A Study On The Role Of OTUB1, A Novel TPP1Interacting Protein, In Telomere Length Regulation

Telomere are specialized structures consist of repetitive DNA fragments andspecific protein complexes at the ends of eukaryotic chromosomes. It can preventlinear chromosomes from fusion and DNA damage responses to maintain genomicintegrity. Telomere is protected by six core proteins called shelterin (TRF1, TRF2,TIN2, RAP1, TPP1and POT1) and other associated regulatory proteins. The telomereassociated regulatory proteins along with six core telomere proteins, form acomplicated network and cooperatively participate in telomere regulation anddynamics.A novel telomere related regulatory protein-OTUB1was found by PICh(proteomics of isolated chromatin segments). OTUB1is a deubiquitin enzyme,belongs to the cysteine enzyme family which contains ovarian tumour (OTU) domain.It has been reported that many different substrates were regulated by OTUB1. Byimmunofluorescence experiments, we confirmed its location on telomeres. Depletionof endogenous OTUB1expression by small hairpin RNA increased the level of DNArepair protein53BP1gathered on genomic DNA，not specifically on telomeres.Overexpression of OTUB1mutant m2(1-157amino acids deleted) significantlyextended telomere length in HTC75cells，about15bp per population doubling.Overexpression of OTUB1dN (1-40amino acids of the N terminal deleted) alsolengthened telomere10bp per population doubling. In contrast, overexpression of other OTUB1mutants did not change telomere length. These results suggested thatthe N terminal1-157amino acids were very important for OTUB1to maintaintelomere length.Further through BiFC (bimolecular fluorescence complementation) and GSTpull-down experiments, we found an apparent interaction between OTUB1and TPP1.In addition, OTUB1could upregulate the protein level of TPP1, both in the nucleusand cytoplasm. The phenotypes suggest that OTUB1plays an important role intelomere protection and length maintaining. We are planning to investigate TPP1ubiquitination in order to explore the mechanism of OTUB1in telomere regulation.