Profile

The general interest of our laboratory is in how tissue specific expression patterns are established and maintained during development, with particular focus on the role that chromatin plays in these processes. We use a Xenopus egg-based system to assemble chromatin and generate synthetic nuclei in vitro. Synthetic nuclei undergo a single round of semi-conservative replication allowing us to address questions concerning maintenance of gene expression.

There are currently two major projects being pursued in the laboratory.

The first project uses phaseolin, the major seed storage protein in bean (P. vulgaris) as a model for tissue-specific gene regulation in plants. The beta-phaseolin gene is tightly regulated at the transcription level resulting in strict tissue-specific and spatial expression during embryonic development. The highly regulated expression of phaseolin requires chromosomal integration indicating that chromatin structure plays a key role in regulating gene expression. Phaseolin expression coincides with the disruption of a nucleosome which is positioned over three phased TATA boxes. In collaboration with Tim Hall's group at Texas AandM we have used in vitro and in vivo approaches to ascertain specific roles for individual TATA boxes in both location and efficiency of transcription initiation. We are currently modifiying our chromatin assembly system to incorporate plant components in order to dissect the individual steps and/or proteins required for embryonic activation of phaseolin. The long-term goal of this study is to identify both universal and plant-specific mechanisms through which developmental gene expression patterns are established and maintained.

The second project is aimed at understanding the molecular processes leading to cell-type specification during liver development. Specifically, we have initiated a study to delineate the respective roles of two tissue-specific transcription factors, Foxa1 and Foxa2, in targeting liver-specific genes for activation. Targeting a gene for expression requires that a transcription factor occupy its binding site in silent chromatin prior to gene activation. To model Foxa activity in vitro, we have used the alpha-fetoprotein gene whose liver specific expression is regulated by the Foxa transcription factors. In vitro chromatin assembly assays suggest that Foxa2 binds with a higher affinity than Foxa1 within a silenced chromatin environment. These data together with transgenic mouse studies suggest that Foxa2 may play a role in potentiating liver gene expression, while Foxa1 may be primarily responsible for maintaining previously established expression patterns. We are continuing to use in vitro assays in combination with RNAi and transient transfections to elucidate the mechanism(s) by which the Foxa factors target liver-specific genes for activation.

Lastly, in a newly established project, we have begun to explore the role that epigenetic mechanisms such as chromatin remodeling and DNA methylation, play in environmental adaptation of plants. The predominantly sessile nature of plants necessitates an ability to adapt to rapidly changing environmental conditions. The remarkable developmental plasticity that plants exhibit strongly suggest the existence of chromatin-mediated mechanisms for altering established gene expression programs. We have chosen Arabidopsis as a model system to study the role of epigenetic mechanisms in adaptation due to the public availability of transgenic lines harboring targeted disruptions of individual chromatin regulatory genes. Our initial study will examine the relative abilities of Arabidopsis chromatin remodeling mutants to adapt to artificially-induced abiotic variables including high salinity and cold temperatures.