Norepinephrine transporter gene silencing by MeCP2 in postural tachycardia syndrome

Abstract

The Postural Tachycardia Syndrome (POTS) is characterized by the clinical symptoms of orthostatic intolerance, light-headedness, tachycardia and syncope or near syncope with assumption of upright posture. While the aetiology remains largely unknown, faulty neuronal reuptake of the sympathetic nervous system signaling neurotransmitter has been implicated. The action of norepinephrine (NE) is terminated, in part, by its uptake into presynaptic noradrenergic neurons by the plasma-membrane NE transporter (NET) which is encoded by the SLC6A2 gene. The effectiveness of NE reuptake relies on the capacity of NET to tightly recapture NE released by sympathetic nerves, this being approximately 90% for the heart. Since inter-individual variation in NET function is likely to be common and, in light of the clinical importance of the transporter’s central role in NE regulation, rather than querying genetic variation, we investigated gene-environment interactions to determine which biological processes may be prime targets for SLC6A2 regulation.

Experimental evidence from our group has previously shown chromatin-modifying events associated with SLC6A2 repression may augment epigenetic regulation in POTS. SLC6A2 repression is associated with substitution of active histone modifications with repressive modifications and binding of Methyl CpG binding protein-2 (MeCP2) co-repressor complex. MeCP2 is commonly assumed to function mainly as a silencing factor at methylated DNA sequences. Interestingly, DNA methylation is unremarkable between the case and control cohort. In context to these findings and given that non-coding RNAs (ncRNAs) interactions can induce structural changes to chromatin to regulate transcription, we hypothesized that MeCP2 binding is dependent on its interaction with ncRNAs. The purpose of this study was to investigate SLC6A2 silencing with the specific aim to understand epigenomic regulation as a mean to reactivate expression.

We developed a novel RICh-seq (RNA of Isolated Chromatin combined with sequencing) method to identify SLC6A2 promoter bound RNAs. Analysis of RICh-seq data identified let-7i associated with SLC6A2 promoter with elevated expression in POTS patients. We show that let-7i miRNA interacts with MeCP2 co-repressor to silence SLC6A2 activity in POTS subjects. Furthermore, we demonstrate that in POTS subject’s histone modifying drugs such as histone deacetylase inhibitors and inhibitor for EZH2 (Enhancer of zeste homologue 2) restores epigenetic modifications associated with SLC6A2 gene expression. Our results represent the first pre-clinical description for the gain of NET function in POTS and a previously unknown target of pharmacological therapy.