Demethylating proteins identified

Different cells in your body only need certain genes in your genetic code to be active, keeping certain genes silent through a process called methylation and activating them through a process called demethylation. Until recently, the mechanistic processes behind demethylation were unclear, and the components of this crucial action were not known.

Researchers at the Hopkins Medical School have discovered the proteins and processes involved in DNA demethylation, one of the processes essential for regulation of DNA expression. In an article published this month in Cell, the group, which consisted of members of the Institute for Cell Engineering at the medical school, detailed their work and results involving human kidney cells and rodent brain cells.

By controlling the expression of certain proteins in these cells, the group was able to isolate and determine the functions of specific proteins and map out their processes and how they fit together into the demethylation mechanism. One of these proteins, TET1, is a hydroxylase that specifically targets 5-methylcytosine (5mC), a specific methyl group on cytosine bases.

Through the addition of a hydroxyl group to these methylated cytosine residues, TET1 promotes processes catalyzed by other proteins that will ultimately lead to the demethylation of a stretch of DNA. TET1 hydroxylates 5mC bases to 5-hydroxymethylcytosine (5hmC), which is then acted upon by the proteins deaminase, glycosylase and BER.

Through their actions, 5hmC bases are converted to 5-hydroxymethyluradine (5hmU) by deaminase, which are then converted to cytosine bases. Thus, the demethylation of methylated DNA along cytosine residues is completed.

The group examined the demethylation process in mouse neurons using a process called electroconvulsive stimulation, which is where an electrical signal is used to stimulate activity in neurons.

“ECS is a method to activate neurons throughout the whole brain. In a previous study, we found that ECS could lead to demethylation of two neurotrophic factor genes,” Junjie Guo, a neuroscience graduate student at the Hopkins Medical School and lead author of the study, wrote in an email with The News-Letter. “In the current study, we asked whether TET1 could play a role mediating this process.”

In addition to using ECS to elicit a response in neurons, Guo and his colleagues relied on the overexpression and knock down of specific genes to determine what roles certain proteins played in the demethylation process. While overexpression involves a stimulation of the machinery in the cell to produce an abundance of the target protein, knock down involves inhibition of the expression of this protein to decrease the amount produced to the point where their functions can be observed.

Methylation of DNA blocks processes in the cell that read and express genes contained within specific stretches of DNA where the sequence encoded in the gene is used to produce a protein, or prevents other sequences that have regulatory roles from being read.

The demethylation process allows previously silenced DNA to become actively expressed again, which has important impacts in processes such as learning and memory as well as diseases such as cancer, neurological degeneration and psychiatric disorders. Although demethylation occurs in about five percent of human cells naturally, these events do have an important impact that is now understood better at the molecular level within the cell.

While methylation is a fairly robust way of silencing genes which are not used in certain cells once they are differentiated, demethylation is critical for precursor cells such as stem cells, which are able to differentiate into specific cells that comprise many different systems and tissues in the body.

With regards to research, Guo notes that an understanding of the demethylation mechanism has important implications in the development of stem cells for medicine. “For stem cell research, we also know dynamic regulations in the epigenome including the DNA methylome control cell differentiation/reprogramming. Knowing the mechanism means we can better control these processes,” he wrote. “In particular, hydroxymethylation is most abundant in pluripotent stem cells, and has been reported to play important roles in self-renewal and differentiation of stem cells.”