We recently identified over 4,000 large intergenic non-coding RNA genes (lincRNAs) including hundreds of lincRNAs that interface with and influence epigenetic regulatory factors across a wide-spectrum of cellular processes and disease states. For example, we discovered that p53 directly regulates a lincRNA that is required for proper localization of chromatin factors to mediate p53 dependent cellular apoptosis in response to DNA damage. The p53 pathway plays a central role in response to DNA damaging agents including environmental hazards that can ultimately lead to cellular transformation. Thus, lincRNAs may herald a new paradigm in understanding the link between exposure to environmental hazards, epigenetic regulation and human health. Here we propose a multifaceted experimental and computational approach to identify lincRNAs that modulate p53-dependent epigenetic regulation, their functional roles and their biochemical mechanisms.
Aim 1) To identify chromatin-associated lincRNAs and epigenetic landscapes regulated by p53 upon exposure to DNA-damaging agents. We will expose primary human fibroblasts to a suite of DNA damaging reagents that are environmental hazards and identify the p53 regulated transcriptome, epigenome and their interactions. Specifically, we will identify p53-regulated lincRNAs using RNA-sequencing, epigenetic states using chromatin immunoprecipitation sequencing and the lincRNAs physically associated with chromatin factors using RNA immunoprecipitation sequencing technologies.
Aim 2) To examine cellular and epigenetic phenotypes associated with lincRNA modulation. Here we will perform a loss-of-function screen to identify lincRNAs that perturb p53 dependent epigenetic and cellular phenotypes. We will use and cell sorting and colorimetric assays to examine perturbations in the regulation of cell-cycle and cellular apoptosis upon lincRNA depletion. We will also monitor lincRNA regulated epigenetic changes lincRNAs using a high-throughput and cost-effective approach termed "ChIP-string".
Aim 3) To elucidate the biochemical mechanisms of p53-regulated lincRNAs. Here we will dissect the biochemical mechanisms of lincRNAs that perturb epigenetic regulation of the p53 dependent DNA damage response. We will carry out RNA-pull down assays to identify proteins associated with each lincRNA, deletion mapping to identify the sites of RNA-protein interactions of these complexes. We will also identify the DNA sites that are directly interacting with lincRNAs using a new approach of RNA based immunoprecipitation assay. These datasets will then be used for numerous computational analyses to find common sequence and structural motifs within lincRNAs that may drive their epigenetic and cellular regulatory roles.

Public Health Relevance

Our research aims to understand a universal problem in human health: How does the same genome present in every cell take on alternate identities that orchestrate distinctive cell states and how are these states misregulated in diseases? We recently discovered a novel class of large intergenic non-coding RNAs (lincRNAs) that have the ability to regulate cellular identity and are misregulated in human cancers and DNA damage pathways. Thus, we seek to establish the fundamental principles and mechanisms by which lincRNAs regulate cellular identity and their roles in human health. The ultimate goal of these innovative experimental and computational approaches is to develop novel RNA based therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES020260-03
Application #
8513990
Study Section
Special Emphasis Panel (ZRG1-GGG-M (50))
Program Officer
Chadwick, Lisa
Project Start
2011-09-20
Project End
2016-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
3
Fiscal Year
2013
Total Cost
$395,986
Indirect Cost
$83,300
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Sun, Lei; Goff, Loyal A; Trapnell, Cole et al. (2013) Long noncoding RNAs regulate adipogenesis. Proc Natl Acad Sci U S A 110:3387-92
Trapnell, Cole; Hendrickson, David G; Sauvageau, Martin et al. (2013) Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol 31:46-53