WEST LAFAYETTE, Ind. – Purdue University and Indiana University School of Medicine scientists were able to force an epigenetic reaction that turns on and off a gene known to determine the fate of the neural stem cells, a finding that could lead to new therapeutics in the fight against select cancers and neural diseases.
Joseph Irudayaraj, a Purdue professor of agricultural and biological engineering, and Feng Zhou, a professor and neuroscientist at the Indiana University School of Medicine, have developed an optogenetic toolbox that brings together proteins and enzymes that methylate or demethylate a gene called Ascl1. Alteration of the methylation pattern in a specific gene with the optogenetic proteins would allow scientists to turn that gene on or off and produce desirable neurons among other cell types.
“If we can alter the epigenetic state at a specific location of a gene, then we can turn that gene on or off for personalized medicine,” Irudayaraj said.
The findings, published in the journal Nature Scientific Reports, have implications for a number of diseases and maladies.
“By the ability of determining the fate of stem cells, one day it may be applied to produce neurons in Down syndrome, or reduce malignancy of glioma, a cancer in the brain,” Zhou said. “By altering the methylation marks at a specific location of the gene, we have shown that the state of a cell can be altered.”
Epigenetics is the study of changes in chemical modifications on top of a gene based on external or environmental factors rather than changes in a DNA sequence. Optogenetics involves the utilization of light-sensitive proteins to alter the genetic or epigenetic profile in a cell or organism.
The researchers’ findings detail the ability to modify the methylation profile of the Ascl1 gene in a site-specific manner, thereby controlling gene expression. DNA methylation involves adding a methyl group to the cytosine base of DNA, utilizing a family of enzymes called DNA methyltransferases (DNMTs). DNA demethylation is the removal of a methyl group from the cytosine bases using enzymes called Ten-Eleven Translocation, or TET.
Irudayaraj and his team attached these cytosine-modifying enzymes DNMT3A/TET to light-sensitive protein pairs to demonstrate site-specific methylation/demethylation. Zhou and his team introduced those light-sensitive proteins into neural stem cells and found that when they shined a blue light, the methylation modifying enzyme DNMT3A/TET and the gene target came together, adjusting the methylation of the gene.
“It’s almost like putting a worm on a hook, and putting it in the water to catch a fish when it comes along. Once the light goes on, the hook and the fish come together and you catch the fish,” Irudayaraj said.
The ability to activate or deactivate a gene, specifically those that suppress or promote a disease condition, could be a valuable tool for cancer therapeutics as well. The team plans to take the findings, done on neural stem cells, to mouse model systems.
“We want to apply this to therapeutics or toxicology,” Irudayaraj said. “Essentially the applications are very broad. It can also include nervous system malfunctions, including addiction.”
The W.M. Keck Foundation and the National Institutes of Health funded the research.
Writer: Brian Wallheimer, 765-532-0233, firstname.lastname@example.org
Epigenetic Editing of Ascl1 Gene in Neural Stem Cells by Optogenetics
Chiao-Ling Lo1, Samrat Roy Choudhury2, Joseph Irudayaraj2 & Feng C. Zhou1,3
1 Department of Anatomy & Cell Biology, Indiana University School of Medicine.
2 Bindley Bioscience Center, Department of Agricultural & Biological Engineering, Purdue University.
3 Stark Institute of Neuroscience Research, Indiana University School of Medicine.
Enzymes involved in epigenetic processes such as methyltransferases or demethylases are becoming highly utilized for their persistent DNA or histone modifying efficacy. Herein, we have developed an optogenetic toolbox fused to the catalytic domain (CD) of DNA-methyltransferase3A (DNMT3A-CD) or Ten-Eleven Dioxygenase-1 (TET1-CD) for loci-specific alteration of the methylation state at the promoter of Ascl1 (Mash1), a candidate proneuron gene. Optogenetic protein pairs, CRY2 and CIB1 were fused to the Ascl1 promoter specific Transcription Activator-Like Element (TALE) DNMT3A-CD or TET1-CD, respectively. A differentially methylated region at the Ascl1 promoter, isolated from murine dorsal root ganglion (hypermethylated) and striated cells (hypomethylated), was targeted with these optogenetic-epigenetic constructs. Optimized blue-light illumination triggered the co-localization of TALE constructs with DNMT3A-CD or TET1-CD fusion proteins at the targeted site of the Ascl1 promoter. We found that this spatiotemporal association of the fusion proteins selectively alters the methylation state and also regulates gene activity. This proof of concept developed herein holds immense promise for the ability to regulate gene activity via epigenetic modulation with spatiotemporal precision.