Date of Award

January 2015

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Jeffrey Bender

Subject Area(s)

Molecular biology

Abstract

IDENTIFICATION OF A LONG NON-CODING RNA REGULATING CHOLESTEROL METABOLISM. Thomas Candler Gilliland, Tamer Sallam, Marius Jones, and Peter Tontonoz. Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA. (Sponsored by Jeffrey Bender, Division of Cardiology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT.)

The liver X receptors (LXRs) are nuclear receptors that regulate sterol homeostasis and inflammation. Long non-coding RNA (lncRNA) is a class of non-coding transcript that has recently been shown to have important functions in numerous settings, frequently through epigenetic modifications that alter target gene transcription. However, their potential function in sterol metabolism has not been investigated. We hypothesized that epigenetic mechanisms underlie some degree of the physiologic regulation of sterol biosynthesis and identified an LXR-regulated lncRNA with a role in cellular sterol homeostasis. To screen for novel LXR-regulated lncRNAs, we treated mouse primary hepatocytes with or without the synthetic LXR agonist GW3965. RNA expression was analyzed by either RNA-seq or cDNA array; analysis enriched for transcripts greater than two-fold induced by treatment. Both screens identified a Liver-Expressed, LXR-Induced Sequence (LEXIS) as the most highly induced transcript (>35-fold induction). Regulation of LEXIS by LXR in mouse primary hepatocytes was confirmed by quantitative PCR. LEXIS was transcribed as multiple isoforms and independently of the nearby gene ABCA1. A predominant isoform was identified using 5' and 3' rapid amplification of cDNA ends. CHIP-seq analysis revealed binding sites the for LXR/retinoid X receptor (RXR) heterodimer within the LEXIS promoter region. Additionally, LEXIS was regulated by LXR in vivo, as treating C57BL/6 mice with LXR agonist by oral gavage resulted in LEXIS induction in many tissues. To test the physiologic relevance of hepatic LEXIS in sterol homeostasis, adenovirus designed to overexpress LEXIS or GFP was injected by tail vein into 12-week old C57BL/6 mice (n = 6-8 per group) maintained on chow diet. Cholesterol and triglyceride levels were measured on day six, and hepatic gene expression was analyzed by quantitative PCR and micro-array. Overexpression of the predominant LEXIS isoform resulted in a marked reduction in circulating cholesterol. Gene expression analysis revealed that LEXIS overexpression suppressed cholesterol biosynthesis pathway genes downstream of, and including, sterol regulatory element binding protein 2 (SREBP2). A reciprocal in vivo experiment using adenoviral shRNA-mediated LEXIS knockdown in liver led to a significant increase in circulating cholesterol. LXR gene products drive the cellular response to cholesterol overload. In contrast, limiting sterol conditions lead to activation of SREBP2 and its downstream targets to increase cholesterol biosynthesis. Here we show that the LXR-dependent lncRNA LEXIS mediates crosstalk between these pathways by downregulating cholesterol biosynthesis when LXR is activated, perhaps by altering SREBP2 transcription or processing. Further work will seek to characterize the precise mechanism by which LEXIS affects SREBP2 and cholesterol biosynthesis.

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