Glucocorticoids are critical components of combination chemotherapy for treating acute lymphoblastic leukaemia (ALL), although mechanisms of glucocorticoid resistance appear to be multifaceted. The pro-apoptotic BIM gene is important for mediating glucocorticoid-induced apoptosis in normal and malignant lymphoid cells. Previous work in our lab showed that a BIM intronic glucocorticoid-receptor binding region (IGR) acts as an enhancer for regulating BIM gene expression and is silenced in glucocorticoid-resistant ALL cells. CTCF, a DNA architectural protein, showed enriched binding at the BIM IGR in glucocorticoid-sensitive ALL cells. Here, we investigated the role of CTCF at the BIM IGR in regulating glucocorticoid-induced BIM expression.
First, we determined CTCF binding sites at the BIM IGR. MEME-ChIP analysis of ChIP-seq data derived from a glucocorticoid-sensitive ALL xenograft (ALL-54S) treated in vivo in immune-deficient NSG mice with dexamethasone (a synthetic glucocorticoid drug) identified a CTCF motif, which led to the identification of two CTCF binding sites. Interestingly, this CTCF motif is different to the published CTCF motif in the ENCODE ChIP-seq datasets, which suggested a third CTCF binding site. The BIM IGR sequences with wild-type or mutated CTCF binding sites were inserted into the pGL2P luciferase reporter vector to conduct luciferase reporter assays in the glucocorticoid-sensitive human ALL cell line, Nalm6. Our results showed that mutating any of the three CTCF binding sites individually did not affect dexamethasone-induced luciferase expression. However, when all three CTCF binding sites were mutated at the same time, dexamethasone-induced luciferase expression was significantly reduced. Furthermore, performing CRISPR/Cas9 mediated gene knockout (KO) studies in Nalm6 cells, we found that KO of the 50 bp CTCF binding site at the BIM IGR or the whole 1 kb BIM IGR region in Nalm6 cells diminished BIM induction following dexamethasone treatment.
In conclusion, we have demonstrated that CTCF plays an important role in the regulation of glucocorticoid-induced BIM transcription, which reveals a novel epigenetic mechanism associated with glucocorticoid resistance in paediatric ALL cells.