Background on HATs and Histone Acetylation
My current research investigates the biological roles of the histone acetyltransferase (HAT) enzyme GCN5 and the associated factors ADA2a and ADA2b in the model plant Arabidopsis thaliana. These factors are part of a class of proteins that can be referred to as transcriptional coactivators. GCN5 can covalently modify histones (chromatin proteins) by catalyzing the addition of acetyl group to specific lysine residues, a process which is facilitated by the ADA2 proteins. This modification is hypothesized to affect histone-DNA contacts or provide binding sites for other factors involved in regulating transcription (the histone code hypothesis), but the exact biochemical effects of histone acetylation are still not well understood. However, there is a well-established correlation between acetylation of histones and activation of gene transcription. Misregulation of histone acetylation states has been implicated in cancer and developmental disorders.

Background on Arabidopsis
Why study these chromatin modifying factors in Arabidopsis?

First, a little bit of background. Arabidopsis is a modest little flowering plant, in the Brassica family, related to plants you are probably more familiar with such as broccoli and cauliflower. Arabidopsis has emerged as an experimental model for a number of reasons, including:

• It's easily grown in the laboratory and is a good genetic system, with a relatively short generation time of ~6 weeks and a single individual producing hundreds of seeds.
• It's easy to transform, allowing for the production of transgenic plants.
• Its genome has been fully sequenced, which along with many other community resources facilitates many aspects of molecular and genetic research.

Background on Our Research
In the lab of Dr. Steven Triezenberg at Michigan State University, my colleague Dr. Kostas Vlachonasios (now at Aristotle University of Thessaloniki, Greece) and I took a reverse genetics approach to understanding the function of transcriptional coactivators in Arabidopsis. We identified mutations in genes encoding the HAT GCN5 and associated factors and studied the resulting phenotype of mutant plants. Plants containing disruptions in either the GCN5 gene or the ADA2b gene displayed dramatic effects with respect to their overall growth (see photos below). These mutants also exibited developmental problems in a variety of plant organs, perhaps most notably defects in their flowers which render the mutants plants infertile. Interestingly, the gcn5 and ada2b mutant plants share some aspects of their phenotype but each mutant also displays unique defects, which suggests that the GCN5 and ADA2b proteins may function together as well as separately to regulate gene expression in plants.

In Arabidopsis, there are two genes encoding proteins that resemble the well-characterized ADA2 transcriptional coactivator from yeast and other organisms. Mutations in one of these genes, ADA2b, lead to dwarf plants with a number of developmental problems as mentioned above. However, disruptions of ADA2a do not result in any dramatic phenotypic effect under our normal growing conditions. Why? One area of current research involves exploring the underlying distinctions that result in different biochemical activities of these proteins.

See the following publications for more background information:
*denotes undergraduate co-author
Vlachonasios, K.E., M.F. Thoamshow, and S.J. Triezenberg. 2003. Disruption Mutations of ADA2b and GCN5 Transcriptional Adaptor Genes Dramatically Affect Arabidopsis Growth, Development, and Gene Expression. Plant Cell 15: 626-638.

Hark, A.T., K.E. Vlachonasios, K.A. Pavangadkar, S. Rao, H. Gordon*, I.-D. Adamakis, A. Kaldis, M.F. Thomashow, and S.J. Triezenberg. 2009. Two Arabidopsis orthologs of the transcriptional coactivator ADA2 have distinct biological functions. Biochimica et Biophysica Acta 1789: 117-124.

While chromatin modifiers have been shown to have global effects on gene expression, identification of direct molecular targets has been more limited. In the case of Arabidopsis GCN5, a handful of targets have been identified in vegetative tissue while only a few floral transcripts affected by loss of GCN5 function have been determined. Projects 2 and 3 described below ultimately strive to uncover direct molecular targets, permitting a more complete understanding of GCN5’s role in flower and trichome development as well as contributing to general knowledge of the mode of action of GCN5 across eukaryotes.

Current Projects
(1) Construction of ADA2b transgenes designed to test the function of various protein domains. Using recombinant DNA technology (cloning), we are in the process of constructing a series of ADA2b transgenes. Following transformation into Arabidopsis plants mutant for ada2b, we can identify which constructs rescue the phenotype and therefore infer which domains of ADA2b are essential for biological activity.

(2) Studies of the reproductive defects in transcriptional coactivator mutants. Original studies showed that gcn5 as well as ada2b mutant plants were infertile and that mature flowers displayed altered morphology.  Dr. Elizabeth McCain and I along with several research students have embarked on a collaborative project to characterize the specific differences that arise throughout floral development in gcn5 mutants using scanning electron microscopy (SEM).  We are also interested in looking at GCN5 localization within the flower. This work may ultimately help us to define which genes are regulated by GCN5 and/or ADA2a/b.

(3) More recently, we have also begun using SEM to compare trichome structure between gcn5 mutant and wildtype plants in collaboration with Dr. McCain.

In addition, previous studies of transcriptional coactivator mutants in response to a variety of environmental conditions or abiotic stressors have been publsihed (Vlachonasios, et al. 2003, Hark, et al. 2009). As part of a collaboration with Dr. Vlachonasios, we might continue to explore these plants' responses to other stresses and/or embark on studies of hormonal signaling in gcn5 and ada2b mutants.

Lab Members