By combining CRISPR technology with a protein designed using artificial intelligence, individual dormant genes were awakened by disabling the chemical “off switches” that silence them. Researchers from the University of Washington School of Medicine in Seattle describe this finding in the journal Cell Reports.
The team fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. They found that targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3).
The technique allows researchers to better understand the role individual genes play in normal cell growth and development, in aging, and in such diseases as cancer, said Shiri Levy, a postdoctoral fellow in UW Institute for Stem Cell and Regenerative Medicine (ISCRM) and the paper’s lead author.
“The beauty of this approach is we can safely upregulate specific genes to affect cell activity without permanently changing the genome and cause unintended mistakes,” Levy said.
The project was led by Hannele Ruohola-Baker, professor of biochemistry and associate director of ISCRM. The AI-designed protein was developed at the UW Medicine Institute for Protein Design (IPD) under the leadership of David Baker, also a professor of biochemistry and head of the IPD.
The new technique controls gene activity without altering the DNA sequence of the genome by targeting chemical modifications that help package genes and regulate their activity—epigenetic markers.
Scientists are particularly interested in epigenetic modifications because not only do they affect gene activity in normal cell function, they can accumulate with time, contribute to aging, and affect the health of future generations as they are passed to children.
In their work, Levy and her colleagues focused on a complex of proteins called PRC2 that silences genes by attaching a small molecule, called a methyl group, to a protein that packages genes called histones. These methyl groups must be refreshed, so if PRC2 is blocked the genes it has silenced can be reawakened.
PRC2 is active throughout development but plays a particularly important role during the first days of life when embryonic cells differentiate into the various cell types that will form the tissues and organs of the growing embryo. However, the specific genetic loci at which PRC2 function is critical for determining cell fate has remained unknown, as there has been no way to target PRC2 inhibition to specific chromosomal locations. A central question in epigenomics and developmental biology, the authors write, is the role PRC2-dependent specific histone 3 lysine 27 methylation (H3K27me3) marks in cell fate decisions. During developmental transitions, the chromatin undergoes extensive epigenetic remodeling at the promoter and enhancer regions, where H3K27me3 marks are added or removed to produce the required decrease or increase in gene expression.
PRC2 can be blocked with chemicals, but using these is imprecise, affecting PRC2 function throughout the genome. The goal of the UW researchers was to find a way to block PRC2 so that only one gene was affected.
