The research in our lab is focused on understanding the regulation of gene expression in a complex plant genome. Our focuses are on understanding the molecular causes and affects of epigenetic regulation of gene expression. While some of the individual research projects in my lab appear to be quite distinct they are tied together by the common theme of understanding epigenetic regulation in maize. The specific research projects occurring in my lab are a reverse-genetic analysis of epigenetic modifiers, elucidation of the cis- and trans-acting factors controlling parental imprinting, analysis of aneuploid syndromes, oligonucleotide microarray technology development and monitoring allele-specific gene expression in heterozygous or aneuploid plants.

I began working towards a reverse-genetics approach to understand epigenetics in maize as a grad student and have continued to work on this project. My initial focus was on the DNA methyltransferase, methyl-CpG-binding domain and Polycomb group genes. I have participated in the effort to clone and characterize these genes. As a post-doc I was part of the Plant functional genomics consortium (www.ChromDB.org) and expanded my focus to include the complete suite of chromatin genes present in maize. The general strategy has been to use sequence-based searches to identify maize EST or GSS sequence tags corresponding to putative epigenetic modifiers. The full-length sequence of the gene is then obtained through RACE or library screening. The gene is then characterized to determine it’s map location, expression pattern and relationship to other sequences in maize and other organisms. We then pursue a genetic strategy to determine the function of the gene. Loss-of-function genetic stocks are created through the generation of transgenic RNAi lines or by identification of transposon insertion alleles. The loss-of-function genetic stocks are then analyzed to determine the role of the putative epigenetic modifier in known epigenetic phenomena. Specifically, my research focuses on determining the role of the chromatin genes in the regulation of imprinting and aneuploid induced gene expression changes.

Analysis of imprinting:

During the course of characterization of the maize Polycomb group proteins I found that one of these genes displays an imprinted pattern of gene expression such that only the allele derived from the maternal genome is expressed in the endosperm tissue. The imprinting of this gene occurs throughout endosperm development but the gene is bi-allelicly expressed in embryonic and vegetative plant tissues. We have begun to use this example of imprinting as a model to dissect the cis and trans acting factors that control imprinted gene expression. We are using the dominant negative transgenic RNAi lines that reduce the expression of putative chromatin genes to perform a reverse-genetic screen to identify trans-acting factors that are required for imprinting. To date we have successfully identified five genes that required for imprinted gene expression in maize. In addition, we are studying the cis-acting sequences that are necessary for imprinting as well as the chromatin modifications that occur to the imprinting gene.

Oligonucleotide microarray technology development:

As a post-doc I began working on a technology development project to enable the use of oligonucleotide microarrays for polymorphism detection (link to website). Oligonucleotide microarrays consist of large numbers (~400,000) oligonucleotides that are attached to a glass surface. Genomic DNA or mRNA from an organism of interest can be labeled and hybridized to the microarray. The amount of hybridization can be monitored by the amount of signal coming from a specific location. Due to the short length (25-30bp) of the oligonucleotides the hybridization kinetics can be significantly altered by the presence of even a single base pair difference (or SNP). We have been studying the parameters for optimizing oligonucleotide microarray design, hybridization and interpretation to allow the use of microarrays to detect polymorphisms in maize genomic DNA or mRNA. Enabling this technology will allow for high-throughput mutation detection, mapping, genome structure analysis and allele-specific expression analysis.

References:

8. Springer NM, Kaeppler SM. (2005) Evolutionary Divergence of Monocot and Dicot Methyl-CpG-Binding Domain Proteins. Plant Physiol. 138(1):92-104.
7. Till B.J., S.H. Reynolds, C. Weil, N.M. Springer, C. Burtner, C.A. Codomo, L.C. Enns, A.R. Odden, K. Young, E. Bowers, E.A. Greene, L. Comai, S. Henikoff . 2004. Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol. 4(1):12.
6. Plant functional genomics consortium. 2005. Transgene-induced RNA interference as a Tool for Plant Functional Genomics. In RNA Interference Methods in Enzymology eds. John Rossi and David Engelke. Methods Enzymol. 392:1-24.
5. Springer N. M., W. B. Barbazuk. 2004. Utility of different gene-enrichment approaches towards identifying and sequencing the maize gene space. Plant Physiol 136:3023-33.
4. Springer N.M., C.A. Napoli, D.A. Selinger, R. Pandey, K.C. Cone, V.L. Chandler, H.F. Kaeppler, S.M. Kaeppler. 2003. Comparative Analysis of SET Domain Proteins in Maize and Arabidopsis Reveals Multiple Duplications Preceding the Divergence of Monocots and Dicots. Plant Physiol. 132:907-925
3. Springer, N.M., O. Danilevskaya, P. Hermon, T. Helentjaris, R.L. Phillips, H.F. Kaeppler, S.M. Kaeppler. 2002. Sequence Relationships, Conserved Domains, and Expression Patterns for Zea mays Homologs of the Drosophila Polycomb Group Genes E(z), esc, and E(Pc). Plant Physiol. 128: 1332-1345
2. Papa, C.M., N.M. Springer, M.G. Muszynski, R. Meeley, and S.M. Kaeppler. 2001. Maize chromomethylase Zea methyltransferase2 is required for CpNpG methylation. Plant Cell 13: 1919-1928.
1. *Cao, X, N.M. *Springer, M.G. Muszynski, R.L. Phillips, S. Kaeppler, and S.E. Jacobsen. 2000. Conserved plant genes with similarity to mammalian de novo DNA methyltransferases. Proc. Natl. Acad. Sci. 9:4979-4984. *These authors contributed equally to this work