Publication Date

2015-12-14

Availability

Open access

Embargo Period

2015-12-14

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2015-12-01

First Committee Member

Claes R. Wahlestedt

Second Committee Member

Mohammad A. Faghihi

Third Committee Member

Peter Buchwald

Fourth Committee Member

Arun Malhotra

Fifth Committee Member

Vladlen Z. Slepak

Sixth Committee Member

Sapna Deo

Abstract

Long noncoding RNAs (lncRNAs) regulate chromatin remodeling through their interactions with epigenetic enzymes during development and disease. The inhibition of the natural antisense transcript of Brain-derived neurotrophic factor (BDNF-AS), results in BDNF promoter de-repression and transcriptional upregulation, both in vitro and in vivo. Recently, we showed that BDNF-AS interacts with the histone methyltransferase enhancer of zeste homolog 2 (EZH2) to suppress BDNF mRNA and protein expression. BDNF is an important neurotrophin that is required for neural development and maintenance of the nervous system. Dysregulation of BDNF occurs in a number of neurological disorders, including: Alzheimer’s Disease, Parkinson’s Disease, Rett syndrome, and amyotrophic lateral sclerosis. Previous attempts to upregulate BDNF by administering the recombinant form in various parts of the central nervous system have failed, mostly due to the challenge of delivering BDNF to the correct cells and neural networks. Our approach to upregulating BDNF by modulating its interaction with an epigenetic enzyme is a highly specific target with potential therapeutic value. To achieve this, we developed a novel pharmacological assay to characterize the interaction between long noncoding RNAs and their epigenetic targets using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) technology. With this assay, we are able to quantify lncRNA-protein interactions rapidly for the purpose of high throughput screening, enabling drug discovery efforts for this novel class of drug targets. In this work, we present our assay development and screening findings, including the identification of potential small molecule modulators of lncRNA-protein interactions. Furthermore, we describe the application of this lncRNA-protein interaction assay to detect RNA requirements for EZH2 recruitment, a much debated and important question that lingers in the field. From our work, it is evident that BDNF-AS has several regions of RNA that are required for EZH2 recruitment, potentially due to the importance of this transcript in regulating BDNF. This work describes exploratory drug discovery for a novel class of drug targets as well as applications to understand the basic biochemistry governing lncRNA-protein interactions.

Keywords

noncoding RNA; epigenetics; drug discovery; genomics; BDNF; EZH2

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