Identification Of The Nuclear Localization Signals Within The Mouse TET1 Protein

Epigenetics, which is defined as the investigation of heritable changes in gene expression that occur without a change in DNA sequence, play a critical role in stem cells renewal, maintenance and differentiation into specific cell lineages. DNA methylation is one of the main epigenetic modifications that can induce repressive of gene expression. The ten-eleven translocation(TET) family proteins are the recently identified proteins that could catalyze the conversion of 5-methylcytosine(5mC) of DNA to 5-hydroxymethylcytosine(5hmC). This conversion induces demethylation of CpG islands in target gene promoters and subsequently transcriptional activation.The nuclear pore complex(NPC) is in charge of the exchange of macromolecules between the nucleus and the cytoplasm of eukaryotic cells. Small compounds can get into the nucleus through the NPC freely or by passive diffusion. However, those proteins whose molecular weights are larger than 50 kDa can only get into nucleus by active transport. These active transport proteins must contain one or more special signals, namely NLSs. Presently, NLSs are classified into three categories, including classical NLSs(cNLSs), which can be recognized by importin-α and importin-β1, PY-NLSs, which can be recognized by importin-β2, and some other NLSs.TET1 acts as a transcription factor located in the nucleus. The TET1 protein is a 2039-amino-acid DNA demethylase that is predicted to possess eighteen nuclear localization signals(NLSs), thirteen monopartite and five bipartite. In this paper, we used a series of green fluorescent protein-tagged and mutation constructs to identify two NLSs of mouse TET1, one NLS(KKRK) embedded between amino acid 603 and 606, and the other NLS(RKRR) embedded between amino acid 1720 and 1723. They facilitates the cytoplasmic protein’s translocation into the nucleus.The main results are as follows:1. Mouse TET1 was cloned into vector pCMV-HA-TET1 to overexpress TET1 with the total RNA of J1 mESCs. Transfection was carried out using the X-tremeGENE HP DNA Transfection Reagent(Roche, USA) according to the manufacturer’s procedure. Immunofluorescent staining revealed that TET1 localized to the nucleus of cells, this results indicated that TET1 must contain one or more functional NLSs.2. Eighteen predicted NLSs were identified within the TET1 protein using PSORT II(http://psort.hgc.jp/form.html). Deletion constructs of TET1 were prepared to determine which part could direct EGFP to the nucleus. Besides, each one contained at least one of these NLSs. All fusion proteins were too large for nuclear pore complex(NPC) transport of small molecules through passive diffusion. In contrast to EGFP alone, which was distributed diffusely in both the cytoplasm and the nucleus, the fusion protein EGFP-TET1-275-862, and EGFP-TET1-1463-1885 localized to the nucleus of cells. These results indicated that the functional NLSs present in TET1 were likely to be embedded in these two fragments.3. To determine whether these NLSs were functional, truncation experiments and site-directed mutagenesis were performed. With a series of green fluorescent protein-tagged and mutation constructs, we identified two functional NLSs of mouse TET1. TET1 possesses two conserved domains, a CXXC domain and a catalytic domain. Interestingly, 603KKRK606 locates in the CXXC domain, and 1720RKRR1723 locates in the catalytic domain. The TET1 proteins of Mus musculus, Rattus norvegicus, Homo sapiens, Macaca nemestrina, Bos taurus and Sus taurus were compared relying on bioinformatics. Multiple sequence alignment of the KKRK region and the RKRR region with TET1 orthologous proteins showed that 603KKRK606 is highly conserved while 1720RKRR1723 is not that conserved among the species examined. However, alignment of the KKRK region and the RKRR region with the other two members of the TET family showed that KKRK or RKRR is not conserved at all.Above all,we constructed the eukaryotic expression vector pCMV-HA-TET1. And we identified two functional NLSs of mouse TET1, 603KKRK606 locates in the CXXC domain, and 1720RKRR1723 locates in the catalytic domain. And 603KKRK606 is highly conserved while 1720RKRR1723 is not that conserved among the species we examined. The results provide new insight and theoretical basises for the research of mechanisms of TET1 nuclear transport.