SGER: Development of an In Vitro Chromatin Assembly and Transcription System for Plant Genes

University Of Washington, Seattle WA

Investigators

Abstract

Studies of both plant and animal genes have illustrated the key role that chromatin and chromatin modulators play in regulating gene expression during development. Correct spatial and developmental expression of phaseolin, the major seed storage protein in bean, requires chromosomal integration. Chromatin structural analyses have established that a nucleosome is rotationally positioned over the phaseolin promoter in non-seed tissue, thereby blocking transcription initiation. This repressive chromatin structure is disrupted prior to beta-phaseolin gene activation in the maturing embryo, suggesting a mechanism by which gene expression can be regulated during development. However, the role that tissue-specific transcription factors and chromatin modulators play in the transition from a silenced to an active phaseolin gene has not been elucidated. The overall aim of this project is to develop an in vitro chromatin assembly and transcription assay to study chromatin-mediated regulation of plant genes. To achieve this goal, the PI will combine existing animal in vitro chromatin assembly systems and a previously developed plant in vitro transcription assay with purified plant transcription factors to reconstitute phaseolin gene regulation in vitro. The three specific aims are 1) to reconstitute physiological chromatin on the phaseolin gene in vitro, 2) to transcribe the phaseolin gene in vitro using a tobacco cell transcription extract, and 3) to reconstitute hormonal and tissue-specific regulation of phaseolin gene expression in vitro. Results from these studies will provide definitive evidence that the chromatin assembly/transcription system faithfully recapitulates the endogenous chromosomal context and tissue-specific expression pattern of a plant gene in vitro. If successful, this in vitro system may provide a universal tool for elucidating the mechanisms driving chromatin-mediated gene regulation in plants. An increased understanding of how chromatin context influences gene expression during plant development will greatly facilitate the engineering of stable transgenic plants for both commercial and agricultural use.