NET silencing by let-7i in postural tachycardia syndrome
Abdul Waheed Khan, … , Murray D. Esler, Assam El-Osta
Abdul Waheed Khan, … , Murray D. Esler, Assam El-Osta
Published March 23, 2017
Citation Information: JCI Insight. 2017;2(6):e90183. https://doi.org/10.1172/jci.insight.90183.
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Categories: Research Article Cardiology

NET silencing by let-7i in postural tachycardia syndrome

Abstract

While strongly implicated in postural tachycardia syndrome (POTS), considerable controversy exists regarding norepinephrine transporter (NET) loss of function. POTS is characterized by the clinical symptoms of orthostatic intolerance, lightheadedness, tachycardia, and syncope or near syncope with upright posture. Abnormal sympathetic nervous system activity is typical, of a type which suggests dysfunction of the NET, with evidence that the gene responsible is under tight epigenetic control. Using RNA of isolated chromatin combined with massive parallel sequencing (RICh-seq) we show that let-7i miRNA suppresses NET by methyl-CpG-binding protein 2 (MeCP2). Vorinostat restores epigenetic control and NET expression in leukocytes derived from POTS participants.

Authors

Abdul Waheed Khan, Mark Ziemann, Susan J. Corcoran, Harikrishnan K.N, Jun Okabe, Haloom Rafehi, Scott S. Maxwell, Murray D. Esler, Assam El-Osta

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Figure 4

RNA-dependent MeCP2 binding at the NET promoter.

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RNA-dependent MeCP2 binding at the NET promoter. (A) Methyl-CpG-binding ...
(A) Methyl-CpG-binding protein 2 (MECP2) shRNA knockdown in HeLa cells (n = 3). Relative expression was measured against ACTB using quantitative reverse transcription PCR (qRT-PCR). (B) Norepinephrine transporter (NET) expression in MECP2-depleted cells (n = 3). Relative expression was measured against ACTB using qRT-PCR. (C) ChIP assay using an anti-MeCP2 antibody indicates binding at the NET promoter relative to IgG antibody (n = 3). (D) Schema of MeCP2 ChIP protocol followed by RNase treatment (ChIP-RNase) and detection of NET by qPCR. Briefly, HeLa cells were crosslinked (1% formaldehyde) and soluble chromatin prepared by sonication. Anti-MeCP2 antibody was used to perform ChIP. Immunopurified complexes were treated with RNase A and unbound chromatin removed by washing. Remaining DNA was purified and NET detected using qPCR. (E) ChIP-RNase assay shows that MeCP2 binding on the NET promoter is RNA dependent (n = 3). (F) ChIP-RNase assay shows that MeCP2 binding at the IL6 promoter is not RNA dependent (n = 3). Data are the mean ± SD. **P ≤ 0.01 by Student’s t test.