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Endocrine Abstracts (2014) 34 P291 | DOI: 10.1530/endoabs.34.P291

SFEBES2014 Poster Presentations Pituitary (36 abstracts)

Chronic glucocorticoid exposure inhibits expression of the pomc activator, tpit, by inducing de-novo DNA methylation

Georgia Bakirtzi & John Newell-Price


The University of Sheffield, Sheffield, UK.


Introduction: The HPA axis is essential for mammalian life. Glucocorticoids are commonly administered and long-term HPA axis suppression is a major clinical problem. Previous experiments in our lab have shown that long-term treatment with glucocorticoids cause silencing of tpit, a pituitary-specific master regulator of the key player in HPA axis regulation – propiomelanocortin (pomc). Even after withdrawal of treatment tpit expression is silenced.

Hypothesis: Chronic glucocorticoid exposure causes de novo DNA methylation of tpit inhibiting its expression and so influencing HPA axis regulation even after withdrawal of long-term glucocorticoid treatment.

Methods: Methylation patterns of tpit were studied by bisulphite sequencing in murine POMC-expressing (AtT20) and non-POMC expressing (3T3-L1) cell lines. A tpit putative promoter region was cloned into a CpG-free vector in order to study the effect of specific CpG methylation.

Results: The region of DNA immediately 5’ to the transcription start site of tpit is methylated in the non-POMC expressing 3T3-L1 cell line, but unmethylated in AtT20 cells. Following chronic glucocorticoid exposure this region becomes de-novo methylated in AtT20 cells. A 410 bp region of DNA corresponding to this area has promoter activity as assessed in transient transfection experiments in AtT20 cells using a reporter construct that lacks CpG sites in the backbone. Specific methylation of the seven CpG sites alone in the ‘promoter’ caused inhibition of expression.

Conclusion: Chronic glucocorticoid exposure causes de novo methylation and silencing of tpit expression. These data have important implications for use of epigenetic modifying agents after chronic glucocorticoid therapy to allow recovery of the HPA axis.

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