Interaction between transcription factor p53 tumor suppressor and p60-katanin (KATNA1) promoter

Cytoskeleton is a network of filamentous structures which are spread throughout the cytoplasm. Cytoskeleton play roles in vital functions such as movement of the cells, cell shape, mitosis, cytokinesis, intracellular transport of molecules. Cytoskeleton consists of three distinct polymeric fibers. Microfilaments are required for maintenance of cell shape, cytokinesis, separation of the dividing cell into two and cell surface preparation for adherence. Other component of cytoskeleton, intermediate filaments provide mechanical strenght and mechanical linkage. The final member of cytoskeleton is microtubules that have crucial roles such as drawing the chromosomes apart in mitosis, shaping morphological structure, intracellular traffic to carry vesicules, cell motility, formation of axons and dentrites and therefore, the neuronal structure. Microtubules are structural polymer proteins and consist of α-tubulin and β-tubulin heterodimers. Microtubules are dynamic structures resulting from rapid polymerization and depolymerization of tubulin monomers by a process known as "dynamic instability". Reconfiguration of the microtubules by these mechanisms is responsible for microtubule growing and shrinking. When dynamic instability, speed and time for branching for special neuronal morphology are considered, it can be easily understood that cells need another mechanism to explain the movement of intracellular microtubules. "Cut and run" model proposes that microtubules are cut into small pieces by severing enzymes; katanin, spastin and fidgetin. These enzymes are members of AAA family of ATPases. Because microtubules lose their ability to move when they are long, microtubule severing is important in terms of movement capacity. Katanin and spastin have same working mechanism, but, they provide different severing approach. Spastin encoded by SPG4 gene. Katanin is a heterodimer of p60-katanin encoded by KATNA1 gene and p80-katanin encoded by KATNB1 gene. p60-katanin has the enzymatic activity and it has AAA ATPases region but p80-katanin enzyme do not have the enzymatic activiy. According to studies, katanin is associated with neurologic diseases. Katanin is ubiqutiously expressed in nervous system and its inhibition or overexpression impairs axon formation. Thus, it is clearly understood that katanin has vital role for microtubule reorganisation and axon growing. Hypophosphorylated form of tau protein normally provides stabiliziation of microtubules and protects microtubules from the severing enzymes. Hyperphosphorylation of tau results in dissociation of tau from microtubules and thus, microtubules become accesible for severin enzyme katanin. The enzymatic activity of katanin is proportional to level of enzyme so regulation of katanin protein level via transcription mechanism comes into prominence. p53 is important for many cellular mechanisms as a tumor supressor in processes such as cell cycle regulation, metabolism, apoptosis, DNA replication, immunity responses, proliferation and differentiation. In our previous study, we have shown that neuronal processes were retracted upon PKC activation following increases in both p60-katanin and p53 levels in neurons. This result led us to analyze changes in p60-katanin, which is organizing the neuronal cytoskeleton. We showed that cells which have enhance p60-katanin protein level also had increase in p53 transcription factor, which is related on neuronal differentiation. In this study, we aimed to identify regulatory DNA sequences of KATNA1 gene and possible regulation of p60-katanin by p53 trasnscription factor. For this purpose, we first decided to characterize regulatory regions of KATNA1 gene. Primarly, the putative transciptional regulatory regions of p60-katanin were identified by bioinformatic methods. p60-katanin regions (336 bp promoter, 448 bp 5'UTR, 784 bp promoter + UTR, 2682 bp intron + UTR and 3000 bp promoter + UTR + intron) were cloned for the identification of their regulatory activities on p60-katanin expression by Luciferase assay. Then, p60-katanin promoter, p60-katanin UTR, p60-katanin promoter + UTR and p60-katanin promoter + p60-katanin UTR + p60-katanin intron regions were amplified by PCR and after required restriction all regions prepared. We identified that 5'-UTR region enhanced transcriptional activity of the reporter gene, but not putative promoter region and the presence of the intron decreased the activity. Thus, expression of katanin-p60 seems to be regulated via 5'-UTR, and intron-1 might have repressor elements. When potential gene regulatory regions were analyzed by bioinformatics software, presence of a CpG island that comprising promoter and 5'-UTR regions (from -615 to -918 bp) was identified. Next, we identified p53 consensus sequence on KATNA1 gene bioinformatically. Therefore, it is thought that KATNA1 gene could possibly be regulated by p53 transcription factor. We started with analyzing the binding of p53 to the corresponding promoter region. To further confirm the specificity of the p53 binding to KATNA1 promoter was confirmed by Chromosome Immunoprecipitation (ChIP) and also by Electrophoretic Mobility Shift Assay (EMSA) using oligonucleotides including related transcription factor binding sites. On the other side, our observation indicated that, p53 acts as an activator or repressor KATNA1 gene promoter. The result for KATNA1 gene was confirmed in mRNA level by performing real time PCR.