Once ovulated, the terminally differentiated mammalian oocyte will die if it does not bind and fuse with a sperm. If fertilization occurs, however, maternal gene products orchestrate the transformation of the egg into a totipotent zygote within hours. The main focus of my laboratory is to identify and study novel, highly-abundant, and egg-specific molecules that are involved in this reprogramming process. These aims are being carried out by first utilizing two-dimensional electrophoresis, tandem mass spectrometry, and bioinformatics to scan the mouse egg proteome for previously uncharacterized molecules that are likely to be functionally-relevant and oocyte-specific. The open reading frames of these genes are then cloned, and the encoded proteins are expressed and used to generate mono-specific antibodies. The antibodies, recombinant proteins, and anti-sense oligonucleotides are then tested in in vitro assays to investigate the molecule's function. We are now utilizing an RNA interference approach to further investigate maternal effect gene function. Given that cellular differentiation is largely controlled by transcriptional regulation, chromatin remodeling will likely play a central role in this reprogramming process. Covalent histone modifications or "marks" provide an attractive storage mechanism for mitotically and meiotically heritable information. By regulating access to underlying DNA, these modifications can dictate correct spatial and temporal gene expression patterns during cellular differentiation. My laboratory has recently begun to investigate the role of specific histone modifications (and the enzymes that perform these modifications) in dictating egg and early embryonic gene expression patterns. We hypothesize that specific histone modifications involved in directing gene expression during germ-cell differentiation will be removed from the chromatin template during reprogramming and that these marks will be replaced by new modifications required to direct early embryonic gene expression. To begin to test this hypothesis, we have been investigating changes in global levels of specific histone marks during oocyte maturation and throughout pre-implantation mouse development. Results show that the modifications can be classified into two strikingly different categories:

1. stable 'epigenetic' marks [e.g. histone H3 lysine 9 methylation, histone H3 lysine 4 methylation, and histone H4/H2A serine 1 phosphorylation], and

2. dynamic and reversible marks [e.g. hyperacetylated histone H4, histone H3 arginine 17 methylation, and histone H4 arginine 3 methylation].
   

We will soon begin performing anti-modified histone antibody chromatin immunoprecipitation followed by microarray analysis of the underlying DNA to identify which egg and early embryonic genes are being regulated by the dynamic histone modifications and to more rigorously test the hypothesis that the "resetting" of these histone modifications leads to changes in gene expression patterns during the egg to embryo transition.

Email: scc2003@med.cornell.edu