Title page
ACKNOWLEDGEMENT
Contents
ABBREVIATION 8
ABSTRACT 9
Ⅰ. Introduction 10
Ⅱ. Materials and methods 13
Ⅱ.1. Cell lines and culture condition 13
Ⅱ.2. In vivo ubiquitination assay 13
Ⅱ.3. In vivo co-immunoprecipitation for neddylation, sumoylation, and ISGylation assays 13
Ⅱ.4. Treatment of MG132 14
Ⅱ.5. Modification in Nanog sequence 14
Ⅱ.6. Reverse transcriptase-polymerase chain reaction (RT-PCR) 14
Ⅱ.7. Binding assay for Oct-3/4 and DUB-X 15
Ⅱ.8. DUB activity assay 15
Ⅲ. Results 16
Ⅲ.1. Nanog contains the putative PEST motif 16
Ⅲ.2. In vivo polyubiquitination of Nanog 18
Ⅲ.3. Effect of MG132 treatment on the Nanog expression 20
Ⅲ.4. Ubiquitination of PEST motif deletion form (ΔNanog) 22
Ⅲ.5. Screening of 55 DUB genes with the overexpression of Oct-3/4 in mES cells (ZHBTc4 cells) 24
Ⅲ.6. Interaction of DUB-X with Oct-3/4 in 293T cells 27
Ⅲ.7. The regulation of Oct-3/4 by DUB-X activity 29
Ⅳ. Discussion 31
Ⅴ. Conclusions 33
References 35
Summary in Korean 40
Figure 1. Identification of the PEST motif in Nanog sequences. PEST algorithm (https://emb1.bcc.univie.ac.at/) was used to identify the possible PEST motif sequence within the Nanog protein sequence. Amino acid sequences are shown for human Nanog (hNanog) and mouse Nanog (mNanog). 17
Figure 2. In vivo ubiquitination of Nanog. (A) Ubiquitination of Nanog was investigated by co-immunoprecipitation assay. Whole cell lysate (WCL) from COS-7 cells (lane 1), COS-7 cells transfected with pcDNA3.1-hyg-Flag- Nanog (lane 2), COS-7 cells transfected with pMT123-HA (lane 3), and... 19
Figure 3. (A) Effect of MG132 treatment on the Nanog expression. Overexpression of Nanog in COS-7 cells treated with a proteasome inhibitor MG132 revealed that Nanog protein level is increased. Mock (lane 1). COS-7 cells transfected with pcDNA3.1-Flag-hNanog (lane 2), COS-7 cells transfected... 21
Figure 4. (A) Deletion of the PEST motif in Nanog (ΔNanog). The structural architecture represents the Nanog full-length and the ΔPEST motif. (B) Flag-Nanog and Flag-ΔNanog constructs were analyzed by immunoblotting with an anti-Flag antibody and Western blotting with an anti-ubiquitin... 23
Figure 5. (A) ZHBTc4 cells transfected with a Tc-regulated Oct-3/4 transgene. In ZHBTc4 cells, induction of transgene was obtained for 48 hrs with the addition of Tc. Western blot analysis using an anti-Oct-3/4 antibody indicates the overexpression of Oct-3/4. (B) We designed 55 sets of DUB... 26
Figure 6. Oct-3/4 interacts with DUB-X in vivo. Co-immunoprecipitation of Oct-3/4 and DUB-X in 293T cells. Myc-tagged Oct-3/4 and myc-tagged DUB-X expressed in the 293T cells were confirmed by immunoboltting with an anti-myc antibody. Whole cell lysates (WCL) were immunoprecipitated with... 28
Figure 7. Myc-tagged Oct-3/4 was expressed in combination with HA-tagged ubiquitin and myc-tagged DUB-X in 293T cells. Ubiquitination of recombinant Oct-3/4 in 293T cells. Oct-3/4 expressed with or without the co-expression of DUB-X and DUB-X siRNA. WCL was confirmed by immunoblotting with an... 30
Figure 8. Ubiquitin-proteasome pathway of ES cell specific transcriptional factors. An ES cell specific transcription factor, Nanog, determines the fate of ES cells by specific functions for pluripotency and self-renewal. Moreover, we identified an interesting deubiquitinating enzyme, DUB-X, which directly... 34