Shaking Up Genome Regulation by Considering Oil/Water-Like Interactions

Gene expression is regulated in part by liquid-liquid phase separation, which has a role in organizing heterochomatin domains. The mouse fibroblast cells shown in this image express HP1alpha, the human version of heterochromatin protein 1a. Notice the presence of liquid droplets in the nucleus, which are visible thanks to a technique that highlights edges between two liquid phases. [Amy Strom/Berkeley Lab]

In the mixed-up world of gene silencing, it’s not exactly clear why some genomic regions are hard to access. These regions, it has been suggested, may simply be too tightly packed to permit the passage of regulatory proteins needed for functions such as DNA repair. Tightly packed DNA, however, doesn’t always behave as expected. For example, heterochromatin has been known to exclude small proteins while admitting large ones.

Such anomalous behavior naturally attracts the attention of scientists. Eager to resolve the problems accompanying the compaction explanation for the silencing of heterochromatin, scientists based at Lawrence Berkeley National Laboratory decided to consider an alternative mechanism. It turns out to be the same one that accounts for the separation of oil and water.

A Berkeley Lab team led by Gary Karpen, a senior scientist specializing in biological systems and engineering, uncovered evidence that heterochromatin organizes large parts of the genome into specific regions of the nucleus using liquid-liquid phase separation, a mechanism well-known in physics but whose importance for biology has only recently been revealed.

Details appeared June 21, 2017 in the journal Nature, in an article entitled, “Phase separation drives heterochromatin domain formation.” The article suggests that phase separation, a phenomenon already known to have biological relevance in giving rise to diverse non-membrane-bound nuclear, cytoplasmic, and extracellular compartments, also mediates the formation of heterochromatin domains.

“We show that Drosophila HP1a protein undergoes liquid–liquid demixing in vitro, and nucleates into foci that display liquid properties during the first stages of heterochromatin domain formation in early Drosophila embryos,” wrote the article’s authors. “Furthermore, in both Drosophila and mammalian cells, heterochromatin domains exhibit dynamics that are characteristic of liquid-phase separation, including sensitivity to the disruption of weak hydrophobic interactions, and reduced diffusion, increased coordinated movement, and inert probe exclusion at the domain boundary.”

Essentially, the researchers observed two non-mixing liquids in the cell nucleus: one that contained expressed genes, and one that contained silenced heterochromatin. They found that heterochromatic droplets fused together just like two drops of oil surrounded by water.

In lab experiments, researchers purified heterochromatin protein 1a (HP1a), a main component of heterochromatin, and saw that this single component was able to recreate what they saw in the nucleus by forming liquid droplets.

“Chromatin organization by phase separation,” noted Amy Strom, study lead author and a graduate student in Karpen's lab, “means that proteins are targeted to one liquid or the other based not on size, but on other physical traits, like charge, flexibility, and interaction partners.”

The authors of the Nature article concluded that the heterochromatic domains form via phase separation mature into structures that include liquid and stable compartments. They also proposed that emergent biophysical properties associated with phase-separated systems are critical to understanding the unusual behaviors of heterochromatin, and how chromatin domains in general regulate essential nuclear functions.

“The importance of DNA sequences in health and disease has been clear for decades, but we only recently have come to realize that the organization of sections of DNA into different physical domains or compartments inside the nucleus is critical to promote distinct genome functions,” commented Dr. Karpen.

The Berkeley Lab study, which used fruit fly and mouse cells, will be published alongside a companion paper in Nature led by UC San Francisco researchers, who showed that the human version of the HP1a protein has the same liquid droplet properties, suggesting that similar principles hold for human heterochromatin.

Interestingly, this type of liquid-liquid phase separation is very sensitive to changes in temperature, protein concentration, and pH levels.

“It's an elegant way for the cell to be able to manipulate gene expression of many sequences at once,” commented Strom.

Other cellular structures, including some involved in disease, are also organized by phase separation.

“Problems with phase separation have been linked to diseases such as dementia and certain neurodegenerative disorders,” remarked Dr. Karpen.

He noted that as we age, biological molecules lose their liquid state and become more solid, accumulating damage along the way. Dr. Karpen pointed to diseases like Alzheimer's and Huntington's, in which proteins misfold and aggregate, becoming less liquid and more solid over time.

“If we can better understand what causes aggregation, and how to keep things more liquid, we might have a chance to combat these types of disease,” Strom suggested.

The work is a big step forward for understanding how DNA functions, but could also help researchers improve their ability to manipulate genes.

“Gene therapy, or any treatment that relies on tight regulation of gene expression, could be improved by precisely targeting molecules to the right place in the nucleus,” explained Karpen. “It is very difficult to target genes located in heterochromatin, but this understanding of the properties linked to phase separation and liquid behaviors could help change that and open up a third of the genome that we couldn't get to before.”

This includes targeting gene-editing technologies like CRISPR, which has recently opened up new doors for precise genome manipulation and gene therapy.