Research Highlights
Highlights from the latest articles in epigenomics
Regulation of memory acquisition and extinction by TET1 Evaluation of: Kaas GA, Zhong C, Eason DE et al. TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation. Neuron 79, 1086–1093 (2013); Rudenko A, Dawlaty MM, Seo J et al. Tet1 is critical for neuronal activity-regulated gene expression and memory extinction. Neuron 79, 1109–1122 (2013).
Accumulating evidence indicates that altered gene expression in response to changes in the environment underlies the plasticity in the brain, including learning and memory. Epigenetic modifications, such as DNA methylation and histone acetylation, have been shown to critically regulate gene transcription, providing a candidate mechanism for synaptic and experience-dependent behavioral plasticity. The dynamic changes of epigenetic modifications (e.g., histone acetylation and deacetylation) play key roles in memory formation and extinction [1] . Generation of methylated cytosine (5mC) in DNA was once considered as a stable modification to silence gene transcription. However, recent studies indicated that DNA methylation may undergo rapid changes in vivo. The teneleven translocation (TET) family enzymes, TET1, TET2 and TET3, catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and thus participate in active DNA demethylation [2 ,3] . Guo et al. found that in response to neuronal activation, TET1 mediates demethylation of the Fgf1 and the Bdnf promoters in the brain [4] . However, the potential role of TET1 as well as TET1mediated DNA demethylation in memory formation is not clear.
10.2217/EPI.13.85 © 2014 Future Medicine Ltd
Recent work by Kaas et al. revealed that TET1 exerts function in active DNA demethylation, gene transcription and memory formation [5] . They showed that TET1 is expressed in neurons throughout the hippocampus in the brain and stimulation of neuronal activity by KCl or seizure downregulates its transcription in hippocampal neurons. Overexpression of TET1 in the dorsal hippocampus result in a significant decrease in global level of 5mC and an increase of 5hmC, indicating that TET1 effectively promotes DNA demethylation by converting 5mC to 5hmC. In parallel, upregulation of activity-dependent genes involved in neuroplasticity, such as Fos, Arc, Egr1, Homer1 and Ner4a2 was also detected in the hippocampus overexpressing TET1. Moreover, Kaas et al. investigated the behavioral effects of TET1. Overexpression of TET1 in the dorsal hippocampus does not affect locomotion and anxietylike behavior, but significantly impairs long-term conditioned fear memory. Interestingly, the expression of a catalytically inactive mutant of TET1 also affected gene expression and memory formation, raising the question that if TET1 regulates gene expression and memory via both hydroxylase activity-dependent and hydroxylase activity-independent mechanisms. Earlier research by Zhang and colleagues showed that Tet1-knockout mice exhibit impaired hippocampal neurogenesis and hippocampus-dependent spatial memory [6] . Rudenko et al. recently examined another stain of Tet1-knockout mice generated by depleting exon 4 [7] . They found that ablation of TET1 does not change the level of 5mC but leads to a reduction of
Epigenomics (2014) 6(1), 17–19
Biao Yan1 & Lan Ma*,1 Pharmacology Research Center & the State Key Laboratory of Medical Neurobiology, School of Basic Medical Sciences & Institutes of Brain Science, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China *Author for correspondence: Tel.: +86 21 5423 7522 Fax: +86 21 5423 7643 [email protected] 1
part of
ISSN 1750-1911
17
News & Views Research Highlights 5hmC level in the cortex and hippocampus. Knockout of TET1 does not affect locomotion, anxiety, depression-related behaviors and the acquisition of hippocampus-dependent spatial memory, and conditioned fear memory as determined one day after training. However, the TET1-deficient mice display an abnormal increase of hippocampal long-term depression, and significantly reduced memory extinction in the Morris water maze and fear conditioning tests. Consistent with the observations of Kaas et al. [5] , Rudenko and colleagues revealed that TET1 depletion causes downregulaton of neuronal activityregulated genes, including Npas4, c-Fos, Egr2, Egr4, and Arc in naive mice, and similar results were also obtained after extinction training [7] . Rudenko et al. further showed that Npas4 promoter-exon1 region is hypermethylated and the expression of Npas4 and c-Fos is reduced in the cortex and hippocampus in naive TET1-deficient mice and those performed memory extinction task. Moreover, memory extinction training results in a significant induction of Npas4 and c-Fos expression in the wild-type but not TET1-deficient mice. Taken together, studies by Kaas et al. [5] and Rudenko et al. [7] revealed that TET1 plays an important role in regulation of neuronal activitystimulated DNA demethylation, gene expression, and memory formation and extinction. These studies implicate that TET1 may serve as a potential therapeutic target for the treatment of memory-related disorders, such as posttraumatic stress disorder and drug addiction. TALE-TET1: a powerful tool for site-specific DNA demethylation in the target gene Evaluation of: Maeder ML, Angstman JF, Richardson ME et al. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat. Biotechnol. 31(12), 1137–1142 (2013).
Extensive methylation of cytosine in DNA is correlated with reduced transcription and the dynamics of methylation and demethylation of particular genes play an important role in the individual development, health, and disease [8] . In mammalian cells, DNA methylation is catalyzed by methyltransferases primarily at CpG dinucleotides. It has been demonstrated recently that the TET family of proteins, enzymes that convert 5mC to 5hmC, play a key role in demethylation of DNA. The genetic or pharmacological manipulation of global level of DNA methylation in cell has been used in many studies to demonstrate the role of DNA methylation in regulation of gene expression and cell biology. However, due to
18
Epigenomics (2014) 6(1)
the lack of methylation site-directed methods, it was difficult to determine the causal effects of methylation of specific CpG sites at a particular gene. Zhang et al. reported that TALE proteins secreted by a Xanthomonas bacterium can be engineered to recognize specific DNA sequences [9] . A study by Konermann et al. integrated the customizable TALE DNA-binding domain with a light-sensitive protein and its interacting partner from Arabidopsis to control endogenous histone modification at specific gene promoter, which enables the instantaneous induction of expression of target gene in vivo with light [10] . Recently, Maeder and colleagues developed a method to modulate the methylation status at specific CpG island, introducing a novel way to study the effects of DNA methylation at a particular gene [11] . Maeder et al. fused the TET1 catalytic domain with TALE repeat arrays that had been modified to bind human KLF4 gene, and demonstrated that the expression of TALE-TET1 fusion protein in HeLa cells could significantly decrease the methylation level of targeted KLF4 CpGs. Furthermore, eleven TALE-TET1 proteins targeting to different CpGs of RHOXF2/2B homeobox gene (RHOXF2) were constructed and tested. Two of the constructs that demethylate CpGs in the region most proximal to the RHOXF2 promoter induced high levels of mRNA expression in both the HEK293 and HeLa cells, demonstrating that the DNA methylation of specific CpGs in RHOXF2 promoter regulates RHOXF2 expression. In addition, Maeder and colleagues constructed ten TALE-TET1 fusion proteins to target various sites at human beta-globin gene promoter, and found that although all of them could reduce the methylation level of CpGs near their targets in K562 cells, only four significantly increased HBB mRNA level, suggesting that demethylation of particular methylated CpGs is critical for the activation of HBB expression. This work by Maeder et al. provides a new strategy for the investigation of the functional significance of specific CpG methylation in the context of endogenous gene loci and the validation of programmable DNA demethylation reagents with potential utility for research and therapeutic applications. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
future science group
Research Highlights
References 1
2
3
4
5
Peixoto L, Abel T. The role of histone acetylation in memory formation and cognitive impairments. Neuropsychopharmacology 38, 62–76 (2013). Ito S, Shen L, Dai Q et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011). He YF, Li BZ, Li Z et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307 (2011). Guo JU, Su Y, Zhong C et al. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145, 423–434 (2011). Kaas GA, Zhong C, Eason DE et al. TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation. Neuron 79, 1086–1093 (2013).
future science group
6
Zhang RR, Cui QY, Murai K et al. Tet1 regulates adult hippocampal neurogenesis and cognition. Cell Stem Cell 13, 237–245 (2013).
7
Rudenko A, Dawlaty MM, Seo J et al. Tet1 is critical for neuronal activity-regulated gene expression and memory extinction. Neuron 79, 1109–1122 (2013).
8
Smith ZD, Meissner A. DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14, 204–220 (2013).
9
Zhang F, Cong L, Lodato S et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).
10
Konermann S, Brigham MD, Trevino AE et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).
11
Maeder ML, Angstman JF, Richardson ME et al. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat. Biotechnol. 31(12), 1137–1142 (2013).
www.futuremedicine.com
News & Views
19
Highlights from the latest articles in epigenomics
Regulation of memory acquisition and extinction by TET1 Evaluation of: Kaas GA, Zhong C, Eason DE et al. TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation. Neuron 79, 1086–1093 (2013); Rudenko A, Dawlaty MM, Seo J et al. Tet1 is critical for neuronal activity-regulated gene expression and memory extinction. Neuron 79, 1109–1122 (2013).
Accumulating evidence indicates that altered gene expression in response to changes in the environment underlies the plasticity in the brain, including learning and memory. Epigenetic modifications, such as DNA methylation and histone acetylation, have been shown to critically regulate gene transcription, providing a candidate mechanism for synaptic and experience-dependent behavioral plasticity. The dynamic changes of epigenetic modifications (e.g., histone acetylation and deacetylation) play key roles in memory formation and extinction [1] . Generation of methylated cytosine (5mC) in DNA was once considered as a stable modification to silence gene transcription. However, recent studies indicated that DNA methylation may undergo rapid changes in vivo. The teneleven translocation (TET) family enzymes, TET1, TET2 and TET3, catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and thus participate in active DNA demethylation [2 ,3] . Guo et al. found that in response to neuronal activation, TET1 mediates demethylation of the Fgf1 and the Bdnf promoters in the brain [4] . However, the potential role of TET1 as well as TET1mediated DNA demethylation in memory formation is not clear.
10.2217/EPI.13.85 © 2014 Future Medicine Ltd
Recent work by Kaas et al. revealed that TET1 exerts function in active DNA demethylation, gene transcription and memory formation [5] . They showed that TET1 is expressed in neurons throughout the hippocampus in the brain and stimulation of neuronal activity by KCl or seizure downregulates its transcription in hippocampal neurons. Overexpression of TET1 in the dorsal hippocampus result in a significant decrease in global level of 5mC and an increase of 5hmC, indicating that TET1 effectively promotes DNA demethylation by converting 5mC to 5hmC. In parallel, upregulation of activity-dependent genes involved in neuroplasticity, such as Fos, Arc, Egr1, Homer1 and Ner4a2 was also detected in the hippocampus overexpressing TET1. Moreover, Kaas et al. investigated the behavioral effects of TET1. Overexpression of TET1 in the dorsal hippocampus does not affect locomotion and anxietylike behavior, but significantly impairs long-term conditioned fear memory. Interestingly, the expression of a catalytically inactive mutant of TET1 also affected gene expression and memory formation, raising the question that if TET1 regulates gene expression and memory via both hydroxylase activity-dependent and hydroxylase activity-independent mechanisms. Earlier research by Zhang and colleagues showed that Tet1-knockout mice exhibit impaired hippocampal neurogenesis and hippocampus-dependent spatial memory [6] . Rudenko et al. recently examined another stain of Tet1-knockout mice generated by depleting exon 4 [7] . They found that ablation of TET1 does not change the level of 5mC but leads to a reduction of
Epigenomics (2014) 6(1), 17–19
Biao Yan1 & Lan Ma*,1 Pharmacology Research Center & the State Key Laboratory of Medical Neurobiology, School of Basic Medical Sciences & Institutes of Brain Science, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China *Author for correspondence: Tel.: +86 21 5423 7522 Fax: +86 21 5423 7643 [email protected] 1
part of
ISSN 1750-1911
17
News & Views Research Highlights 5hmC level in the cortex and hippocampus. Knockout of TET1 does not affect locomotion, anxiety, depression-related behaviors and the acquisition of hippocampus-dependent spatial memory, and conditioned fear memory as determined one day after training. However, the TET1-deficient mice display an abnormal increase of hippocampal long-term depression, and significantly reduced memory extinction in the Morris water maze and fear conditioning tests. Consistent with the observations of Kaas et al. [5] , Rudenko and colleagues revealed that TET1 depletion causes downregulaton of neuronal activityregulated genes, including Npas4, c-Fos, Egr2, Egr4, and Arc in naive mice, and similar results were also obtained after extinction training [7] . Rudenko et al. further showed that Npas4 promoter-exon1 region is hypermethylated and the expression of Npas4 and c-Fos is reduced in the cortex and hippocampus in naive TET1-deficient mice and those performed memory extinction task. Moreover, memory extinction training results in a significant induction of Npas4 and c-Fos expression in the wild-type but not TET1-deficient mice. Taken together, studies by Kaas et al. [5] and Rudenko et al. [7] revealed that TET1 plays an important role in regulation of neuronal activitystimulated DNA demethylation, gene expression, and memory formation and extinction. These studies implicate that TET1 may serve as a potential therapeutic target for the treatment of memory-related disorders, such as posttraumatic stress disorder and drug addiction. TALE-TET1: a powerful tool for site-specific DNA demethylation in the target gene Evaluation of: Maeder ML, Angstman JF, Richardson ME et al. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat. Biotechnol. 31(12), 1137–1142 (2013).
Extensive methylation of cytosine in DNA is correlated with reduced transcription and the dynamics of methylation and demethylation of particular genes play an important role in the individual development, health, and disease [8] . In mammalian cells, DNA methylation is catalyzed by methyltransferases primarily at CpG dinucleotides. It has been demonstrated recently that the TET family of proteins, enzymes that convert 5mC to 5hmC, play a key role in demethylation of DNA. The genetic or pharmacological manipulation of global level of DNA methylation in cell has been used in many studies to demonstrate the role of DNA methylation in regulation of gene expression and cell biology. However, due to
18
Epigenomics (2014) 6(1)
the lack of methylation site-directed methods, it was difficult to determine the causal effects of methylation of specific CpG sites at a particular gene. Zhang et al. reported that TALE proteins secreted by a Xanthomonas bacterium can be engineered to recognize specific DNA sequences [9] . A study by Konermann et al. integrated the customizable TALE DNA-binding domain with a light-sensitive protein and its interacting partner from Arabidopsis to control endogenous histone modification at specific gene promoter, which enables the instantaneous induction of expression of target gene in vivo with light [10] . Recently, Maeder and colleagues developed a method to modulate the methylation status at specific CpG island, introducing a novel way to study the effects of DNA methylation at a particular gene [11] . Maeder et al. fused the TET1 catalytic domain with TALE repeat arrays that had been modified to bind human KLF4 gene, and demonstrated that the expression of TALE-TET1 fusion protein in HeLa cells could significantly decrease the methylation level of targeted KLF4 CpGs. Furthermore, eleven TALE-TET1 proteins targeting to different CpGs of RHOXF2/2B homeobox gene (RHOXF2) were constructed and tested. Two of the constructs that demethylate CpGs in the region most proximal to the RHOXF2 promoter induced high levels of mRNA expression in both the HEK293 and HeLa cells, demonstrating that the DNA methylation of specific CpGs in RHOXF2 promoter regulates RHOXF2 expression. In addition, Maeder and colleagues constructed ten TALE-TET1 fusion proteins to target various sites at human beta-globin gene promoter, and found that although all of them could reduce the methylation level of CpGs near their targets in K562 cells, only four significantly increased HBB mRNA level, suggesting that demethylation of particular methylated CpGs is critical for the activation of HBB expression. This work by Maeder et al. provides a new strategy for the investigation of the functional significance of specific CpG methylation in the context of endogenous gene loci and the validation of programmable DNA demethylation reagents with potential utility for research and therapeutic applications. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
future science group
Research Highlights
References 1
2
3
4
5
Peixoto L, Abel T. The role of histone acetylation in memory formation and cognitive impairments. Neuropsychopharmacology 38, 62–76 (2013). Ito S, Shen L, Dai Q et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011). He YF, Li BZ, Li Z et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307 (2011). Guo JU, Su Y, Zhong C et al. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145, 423–434 (2011). Kaas GA, Zhong C, Eason DE et al. TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation. Neuron 79, 1086–1093 (2013).
future science group
6
Zhang RR, Cui QY, Murai K et al. Tet1 regulates adult hippocampal neurogenesis and cognition. Cell Stem Cell 13, 237–245 (2013).
7
Rudenko A, Dawlaty MM, Seo J et al. Tet1 is critical for neuronal activity-regulated gene expression and memory extinction. Neuron 79, 1109–1122 (2013).
8
Smith ZD, Meissner A. DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14, 204–220 (2013).
9
Zhang F, Cong L, Lodato S et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).
10
Konermann S, Brigham MD, Trevino AE et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).
11
Maeder ML, Angstman JF, Richardson ME et al. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat. Biotechnol. 31(12), 1137–1142 (2013).
www.futuremedicine.com
News & Views
19
