Introduction: Long-term repression of the HPA axis is a major side effect of chronic glucocorticoid administration, even after attempted withdrawal of therapy, and complicates management in patients. We have previously shown that the effect of chronic glucocorticoid treatment in vitro is in part mediated by inhibition of expression the POMC transcription factors Neuro D1 and Tpit, and that this is sustained on treatment withdrawal. We have now further investigated the methylation state of Tpit. after chronic glucocorticoids and using RNA interference have analyzed directly the contribution that it makes to POMC expression.
Method: AtT20 cells were cultured with dexamethasone at a dose of 106 M for 40 weeks. DNA was extracted at 5, 20 and 40 weeks and bisulphite converted. The methylation of Tpit was assessed by bisulphite sequencing. siRNAs to Tpit were used to assess the specific contribution made by Tpit to the expression of POMC.
Results: Tpit undergoes de novo methylation after chronic treatment with dexamethasone: after 40 weeks 46% of CpG sites were methylated in the treated group compared with 0% in the non treated groups; at 20 weeks 43%, at 5 weeks there is very minor areas of methylation (only 3 CpG sites in one clone). In separate experiments knockdown of Tpit by RNA interference caused a 60% reduction in expression of POMC, confirming the critical importance of this factor.
Conclusion: We have now shown, for the first time, that glucocorticoid exposure causes de novo methylation of Tpit, associated with silencing of this gene and a dramatic reduction in POMC expression. To our knowledge this is the first example where hormonal exposure causes a DNA epigenetic imprint. Moreover, this may have clinical implications for use of demethylating agents in the context of steroid-withdrawal.