Abstract
Increased fetal hemoglobin (HbF) can alleviate the symptoms and increase the life span of patients with sickle cell disease. The development of new methods to increase HbF in patients has been hampered by the lack of cell line models. Recently a new murine fetal bone marrow (FBM) cell line containing the human β-globin gene locus in the context of a yeast artificial chromosome (βYAC) whose growth is dependent on the chemical inducer of dimerization (CID) AP20187 (Ariad Pharmaceuticals) was described (Blau et al, J Biol Chem280: 36642, 2005). The DNA methyltransferase (DNMTase) inhibitor 5-azacytidine increased γ-globin gene expression in this cell line suggesting that it is a good model system for studying the mechanism of DNA methylation in γ-globin gene silencing. To investigate the role of methylated DNA binding proteins in γ-globin silencing, we transduced this cell line with retrovirus vectors expressing shRNA targeting the methylated DNA binding proteins MBD2 and MBD3. Transduced pools were selected for puromycin resistance and the effect on expression of the target gene and γ-globin determined by real time PCR using the ΔΔCT method. Single cell clones were isolated by limited dilution platings for further analysis. No difference in γ-globin expression was observed between clones derived from the parental cell line and cells transduced with a nonsilencing control vector. MBD3 expression was decreased 70–85% but γ-globin expression was not affected in the selected pool or in 12 clones following transduction wth a vector targeting MBD3. In 2 selected pools transduced with an shRNA vector targeting MBD2, MBD2 expression was decreased 60 and 82% and γ-globin expression increased 3.7 fold and 4.76 fold. Mean expression of γ-globin was 3.83 fold higher in 14 clones that showed >90% decrease in MBD2 mRNA compared to 11 clonal isolates derived from cells transduced with the non-silencing control vector (p<.05). In three clones that showed the greatest induction of γ-globin, the fold change (mean +SD) of ε-, γ-, and β-globin expression was 3.98±1.75, 11.29±4.27 and 1.90±1.34, respectively, demonstrating that MBD2 knockdown increased both ε- and γ-globin expression. The level of MBD2 protein in these clones and in two additional clones that showed >90% decrease in MBD2 mRNA but no induction of γ-globin expression were compared by Western blot analysis. MBD2 was decreased >90% in all clones compared to the parental line. Decreased MBD2 levels were therefore not associated with increased γ-globin expression in all clones. We then compared shRNA-mediated MBD2 knockdown with the DNMTase inhibitor decitabine for effects on ε- and γ-globin expression. Expression of ε-globin was increased 76.0±6.1 fold, γ-globin 131±10.2 fold, and β-globin 3.6±0.38 fold following treatment with decitabine (1 × 10−6 M; 48 hours; n=3). We conclude that decreased expression of MBD2 following transduction of the CID-dependent mouse FBM βYAC cell line with an shRNA vector targeting MBD2 is associated with increased γ-globin expression, confirming previous results of analysis of βYAC-MBD2 knockout mice (Rupon et al, PNAS103: 6617, 2006). However, the increase in γ-globin expression was approximately 10 fold greater following decitabine treatment than was achieved with shRNA-mediated MBD2 knockdown.
Ligatoxin B, a new cytotoxic protein with a novel helix–turn–helix DNA-binding domain from the mistletoe Phoradendron liga