Genome wide analysis of p53's interplay with chromatin structure and its transcriptional program during the cell cycle

During my PhD I focused on two main projects, each dealing with a different aspect of the p53 transcription factor:;1. The interplay between p53 and chromatin: This project was done in collaboration with Yair Field and Dr. Eran Segal. The human transcription factor p53 is a pivotal roadblock against cancer. A key unresolved question is how p53 selects its genomic binding sites in vivo out of a large pool of potential consensus sites. I hypothesized that chromatin may play a significant role in this site selection process. To test this, I used a custom DNA microarray to measure p53 binding at ∼2000 sites predicted to possess high sequence specificity, and identified both strongly-bound and weakly-bound sites. When placed within a plasmid and transiently transfected into human cells with increasing amounts of a plasmid expressing p53, weakly-bound sites become p53-responsive, and these sites also regain p53 binding when their containing plasmid was stably randomly integrated into the genome in human cells. Notably, strongly-bound sites reside preferentially within genomic regions whose DNA sequence is predicted to encode relatively high intrinsic nucleosome occupancy. Using in vivo nucleosome occupancy measurements under conditions where p53 is inactive, I experimentally confirmed this prediction. Furthermore, these p53-bound sites encompassing high nucleosome occupancy were specifically marked by a set of euchromatic modifications in contrast to the unbound sites that reside preferentially in regions marked with heterochromatic modifications. Upon p53 activation, nucleosomes are partially displaced from a relatively broad region surrounding the bound p53 sites, and this displacement is rapidly reversed upon inactivation of p53. Thus, in contrast to the general assumption that transcription factor binding is preferred in sites that have low nucleosome occupancy prior to factor activation, I found that p53 binding occurs preferentially within a chromatin context of high intrinsic nucleosome occupancy. Moreover, human regulatory regions are more generally found to be enriched for nucleosome occupancy as compared to the genome average, and these regulatory regions also display a tendency to be more accessible.;2. p53 transcriptional activity during the different stages of the cell cycle: p53 has a very important role in regulating cell cycle progression. A very interesting question in this respect is whether p53 responds differently to a DNA stress signal imposed at different stages of the cell cycle. Since p53 can lead to various biological outcomes it remains unclear whether p53 can adjust its response to specifically fit the stage at which the cell is currently present. I utilized centrifugal elutriation to obtain fractions of cells corresponding to G1, G1-S, S and G2-M phases of the cell cycle. Each fraction was then either treated with a DNA damaging agent (NCS) or served as control. Samples from all of these fractions as well as from an asynchronous cell population were subjected to expression microarray analysis. To specifically focus on p53-dependent effects, the entire process was performed in MCF7EcoR cells that posses wild type p53, as well as in MCF7sip53 that are depleted of p53. The expression patterns obtained suggest that as a whole, the p53 transcriptional response is similar throughout all the stages of the cell cycle. However, there exist some cases in which p53 selectively regulate specific target genes that are relevant for the particular stage in which the cell is present at the time that it encounters DNA damage. One such case is a cluster of mitotic genes. These genes were found to be specifically repressed at the G2-M phase of the cell cycle in a p53 dependent manner and this most probably is what enables efficient arrest of the cells at this stage prior to mitosis. Further efforts are required to discover the basis that underlies p53's ability to downregulate these sets of genes in a cell cycle specific manner.