The first goal of my project within the Bustos lab is to develop an efficient protocol for the analysis of histone modifications at specific areas of plant tissue chromatin. In brief, the chromatin preparation involves covalently crosslinking the histones to the plant DNA immediately after harvest, breaking open the cells, and sonicating the DNA to reduce the average strand length to between .3 and 3 kb. We then use these chromatin preps to immunoprecipitate the DNA that is attached to the protein of interest using specific antibodies. Once that DNA is recovered, the crosslinks are reversed and DNA is precipitated to use in PCR reactions targeted at the gene of interest.

Once this protocol has been reliably established, we will use this method to attempt to distinguish differences in chromatin structure at regulatory regions of a number of key genes involved in germination between embryonic tissue, vegetative tissue and root tissue harvested from Arabidopsis thaliana and Brassica napus plants. I will be specifically looking at the modification state of Histone 3 using antibodies against acetylated Histone 3 at Lysine 9, Methylated Histone 3 at Lysine 9, and unmodified Histone 3 to determine whether chromatin remodeling plays a role in the regulation of these genes. We hope also to be able to successfully use this ChIP protocol on prostate cell lines given to us by the Bieberich lab to verify a proposed binding site for their transcription factor of interest using antibodies against that specific transcription factor.