Regulating Cells With Designed Proteins

Illustration of precise epigenomic remodeling using computer designed PRC2 inhibitor. Lady Histone’s hair is tightly curled in hair rollers (orange histones) resembling heterochromatin. Selected histones (green) are precisely de-repressed by the de novo designed protein, EBdCas9, reminiscent of euchromatin. Credits: Idea and design (Shiri Levy); Graphical design (Rita Gruber)

Your new laptop can be almost anything you need it to be. Right out of the box, it has the potential to become a business machine, a design studio, or a gaming console. Over its lifetime, the identity of the laptop will be determined by changes that add or enhance features, all without changing its original coding.

The ability to become something specialized from something ordinary makes your laptop a lot like a stem cell – the unprogrammed progenitor cells that give rise to heart cells, skin cells, muscle cells, and almost all other types of cells in our bodies. In biology, the refinements like the ones you make to your laptop would be called epigenetic changes.

Epigenetics are the changes that occur to an organism’s genetic makeup during its development and its life. These changes, which can be caused by environmental or chemical factors, do not alter an individual’s DNA sequence, but do affect the way cells interpret its genetic code and which proteins its cells manufacture. Diet, lifestyle, exposure to sunlight, and aging are all factors that can cause epigenetic changes.

Controlling Cell Fate by Modulating Genes

Naturally, stem cell biologists are eager for a way to regulate epigenetics – to control how cells become functionally different from one another. At home, we might modulate the functionality of a laptop by installing or updating software. At the Institute for Stem Cells and Regenerative Medicine (ISCRM), scientists are attempting to regulate the functionality of cells by repressing or activating particular genes that give cells certain instructions.

Over the last several years, Shiri Levy, an Acting Instructor in the lab of ISCRM Associate Director Hannele Ruohola-Baker, PhD, has spearheaded the development of a tool that is capable of selectively controlling the PRC2 complex – an epigenetic regulator that influences cell fate across multiple stages of development. That tool is a computer-designed protein binder engineered in partnership with the University of Washington Institute for Protein Design (IPD).

Shiri Levy, PhD, Hannele Ruohola-Baker, PhD, and Julie Mathieu, PhD

As a postdoc in the Ruohola-Baker Lab, Levy patented a method for selectively derepressing genes by fusing the computer designed protein binder to an inert CAS9 molecule. This technology is detailed in a study, now published in the journal Cell Reports, that reveals which epigenetic marks are necessary for gene repression.

Each DNA sequence that contains instructions to make a protein is known as a gene, while a genome is the entire set of an organism’s genetic material. The epigenome refers to the sum of chemical compounds and proteins that are responsible for interpreting instructions coded in DNA. Epigenetic marks are impressions left on the DNA when epigenetic factors alter the interpretation of the instructions (but usually not the sequence of DNA).

Ruohola-Baker emphasizes the novelty of the study. “This paper introduces the first of many razor sharp designed proteins to come that are capable of precisely regulating our epigenome, one gene at a time. The capacity to precisely modify epigenome could be revolutionary. The capacity to use artificial intelligence to erase specific epigenomic mistakes associated with aging and disease moves us closer to a new generation of therapies.”

“What we have found could someday be very useful in a clinical setting,” says Levy. “Targeted epigenomic editing provides greater safety and precision compared to genomic editing. Our research shows that once directed to a specific site, the computer design epigenomic binder successfully upregulates gene expression, leading to the desired changes in cell function. Best of all, this is an organic approach in which careful human intervention nudges cells to follow their natural instincts.”

Levy attributes the progress in epigenetic regulation to the marriage of biochemistry and protein design. “If we had just knocked out part of the PRC2 complex, we would not have been able to study the various stages of stem cell development. We needed a clever way to conditionally inhibit genes that would give us more control than we would get with drugs or other genetic manipulation protocols, allowing us to closely observe each step. It’s the difference between watching dominoes fall one-by-one and toppling them all at once.”

Designed Protein “Tweezers”

According to Levy, one of the biggest hallmarks of this work is the discovery of a distant TATA box (a strand of DNA that tells RNA where to look for decodable genetic sequences) on a gene (TBX18) that is normally masked by PRC2. “The ability to use computer-design proteins as “tweezers” to expose core promoter elements for gene regulation will allow us to understand basic biology in high resolution.”

In late 2021, Levy’s effort to control cell fate and gene expression precisely received a major boost in the form of a $245,000 technology commercialization grant from the Washington Research Foundation (WRF). The funding is enabling Levy to explore which applications for the epigenetic regulator system have the most promising therapeutic potential in areas such as regenerative medicine, cancer treatment, and autoimmune disease research. Previous rounds of WRF funding supported pivotal early stages of Levy’s work on this technology.

“Funding, flexibility, and mentorship from WRF helped us move our research forward” says a grateful Levy. “I also want to give a shout out to Hannele and to Julie Mathieu, whose resiliency has always inspired me. They’ve created an inviting environment that nurtures innovation.”


This work was supported by grants from the National Institutes of Health (R01GM097372, R01GM97372-03S1, R01GM083867, 1P01GM081619, R42HG010855, U01CA246503), Department of Defense (PR203328 W81XWH-21-1-0006), American Heart Association (19IPLOI34760143) and the Washington Research Foundation, ISCRM Fellows program, and Brotman Baty Institute (BBI) for Precision Medicine.