Tzung Fu Hsieh

The overall goal of this project is to develop effective management practices to minimize the biological risks associated with the newly developed genetically engineered (GE) tomato. Tomato is a predominantly self-fertilizing crop and also a facultative outcrossing species with a substantial pollen-mediated gene flow (PMGF) rate in the field. The adoption of genetic engineering significantly contributed to crop improvement. The inclusion of GE tomato into the agricultural landscape carries high risks related to the introduction of transgenes into related agricultural and wild relatives. Thus, effective biological containment technologies are needed to prevent PMGF from GE to non-GE tomato. We proposed to engineer tomato to produce CRISPR pollen which will degrade endosperm development in the non-GE plants but not the GE plants which will express a mutated endosperm lethality gene. It is expected that the engineered CRISPR pollen will not interfere with seed production in tomato. By doing this, the newly-developed GE tomato could be used as the background source for genetic engineering of any traits for sustainability. Our goals are intimately tied with the management practices to minimize environmental risks of the newly developed GE tomato, and fits squarely into the BRAG objectives. We expect our results will help develop regulatory and best management practices for future GE tomato plantings. We also expect we will develop an efficient biological containment strategy that is applicable to other GE crops currently or soon-to-be incorporated into non-GE agricultural settings.

Epigenetic modifications play critical roles in gene regulation, development, and diseases. Understanding how epigenetic changes between species occur and how they affect gene regulation has potential to advance our knowledge of regulatory evolution. However, the details of epigenetic evolution are sparse, and how epigenetic evolution correlates with phenotype evolution is poorly understood. The proposed research will test a novel hypothesis that DNA methylation of distinctive cell types in human and non-human primate brains shows variation consistent with brain size evolution. Moreover, validation studies will be performed for specific candidate genes and genomic regions that show DNA methylation and gene expression difference related to brain region differences and species differences. This study will generate novel data to expand our understanding of epigenetic evolution of brains, and to infer functionally important positions of noncoding genomic regions. Furthermore, it will also provide knowledge on how epigenome changes during evolution and how epigenome evolution correlates with phenotype, which is a fundamental yet currently little understood topic.

DNA methylation is critical for processes including genomic imprinting, X-inactivation, transposonsilencing, and genome stability. Maintaining DNA methylation patterns is a result of active DNAmethylation and demethylation processes. Compared to pathways that promote and maintain DNAmethylation, much less is known about the function and regulation of DNA demethylation. In plants,genomic imprinting is established in the gametes by DEMETER (DME) mediated active DNAdemethylation, which is required for seed viability in Arabidopsis. The DME-like 5-methylcytosine(5mC) DNA glycosylases are active DNA demethylases that mediate the remove of 5-mC via baseexcision repair pathway. DME encodes a large polypeptide with multiple conserved domains, and exceptfor the well-characterized glycosylase domain, almost nothing is known about the functions of thesedomains. The proposed research focuses on understanding the function and regulation of the conserveddomains of DME. A novel bipartite model for structural and functional regulation for DME activity isproposed. Understanding how DME evolved to contain modular catalytic and regulatory domains, andelucidating how linker histone H1 assists DME active demethylation will significantly increase ourunderstanding of the active DNA demethylation pathway that has been adopted for reproductive successin plants. The knowledge learned in this application will provide vital information on how epigeneticinformation is established and maintained.