Date of Award

9-16-2010

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Michael Simons

Second Advisor

Frank Giordano

Third Advisor

Lloyd Cantley

Abstract

EPIGENETIC PROGRAMMING OF BLOOD VESSEL IDENTITY. Aaron W. Aday, Lihua J. Zhu, and Nathan D. Lawson. Program in Gene Function and Expression, UMass Medical School, Worcester, MA. (Sponsored by Michael Simons, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT). Recent studies have revealed details of the signaling pathways controlling blood vessel development and function. However, little is known about what controls endothelial cell identity in different blood vessel types. It is important to identify transcriptional control elements that function in endothelial cells in order to examine their roles in differentiation and vascular development. Certain histone modifications can serve as molecular markers for these regulatory elements. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) allows one to identify DNA sequences bound by these histones, and mapping to a reference genome permits localization of putative enhancer and promoter regions enriched for modified histones. By using this technology to identify global epigenetic modifications associated with transcriptional activation in endothelial-expressed genes, one can locate cis-regulatory elements that may play essential roles in controlling cell type-specific gene expression and defining blood vessel identity. In an effort to identify cis-regulatory elements that control endothelial gene expression, we have performed ChIP-Seq on zebrafish embryos. Similar to previous studies, promoters are enriched for modifications such as trimethylation of histone 3 at the fourth lysine residue (H3K4me3). Monomethylation of histone 3 at the same position (H3K4me1) is less strongly enriched at promoter elements and often localizes up and downstream of predicted gene sequences or in intronic regions. In several cases, these corresponding sequences are evolutionarily conserved and map to known transcription factor binding sites. We have also analyzed ChIP-Seq data from endothelial cells isolated from zebrafish embryos by fluorescence-activated cell sorting (FACS), and this vascular dataset has a unique epigenetic signature compared to whole embryos. Finally, we performed in vivo reporter assays and confirmed that some of the candidate enhancer elements identified through ChIP-Seq are able to drive gene expression. Together, these resources will allow us to better understand the transcriptional regulatory networks that are responsible for endothelial cell heterogeneity.

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