Role of Sox2 O-GlcNAc modification in embryonic stem cells

다국어 초록 (Multilingual Abstract)

SRY (sex determining region Y) -box 2, also known as Sox2, is a transcription factor essential for the maintenance pluripotency of embryonic stem cells (ESCs) along with Oct4. O-linked β-N-acetylglucosamine (OGlcNAc) modification reflects cellular nutritional status and regulates pluripotency. Among transcription factors of core components of the pluripotency networks, Oct4 and Sox2 have been known to be modified by O-GlcNAc on threonine 228 (T228) for Oct4 and on Serine 248 (S248) and threonine 258 (T258) for Sox2. Although the role of O-GlcNAc modification on Oct4 has been studied extensively, that of Sox2 is not clear yet.
Here we found that O-GlcNAc modification of Sox2 on T258 is important for the maintenance of ESCs. When endogenous Sox2 was substituted with various O-GlcNAc-defective Sox2 mutants, a T258A point mutation reduces the capacity of Sox2 to maintain ESC self-renewal, whereas A S248A, T258A double mutation restores the capacity, suggesting that posttranslational modifications on two sites play the opposite role. To confirm the role of T258A in more physiological condition, we mutated endogenous chromosome of Sox2 wild type (WT) to T258A using CRISPR-cas9 system. ESCs with one allele Sox2 T258A mutation were prone to differentiate, and ESCs with homologues Sox2 T258A mutation were not obtained. Because O-GlcNAcylation usually controls cellular localization, protein stability, and protein-protein interactions, we investigated whether Sox2 T258A mutation affected those things. Both Sox2 WT and T258A were localized in nucleus and their protein stability is not significantly differ. We purified Sox2-interacting-proteins complex and identified them by liquid chromatography-mass spectrometry. We found about 800 Sox2 interacting proteins. Among them 164 proteins were not found in Sox2 T258A complex, suggesting that the interaction between these 164 proteins and Sox2 is dependent on T258 O-GlcNAcylation. Combined analysis of RNA-sequencing, microarray, and chromatin immunoprecipitation- sequencing data showed that a Sox2 T258A point mutation increased the expression of genes in the lineage-developmental process, which suggests Sox2 suppressed developmental genes T258 O-GlcNAcylation dependently. Of those proteins whose interaction with Sox2 were dependent on T258 O-GlcNAcylation, we found 40 transcription factors, whose roles are negative regulation of gene expression and regulation of embryo development. Among them, we found Otx2, a transcription factor, which has been reported to regulate early stage embryogenesis and embryonic stem cells, co-occupied lineage-specific genes promoters with Sox2 and nucleosome remodeling and deacetylation complex (NuRD) complex. In summary, O-GlcNAcylation of Sox2 T258 is important for the interaction between Sox2 and Otx2, which in turn is important for the suppression of genes in the developmental process.

목차 (Table of Contents)

• I. INTRODUCTION 1
• 1-1. Role of Sox2 in ESCs . 2
• 1-1-1. Sox2: Core regulator of embryonic stem cell pluripotency 2
• 1-1-2. Expression of Sox2 during development. 2
• 1-1-3. Critical role of Sox2 in development 3
• 1-2. Regulation of Sox2 . 5
• 1-2-1. Sox2 protein structure . 5
• 1-2-2. Post-translational modification of Sox2 6
• 1-3. O-GlcNAcylation of Sox2 . 9
• 1-4. Purpose . 11
• II. MATERIALS AND METHODS . 16
• 2-1. Cell culture . 17
• 2-2. DNA construct, chemicals and antibodies . 18
• 2-3. Colony forming assay 18
• 2-4. Immunoprecipitation 18
• 2-5. Immunofluorescence 19
• 2-6. Western blot analysis 19
• 2-7. RNA, DNA precipitation 19
• 2-8. RNA sequencing 20
• 2-9. Real-time PCR . 20
• 2-10. Purification of Sox2 complex 20
• 2-11. Identification of Sox2 interacting Proteins. 21
• 2-11-1. Protein Digestion and Peptide Cleanup . 21
• 2-11-2. LC-MS/MS analysis 21
• 2-11-3. Database search . 22
• 2-12. Flow cytometry analysis and cell sorting . 22
• 2-13. Site mutation of threonine 258 of Sox2 in ESCs by CRISPR-Cas9 23
• 2-14. Statistical analysis 23
• III. RESULTS 25
• 3-1. Sox2 T258A disrupts self-renewal ability in mESCs . 26
• 3-2. WT and T258A Sox2 have no difference in cellular localization and protein stability 30
• 3-3. Sox2 O-GlcNAcylation affects interaction of Sox2 with various proteins 33
• 3-4. Sox2 T258A is upregulated in developmental signature 36
• 3-5. Otx2 and Sox2 suppresses developmental genes in dependent of O-GlcNAcylation . 40
• IV. DISCUSSION 42
• V. REFERENCES . 47
• VI. ABSTRACT IN KOREAN 61