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  • Addiction, Drugs
  • Information from Lay-Language Summaries is Embargoed Until the Conclusion of the Scientific Presentation

    508—Learning and Memory: Epigenetics

    Tuesday, November 12, 2013, 8:00 am - 11:15 am

    508.13: Role of TET1 and 5-hydroxymethylcytosine in cocaine action

    Location: 5B

    *J. FENG1, K. E. SZULWACH2, V. VIALOU1, J. HUYNH1, N. SHAO1, T. LE3, D. FERGUSON1, J. KOO1, P. KENNEDY1, C. DIAS1, H. SONG4, P. CASACCIA1, G. FAN3, L. SHEN1, P. JIN2, E. NESTLER1;
    1neuroscience, Mount Sinai Sch. of Med., New York, NY; 2Dept. of Human Genet., Emory Univ. Sch. of Med., Atlanta, GA; 3Dept. of Human Genet., UCLA, Los Angeles, CA; 4Inst. for Cell Engin., Johns Hopkins Univ. Sch. of Med., Baltimore, MD

    Abstract Body: TET family proteins have been shown recently to oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which then further leads to unmethylated cytosine. This finding provides a novel mechanism of active DNA demethylation. Although 5hmC is most enriched in brain compared with other organs, the influence of 5hmC and TET on complex behavior remains unknown. Here, we first demonstrate that repeated cocaine administration decreased TET1 expression in nucleus accumbens (NAc), a key brain reward structure, in both mice and humans. Mouse behavioral assays after viral-mediated TET1 overexpression or knockdown in NAc indicated that TET1 negatively regulates behavioral responses to cocaine. Through genome-wide 5hmC capture and sequencing, we profiled cocaine-triggered global dynamics in 5hmC in NAc. Besides a robust regulation of 5hmC at distal enhancer regions, we identified major 5hmC changes within coding regions of specific genes. By superimposing such 5hmC changes on transcription changes determined by RNAseq, we found that repeated cocaine-triggered 5hmC alterations enrich both at genes that show steady state expression regulation after cocaine, as well as at genes poised for abnormal transcriptional induction in response to a subsequent cocaine challenge. In addition, these genes are highly clustered in neural functional groups with many known to have pivotal roles in drug addiction. We are now utilizing single base 5hmC sequencing to quantify 5hmC changes on several candidate genes in NAc. We have found that 5hmC alterations at certain genes last up to a month after repeated cocaine exposure. Though 5hmC is known as a transient epigenetic state between methylated and unmethylated cytosine, our findings support a role of 5hmC as a stable epigenetic mark in the brain. In summary, our study reveals a novel epigenetic mechanism of cocaine action, and provides fundamental insight into how 5hmC regulates neural transcription in vivo.

    Lay Language Summary: Our research reveals a novel mechanism of cocaine action. We found that a protein named TET1 opposes the behavioral effects of cocaine. Interestingly, we also recognized that a form of DNA methylation (5-hydroxymethylcytosine or 5hmC), which is catalyzed by the TET1 enzyme, is particularly enriched at genes involved in cocaine action. 5hmC is a recently identified and reversible regulator of gene expression, our study promises to promote the development of novel therapeutic interventions for cocaine addiction.
    Drug abuse and addiction are a major burden to society. It is estimated that 19.5 million people over the age of 12 use illegal drugs in the U.S. The death rate for drug addiction in the U.S. is 19,102 per year. It is estimated that the total cost of addictive disorders approaches half a trillion dollars annually in the U.S. However, the molecular mechanisms of drug addiction remain incompletely defined which hinders the progression of effective clinical therapies.
    Over the past decade, epigenetics has been shown to play pivotal roles in brain development, in normal brain function, and in several brain diseases, including drug addiction. Epigenetics refers to the modification of gene expression without changing the underlying DNA sequence. Epigenetics provides a sophisticated regulatory code to enable individuals to adapt to a dynamic environment. DNA methylation is a major epigenetic mechanism where a methyl group is added to the DNA base, cytosine, and has been shown to predominantly repress gene transcription. Recently, TET proteins have been shown to convert methylated cytosine into 5hmC. Although 5hmC is mostly enriched in the brain, the role of TET proteins and 5hmC in complex behavior as well as brain disease is still unknown.
    Here, we first found that chronic cocaine administration selectively decreased levels of TET1 expression in the nucleus accumbens (NAc), a brain structure important in addiction, in both cocaine-treated mice and human addicts. Other forms of TET were not affected. Mice with TET1 overexpression in NAc demonstrated less drug seeking behavior and TET1 knockdown mice craved drug more, which indicates that TET1 normally acts as a negative regulator of cocaine action.
    Moreover, through genome-wide 5hmC capture and sequencing, we globally profiled cocaine-triggered changes in 5hmC in NAc. We found robust regulation of 5hmC at distal enhancer regulatory regions, as well as within coding regions of specific genes. By superimposing such 5hmC changes on changes in gene expression as determined by RNA sequencing (RNA-seq), we found that cocaine-triggered 5hmC alterations are enriched at genes known to be important in neural function, with many known to have pivotal roles in drug addiction.
    We are now utilizing a novel oxidative bisulfite sequencing method to validate and quantify 5hmC changes with single base resolution on several candidate genes in NAc. We have found that 5hmC alterations at certain genes can be long lasting, which could contribute to the persistent aberrant behavior associated with drug addiction. As a result, these findings support a role of 5hmC as a stable epigenetic mark in the brain.
    In sum, our study reveals a novel epigenetic mechanism of cocaine action. As 5hmC is mostly enriched in brain compared with other organs, our study also provides fundamental insights that will benefit our exploration of this novel epigenetic mark in brain function and diseases. In the future, it will be intriguing to manipulate 5hmC at its regulating sites as exposed from this study to potentially affect drug behavior, thus to pave a new path to addiction therapy.