p60-katanin (KATNA1) gen promotörünün ELK1 ile regülasyonu

Microtubules are highly dynamic cytoskeletal elements, and they are important for chromosome segregation and intracellular transport in mitotic cells as well as neuronal morphogenesis and branching of neurons. Microtubule severing contributes their dynamic nature as a result of leading to depolymerization and polymerization. Katanin has microtubule severing activity and exists as a heterodimeric protein in the cell. There are two subunits of katanin; p60-katanin encoded by KATNA1 and p80-katanin encoded by KATNB1. Microtubule severing is mainly conducted by p60-katanin due to AAA domain and p80-katanin is responsible for enhancing its enzymatic activity. It has been known that katanin severing induces non-centrosomal microtubules in neurons. Also, neuronal branching is associated with microtubule severing. Elk1 is a transcription factor which has a role in cell proliferation, neuronal differentiation and tumor progression. It can act both as transcriptional activator or repressor of a target gene. C domain of Elk1 is known as activation domain while ETS domain and R domain are related with repression. Elk1 has two main post-translational modifications; phosphorylation and SUMOylation. ERK/MAPK phosphorylates Serine 383 and Serine 389 at the C domain. It has been indicated that Lysines located on 230, 249 and 254 at R domain are essential for SUMOylation and point mutations of these amino acids prevent SUMO conjugation. KATNA1 includes M3 motif due to containing TATA-less promoter and this motif corresponds to Elk1 consensus sequence. Besides, in our laboratory, it has been revealed that Elk1 plays a role in KATNB1 gene expression which encodes p80-katanin subunit and SPG4 gene expression which encodes the other microtubule severing protein spastin. The expression mechanisms and regulation of microtubule severing proteins are important for many cellular processes particularly in neurons. In the light of this information, the effect of Elk1 on KATNA1 regulation was investigated in this study. Firstly, the binding site of Elk1 was determined by in vitro and in vivo assays. EMSA was performed using recombinant protein and oligonucleotides that include different binding sites of Elk1 on KATNA1 promoter and 5' UTR. KATNA1 promoter characterization has been previously identified in our laboratory and also 5' UTR is detected as the critical region for KATNA1 transcription. After determining the main binding site, binding was confirmed by ChIP assay. Elk1 regulation on KATNA1 mRNA was investigated through qRT-PCR and it was found that mRNA level was increased due to Elk1 over-expression. However, luciferase reporter assay, Western blot and immunocytochemistry experiments revealed the repressor activity of Elk1 on p60-katanin protein. Also, the effect of Elk1 SUMOylation was investigated by generating point mutations in the R domain as described previously. According to the results of qRT-PCR, luciferase reporter assay, Western blot and immunocytochemistry, no significant change was detected between the effects of wild-type and mutated Elk1. However, it was observed that mutated Elk1 was localized in both nucleus and cytoplasm despite the previous studies which indicate the rapid re-localization of mutated Elk1 to the nucleus. Thus, the SUMOylation levels of both wild-type and mutated Elk1 constructs were investigated through co-immunoprecipitation experiments. It was shown that mutated Elk1 could also be SUMOylated. It has been known that TATA-less promoters have high GC content. Thus, CpG islands in KATNA1 promoter and 5' UTR were investigated through bioinformatic analysis and a CpG island which includes previously identified Elk1 binding site was found. Thus, KATNA1 oligonucleotide was methylated in order to investigate whether methylation is a regulatory mechanism of p60-katanin expression in terms of Elk1 binding ability. EMSA was performed by using both methylated and unmethylated oligonucleotides. Results showed that Elk1 binding ability was decreased due to methylation.