Statement of Research Interests

Impact of histone acetylation on plant development
Background on HATs
Background on Arabidopsis
Background on Our Research
Current Projects

Comparative genomics in Drosophila

Lab Members

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.

The plant on the left is ada2b -/-;
the plant on the right is wildtype.

See the following publication for more background information:
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.

Current Arabidopsis Projects
(1) 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?  Please see the following publication for more background information:
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.

One area of current research involves exploring the underlying distinctions that result in different biochemical activities of these proteins. Using recombinant DNA technology (cloning), we constructed 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 embarked on a collaborative project to characterize the specific differences that arise throughout floral development in gcn5 mutants using scanning electron microscopy (SEM).  Our initial results are reported here:
Cohen, R.*, J. Schocken*, A. Kaldis, K.E. Vlachonasios, A.T. Hark, and E.R. McCain. 2009. The histone acetyltransferase GCN5 affects the inflorescence meristem and stamen development in Arabidopsis. Planta 230: 1207-1221.

Our next step involves utilizing this morphological data to identify and test candidate genes that may serve as targets of GCN5 activity.  We are also interested in looking at GCN5 localization within the flower. These projects should help us to define which floral 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.  Trichomes are a well-studied model of cellular differentiation and this project builds off previous work comparing developmental differences between gcn5 mutant and wildtype plants.

Comparative Genomics Project

This project is part of my collaboration with the Genomics Education Partnership (GEP; gep.wustl.edu), a group based at Washington University in St. Louis and funded by Howard Hughes Medical Institute.  In short, this work in comparative genomics involves using in silico (computer-based) analysis to annotate genes in Drosophila species (defining their start/stop sites,  exon/intron boundaries, etc.).  The underlying biological question is how genome and chromatin organization impacts gene function, which connects to my basic research interests and work of other members of the Hark Lab.  This line of experimentation allows students to be part of a large-scale research project and have their work published while increasing their knowledge of fundamental molecular biology and research tools.

See http://www.gep.wustl.edu/ for more information.

There are opportunities for students to carry out research to address these and related questions, working in the fields of genetics, developmental biology, and molecular biology. I encourage you to contact me if you are interested in doing research in the lab, so that we may talk more about your area(s) of interest and potential projects.

Lab Members

Jenna Kotak '13 (right) is continuing the research on gcn5 trichome patterning in collaboration with Dr. Elizabeth McCain.  Jenna began her work in the Spring 2011 semester; her work in the summer of 2011 was supported by the James R. Vaughn '52 Student Award.		Meredith Colwell '12 worked on several molecular projects in the lab during the summer of 2011 and Fall 2011. Edward Quach '14 (not pictured) has been serving as a lab and research assistant since Fall 2010.  Ed and Cyrus Kuschner '14 (not pictured) will begin work on the GEP project in Spring 2012.
	Lindsey Shantzer '12 (left) and Anne Bertolet '13 (not pictured) joined the lab in Fall 2010.  Both Lindsey and Anne worked on the GEP project in Fall Independent Studies.  Anne continued this work in Spring 2011 while Lindsey shifted her focus to wet-lab work for the Spring 2011-Spring 2012 semesters.
	Elia Wright '10 (left) served as a Research Assistant during the 2009-2010 academic year, piloting expression analysis of ADA2b transgenic lines using Real-time PCR.  Mazen Tolaymat '11 (right) continued this work as an Independent Study in Fall 2010.

Dana Tedesco '11 has carried out Independent Studies during the 2009-2010 and 2010-2011 academic years as well as conducted summer research that was supported by Merck-AAAS.  Dana began by constructing an ADA2b transgene harboring a deletion of a plant-specific domain for use in functional tests.  In collaboration with Dr. Steven Triezenberg and Nathan Lord (both formerly at Michigan State University), Dana also initiated a biochemical project in our lab designed to determine interacting partners of ADA2b and GCN5.

Kelsey Parry '11 joined the lab in Fall 2009 after participating in a summer undergraduate training workshop sponsored by the GEP held at Washington University in St. Louis in August of 2009.  In addition to developing course materials for BIO 152, in the same semester Kelsey annotated a D. erecta fosmid as a independent study project.  In Spring 2010, Kelsey worked on genotyping various ADA2b transgenic lines.

Ashley Kendig '10 (left) worked on a collaborative project with Dr. Elizabeth McCain
during the 2008-2009 academic year.  Ashley's research further investigates trichome patterning in gcn5-1 mutant and wildtype plants using scanning electron microscopy (SEM).  Peter Ashman '11 (right) continued this work, serving as a Research Assistant in Fall 2009.