TET enzymes

Many different proteins bind to particular TET enzymes and recruit the TETs to specific genomic locations. In some studies, further analysis is needed to determine whether the interaction per se mediates the recruitment or instead the interacting partner helps to establish a favourable chromatin environment for TET binding. TET1‑depleted and TET2‑depleted cells revealed distinct target preferences of these two enzymes, with TET1‑preferring promoters and TET2‑preferring gene bodies of highly expressed genes and enhancers.[7]

The three mammalian

EGR1 is another example of a protein that recruits a TET enzyme.^[11] EGR1 has an important role in learning and memory.^[12][13] When a new event such as fear conditioning causes a memory to be formed, EGR1 messenger RNA is rapidly and selectively up-regulated in subsets of neurons in specific brain regions associated with learning and memory formation.^[14] TET1s is the predominant isoform of TET1 that is expressed in neurons.^[15] When EGR1 proteins are expressed, they appear to bring TET1s to about 600 sites in the neuron genome.^[11] Then EGR1 and TET1 appear to cooperate in demethylating and thereby activating the expression of genes downstream of the EGR1 binding sites in DNA.^[11]

TET processivity

TET processivity can be viewed at three levels, the physical, chemical and genetic levels. Physical processivity refers to the ability of a TET protein to slide along the DNA from one CpG site to another. An in vitro study showed that DNA-bound TET does not preferentially oxidize other CpG sites on the same DNA molecule, indicating that TET is not physically processive. Chemical processivity refers to the ability of TET to catalyze the oxidation of 5mC iteratively to 5caC without releasing its substrate. It appears that TET can work through both chemically processive and non‑processive mechanisms depending on reaction conditions. Genetic processivity refers to the genetic outcome of TET‑mediated oxidation in the genome, as shown by mapping of the oxidized bases. In mouse embryonic stem cells, many genomic regions or CpG sites are modified so that 5mC is changed to 5hmC but not to 5fC or 5caC, whereas at many otherCpG sites 5mCs are modified to 5fC or 5caC but not 5hmC, suggesting that 5mC is processed to different states at different genomic regions or CpG sites.^[7]

TET enzyme activity

TET enzymes are

The first step involves the binding of α-KG and 5-methylcytosine to the TET enzyme active site. The TET enzymes each harbor a core catalytic domain with a double-stranded β-helix fold that contains the crucial metal-binding residues found in the family of Fe(II)/α-KG- dependent oxygenases.

The TET proteins also have activities that are independent of DNA demethylation.[17] These include, for instance, TET2 interaction with O-linked N-acetylglucosamine (O-GlcNAc) transferase to promote histone O-GlcN acylation to affect transcription of target genes.^[18]

TET functions

Early embryogenesis

The mouse

In addition, from embryo day 9.5 to 13.5 there is an active form of demethylation. As indicated in the Figure of the demethylation pathway above, two enzymes are central to active demethylation. These are a ten-eleven translocation (TET) methylcytosine dioxygenase and thymine-DNA glycosylase (TDG). One particular TET enzyme, TET1, and TDG are present at high levels from embryo day 9.5 to 13.5,^[22] and are employed in active TET-dependent demethylation during gametogenesis.^[21] PGC genomes display the lowest levels of DNA methylation of any cells in the entire life cycle of the mouse by embryonic day 13.5. ^[23]

Learning and memory

Learning and memory have levels of permanence, differing from other mental processes such as thought, language, and consciousness, which are temporary in nature. Learning and memory can be either accumulated slowly (multiplication tables) or rapidly (touching a hot stove), but once attained, can be recalled into conscious use for a long time. Rats subjected to one instance of contextual fear conditioning create an especially strong long-term memory. At 24 hours after training, 9.17% of the genes in the genomes of rat hippocampus neurons were found to be differentially methylated. This included more than 2,000 differentially methylated genes at 24 hours after training, with over 500 genes being demethylated.^[24] Similar results to that in the rat hippocampus were also obtained in mice with contextual fear conditioning.^[25]

The hippocampus region of the brain is where contextual fear memories are first stored (see Figure), but this storage is transient and does not remain in the hippocampus. In rats contextual fear conditioning is abolished when the hippocampus is subjected to hippocampectomy just one day after conditioning, but rats retain a considerable amount of contextual fear when hippocampectomy is delayed by four weeks.^[26] In mice, examined at 4 weeks after conditioning, the hippocampus methylations and demethylations were reversed (the hippocampus is needed to form memories but memories are not stored there) while substantial differential CpG methylation and demethylation occurred in cortical neurons during memory maintenance. There were 1,223 differentially methylated genes in the anterior cingulate cortex (see Figure) of mice four weeks after contextual fear conditioning. Thus, while there were many methylations in the hippocampus shortly after memory was formed, all these hippocampus methylations were demethylated as soon as four weeks later.

Li et al.^[27] reported one example of the relationship between expression of a TET protein, demethylation and memory while using extinction training. Extinction training is the disappearance of a previously learned behavior when the behavior is not reinforced.

A comparison between

A short hairpin RNA (shRNA) is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference. Mice trained in the presence of TET3-targeted shRNA showed a significant impairment in fear extinction memory.^[27]

Addiction

The nucleus accumbens (NAc) has a significant role in addiction. In the nucleus accumbens of mice, repeated cocaine exposure resulted in reduced TET1 messenger RNA (mRNA) and reduced TET1 protein expression. Similarly, there was a ~40% decrease in TET1 mRNA in the NAc of human cocaine addicts examined postmortem.^[28]

As indicated above in learning and memory, a short hairpin RNA (shRNA) is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference. Feng et al.^[28] injected shRNA targeted to TET1 in the NAc of mice. This could reduce TET1 expression in the same manner as reduction of TET1 expression with cocaine exposure. They then used an indirect measure of addiction, conditioned place preference. Conditioned place preference can measure the amount of time an animal spends in an area that has been associated with cocaine exposure, and this can indicate an addiction to cocaine. Reduced Tet1 expression caused by shRNA injected into the NAc robustly enhanced cocaine place conditioning.

Pain (nociception)

As described in the article Nociception, nociception is the sensory nervous system's response to harmful stimuli, such as a toxic chemical applied to a tissue. In nociception, chemical stimulation of sensory nerve cells called nociceptors produces a signal that travels along a chain of nerve fibers via the spinal cord to the brain. Nociception triggers a variety of physiological and behavioral responses and usually results in a subjective experience, or perception, of pain.

Work by Pan et al.^[3] first showed that TET1 and TET3 proteins are normally present in the spinal cords of mice. They used a pain inducing model of intra plantar injection of 5% formalin into the dorsal surface of the mouse hindpaw and measured time of licking of the hindpaw as a measure of induced pain. Protein expression of TET1 and TET3 increased by 152% and 160%, respectively, by 2 hours after formalin injection. Forced reduction of expression of TET1 or TET3 by spinal injection of Tet1-siRNA or Tet3-siRNA for three consecutive days before formalin injection alleviated the mouse perception of pain. On the other hand, forced overexpression of TET1 or TET3 for 2 consecutive days significantly produced pain-like behavior as evidenced by a decrease in the mouse of the thermal pain threshold.

They further showed that the nociceptive pain effects occurred through TET mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine in the promoter of a microRNA designated miR-365-3p, thus increasing its expression. This microRNA, in turn, ordinarily targets (decreases expression of) the messenger RNA of Kcnh2, that codes for a protein known as K_v11.1 or KCNH2. KCNH2 is the alpha subunit of a potassium ion channel in the central nervous system. Forced decrease in expression of TET1 or TET3 through pre-injection of siRNA reversed the decrease of KCNH2 protein in formalin-treated mice.

References