express due to the LAD’s repressive nature. But if DNA elements with barrier activity are placed alongside the gene, then the gene should still be able to express. Zhao refers to this as a “stress test” for the barrier element’s “anti-silencing” capabilities.

The researchers used a fluorescent protein as their reporter, so that cells where the gene was able to express would glow and could be easily identified. The gene encoding the fluorescent protein was placed in the LAD of cells alongside candidates for barrier DNA elements, and the cells were then screened for fluorescence.

The SHIELD technology was able to quickly screen 1000 candidate elements at once, and using flow cytometry to sort cells with the highest fluorescence expression, the team was able to identify 8 candidates that showed potent barrier activity. The researchers found that three of these elements performed equally or better than the barrier element currently used in medicine and research.

“Barrier DNA elements have not been well characterized before in the human genome compared to other non-coding elements like promoters or enhancers,” said Zhang. “This is partly because there wasn’t a technology available to readily evaluate activity of these DNA elements and screen for barrier activity. The barrier element widely used right now was initially discovered in 1993 and has been in use since then. Our technology will now allow us to identify new barrier elements in a quick and efficient manner, thus expanding the toolbox of such elements for synthetic biologists.”   

Newly identified barrier elements have many potential therapeutic applications. For example, scientists typically use a viral vector to deliver therapeutic genes to patient cells lacking the working gene, but sometimes the therapeutic genes become silenced by the host cell. Barrier elements could be used to protect the therapeutic cargo from being silenced. Zhao says the applications for the new technology extend beyond just therapeutics.

“Our findings have many therapy applications, but they also are important for fundamental studies in the field of biology, particularly for those studying genetic circuits,” said Zhao. “Identifying these barrier elements will give them better tools to work with for their research. Furthermore, the knowledge gained from such screening can help us understand what types of DNA sequences contribute to the establishment of chromatin boundaries in the genome.”   

Zhao also hopes other researchers will use the tool to discover more barrier elements and expand the library, not only in mammals, but in other eukaryotes such as other animals, plants, and fungi, as well using a similar strategy.

The study is published in Nature Communications and was supported by the NIH. The paper can be found at https://doi.org/10.1038/s41467-023-41468-3