Cytosine methylation of DNA is a repressive chromatin mark important for silencing genes during mammalian development, and cancer cells frequently silence tumor suppressor genes by aberrantly methylating their promoters. Genome stability is compromised when DNA methylation patterns are disrupted in cancer or in developmental disease. Despite the critical role of DNA methylation in gene expression and genome stability, the mechanisms guiding de novo DNA methylation remain poorly defined. Oxytricha trifallax is a unicellular eukaryote that performs dramatic genome rearrangements in a developmental process that transforms its micronucleus (MIC) genome into a differentiated macronucleus (MAC). This process is accomplished by the precise elimination of 95% of the genome, and recapitulates the key property of stem cells: self-renewal (one copy of the MIC is retained at all times) and differentiation (MAC formation). Interestingly, my work in the Landweber lab has shown that this organism uses de novo DNA methylation in the elimination process. The fact that most of the Oxytricha genome is methylated and eliminated makes it an attractive model system to understand how methylation is targeted: aberrant methylation and subsequent elimination of the 5% retained DNA would be fatal. In addition, the reproducibility of genome rearrangements in this ciliate makes it a unique model for studying the connection between DNA methylation and genome stability, both of which are relevant to cancer and developmental processes generally.
AIM 1 : Test the hypothesis that methylation induces DNA degradation in Oxytricha through microinjection of artificially constructed, in vitro methylated chromosomes into the MAC of vegetative cells.
AIM 2 : Methyl-DNA immunoprecipitation and high throughput sequencing (meDIP-Seq) will be used to define the endogenous domains of de novo DNA methylation during genome rearrangements in Oxytricha.
AIM 3 : The functional relevance of cytosine methylation in Oxytricha genome rearrangements will be analyzed by use of chemical inhibitors of methyltransferases, followed by deep sequencing the DNA from treated cells.
AIM 4 : Identification of novel methylation pathway proteins by methyl-cytosine chromatin IP (me-ChIP) and mass-spec analysis. Oxytricha trifallax provides an unprecedented opportunity to study the role of DNA methylation in a model system that has evolved an elaborate genome rearrangement process. Currently, the targeting of de novo methylation in human disease is poorly understood, so insights from Oxytricha will be immediately relevant to studies of human health and disease. Any conserved candidate proteins or motifs identified in the ciliate model can be tested in human tissue culture or mouse cancer models for potential oncogenic roles.

Public Health Relevance

Aberrant DNA methylation causes several human developmental diseases and is correlated with genome instability, cancer initiation, and oncogenic progression. However the mechanism by which DNA methylation is targeted to the correct sequences remains obscure. The goal of the proposed research is to use the unique properties of ciliates to uncover new targets for therapeutic drug development. Ultimately, the ability to correct errors of DNA methylation will allow us to prevent or treat human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM099462-01A1
Application #
8397434
Study Section
Special Emphasis Panel (ZRG1-F08-Q (20))
Program Officer
Janes, Daniel E
Project Start
2012-08-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
1
Fiscal Year
2012
Total Cost
$52,190
Indirect Cost
Name
Princeton University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08544
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Lindblad, Kelsi A; Bracht, John R; Williams, April E et al. (2017) Thousands of RNA-cached copies of whole chromosomes are present in the ciliate Oxytricha during development. RNA 23:1200-1208
Bracht, John R (2014) Beyond transcriptional silencing: is methylcytosine a widely conserved eukaryotic DNA elimination mechanism? Bioessays 36:346-52
Chen, Xiao; Bracht, John R; Goldman, Aaron David et al. (2014) The architecture of a scrambled genome reveals massive levels of genomic rearrangement during development. Cell 158:1187-1198
Swart, Estienne C; Bracht, John R; Magrini, Vincent et al. (2013) The Oxytricha trifallax macronuclear genome: a complex eukaryotic genome with 16,000 tiny chromosomes. PLoS Biol 11:e1001473
Bracht, John R; Fang, Wenwen; Goldman, Aaron David et al. (2013) Genomes on the edge: programmed genome instability in ciliates. Cell 152:406-16
Bracht, John R; Perlman, David H; Landweber, Laura F (2012) Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax. Genome Biol 13:R99
Fang, Wenwen; Wang, Xing; Bracht, John R et al. (2012) Piwi-interacting RNAs protect DNA against loss during Oxytricha genome rearrangement. Cell 151:1243-55