Abstract

p53 and NF-kB families of Transcription Factors (TFs) are among the most studied proteins in tumour biology, typically known to function as antagonists, although recent studies identified example of positive, even cooperative interactions. p53 and NF-kB act as dimer or dimer of dimers, bind cis regulatory elements (referred herein as Response Elements REs) of which multiple versions exist in the genome, and coordinate very large networks of target genes, through highly regulated transactivation specificities. TAp53 is a tumour suppressor activated upon genotoxic and physiological stress and involved primarily in deciding senescence, cell cycle arrest or apoptosis as cell fate; the NF-kB proteins are involved in cell survival, proliferation and innate immunity responses. In our study we tried to elucidate in detail the intrinsic nucleotide preferences of p53 and NF-kB as sequence specific TFs and their impact on transactivation specificity. Selecting various in vivo validated cognate REs and testing various ad-hoc sequence permutations, we evaluated the role of identity and positioning of nucleotides in transactivation potential and specificity. To this aim different transcription assays were used, starting from a defined assay in yeast where p53 or NF-kB protein levels and the sequence of the RE are the only variables. With human wild type p53, I tested various REs used in co-crystallization studies probing nucleotide positions that are not directly contacted by the p53 DNA binding domain, to explore the effect of DNA conformational shifts on transactivation. Also, we investigated the effect on strength and direction of p53-induced transcription of changes in the nucleotides flanking an RE, selected based on torsional flexibility measurements. For the NF-kB family, relA/p65 and NFkB1/p50 were tested as single proteins or when co-expressed using a panel of REs selected based on different DNA binding affinities. The correlation between DNA binding and transactivation potential was examined. Further, the negative modulator IkB-alpha was co-expressed and its impact measured as a function of NF-kB protein type, expression level, or RE being tested. Both for p53 and NF-kB studies, we confirmed that the hierarchical organization of nucleotides within REs observed with yeast was reasonably well conserved in A549, H1299 or MCF7 human cells using transient transfection and/or treatments to activate endogenous p53 or NF-kB. Finally, I contributed to an ongoing study focusing on the interplay between p53 and NF-kB at the transcriptional level. Using microarrays and quantitative PCR we had observed highly synergistic expression of a group of genes involved potentially in metastasis, cell growth and proliferation upon combined treatment of MCF7 cells with doxorubicin, a chemotherapeutic agent, and TNF-alpha, an inflammatory cytokine. I have studied regions of the promoters of several such synergistic genes carrying putative p53 and NF-kB binding sites to study cis-mediated regulation of gene expression. This led to the investigation of cell type specific effects and the contribution of cofactors in the transcriptional synergy. Thus the main goals of my thesis work have been: 1. To evaluate the hierarchy of nucleotides in the DNA code read by p53 and NF-kB as sequence-specific transcription factors. 2. Investigate the conservation of transactivation capacity and specificity between yeast and mammalian systems for the tested panel of REs. 3. Estimate the contribution of physical properties of DNA (torsional rigidity or flexibility) contiguous to a p53 RE in influencing the strength and the direction of transcription. 4. Explore the molecular mechanisms underlying transcriptional synergy in response to Doxorubicin and TNF alpha treatment.