Establishment of a Volvox chromatin immunoprecipitation (ChIP) platform to analyze the function of the master cell-fate determination regulator RegA
Alicia D. Howard1, Daniela Strenkert2, Michael Schroda2, and Stephen M. Miller1
1) Dept. of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250
2) Max Planck Institute for Molecular Plant Physiology, Potsdam-Golm 14476, Germany
Volvox carteri possesses two cell types: large, reproductive gonidia and small, motile somatic cells that resemble Chlamydomonas in morphology but do not divide. The regA gene is expressed only in somatic cells and is essential for maintaining them in a terminally differentiated state. regA encodes a putative transcriptional repressor (RegA) with a VARL domain, which resembles the DNA-binding SAND domain of several animal and plant transcription factors. RegA is hypothesized to repress growth and reproduction in somatic cells by binding (and repressing) the promoters of nuclear-encoded chloroplast protein genes (NECPGs), since NECPG transcripts do not accumulate in wild type somatic cells but are abundant in somatic cells of regA mutants. As a first step toward testing this hypothesis we have developed a chromatin immunoprecipitation (ChIP) platform for Volvox and are using it to assay occupancy of histones in the upstream regions of NECPGs and several other genes. We have optimized a number of experimental parameters, including DNA-protein crosslinking, chromatin shearing, antibody incubations, and real time PCR conditions. As a first test of our platform we examined occupancy of histone H3 at the hsp70A and control promoters under ambient and heat shock conditions. Consistent with previous results obtained for heat shock gene promoters in yeast and Chlamydomonas, our preliminary results indicate that heat shock induces marked displacement of histones from the hsp70A promoter. We are now using our ChIP protocol to analyze histone occupancy and the state of histone modifications at NECPGs in wild type versus regA- strains, and in somatic cells versus gonidia, with the goal of learning more about how somatic cell fate is established/maintained. Progress toward these goals will be reported.
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