Epigenetic modification dependent DNA methyltransferases (DNMTs) play an important role in tissue- and stage-specific gene regulation and normal mammalian development. In this study, we show that DNMTs are expressed at different levels during hematopoietic stem cell (HSC) differentiation to proerythrocytes. DNMT1, DNMT3A, and DNMT3B were highly expressed at day 7 after differentiation. We used specific siRNA as a tool to probe the relationship between the expression of DNMTs and erythropoietic differentiation. When introduced siRNA of DMNT1 and DMNT3b in human CD34+ cells, these more differentiated into erythrocytes. This was confirmed by glycophorin A (GPA) positive cell analysis and globin gene expression. GPA+ cells increased up to 20～30%, and γ- and ε-globin genes increased in siRNA transfected cells. Therefore, our data suggest that suppression of DNA methylation can affect positively differentiation of HSC and may contribute to expression of erythrocyte lineage genes including GPA and globins.

ABSTRACT   INTRODUCTION    Isolation and Culture Conditions of Human CD34+ Cells    Cell line and Erthropoiesis Induction    Construction of siRNA Vectors for DNMT1, DNMT3b and Transfection in K562 and Human CD34+ Cells    Fluorescence Activated Cell Sorter(FACS) Analysis of Antigen Expression    Reverse-Transvcription (RT) PCR and Quantitative Real-Time PCR    Western Blotting    Statistical analysis   RESULTS    Differentiation of Human CD34+ Cells Originationg from Human UCB into Proerythrocytes    Expression of DNMTs during HSC Differentiation    Increment of Erythropoiesis in K562 and Human CD34+ Cells that Suppressed DNMTs   DISCUSSION   ACKNOWLEDGMENT   REFERENCES

• Seok-Ho Kim(Department of Oral Biochemistry, The 2nd Stage of KB21 Program for Dental School, Dental Science Research Institute)
• Hee-Young Yang(Department of Oral Biochemistry, The 2nd Stage of KB21 Program for Dental School, Dental Science Research Institute)
• Dong Kee Jeong(Department of Animal Science & Biotechnology, College of Applied Life Science, Cheju National University)
• Sang Ryeul Lee(Department of Oral Biochemistry, The 2nd Stage of KB21 Program for Dental School, Dental Science Research Institute)
• Zac Young Ryoo(School of Life Science and Biotechnology, Kyungpook National University)
• Tae-Hoon Lee(Department of Oral Biochemistry, The 2nd Stage of KB21 Program for Dental School, Dental Science Research Institute) Corresponding author