The long-range goal of the proposed studies is to determine how chromatin modulates gene expression during embryogenesis. The focus is on the role of two histone modifications that are associated with developmental control genes. Trimethylation of lysine 27 of histone H3 (H3K27me3) is commonly associated with silenced genes, whereas trimethylation of lysine 4 of histone H3 (H3K4me3) is commonly associated with genes that are active or permissive to be activated. Studies in pluripotent embryonic cells have revealed that both these marks are associated with developmental control genes before these genes are expressed. These bivalent H3K4me3/H3K27me3 marks have been proposed to contribute to the quantitative, temporal or spatial precision of developmental gene expression, but the field lacks a technology that would allow a direct test of these hypotheses. This project aims to (1) develop a method to induce specific histone modifications at defined genes and (2) apply this technology to determine the role of bivalent histone marks during zebrafish embryogenesis. The method involves the design of zinc finger fusion proteins that have histone methyltransferase or demethylase activities. These proteins will be targeted to defined genes to change H3K4 or H3K27 methylation. The technology will then be used to analyze the consequences of disrupting or introducing H3K4me3 or H3K27me3 marks at specific genes. Since specific histone modifications accompany developmental and physiological states in all eukaryotes, developing a technology for the gene-specific modulation of histone marks would help elucidate the role of such modifications in numerous settings. The proposed technology could also have direct impact in studying and modulating disease processes. For example, abnormal histone modifications have been implicated in several cancers. Local manipulation of histone modifications might help to determine which of the many misregulated genes are responsible for tumor formation. It might even become possible to use the proposed technology to specifically activate disease-protective genes or repress disease-inducing genes.

Public Health Relevance

This project addresses how the myriad of different cell types form during animal development. Abnormal regulation of this process leads to birth defects and cancer, whereas methodical manipulation of this process can help understand and treat degenerative diseases.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21HD072733-01
Application #
8320462
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Coulombe, James N
Project Start
2012-03-15
Project End
2014-02-28
Budget Start
2012-03-15
Budget End
2013-02-28
Support Year
1
Fiscal Year
2012
Total Cost
$249,201
Indirect Cost
$99,201
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Zhang, Yong; Vastenhouw, Nadine L; Feng, Jianxing et al. (2014) Canonical nucleosome organization at promoters forms during genome activation. Genome Res 24:260-6
Gagnon, James A; Valen, Eivind; Thyme, Summer B et al. (2014) Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One 9:e98186
Vastenhouw, Nadine L; Schier, Alexander F (2012) Bivalent histone modifications in early embryogenesis. Curr Opin Cell Biol 24:374-86