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. 2001 Apr 17;20(8):1974-83.
doi: 10.1093/emboj/20.8.1974.

Glucocorticoid-induced DNA demethylation and gene memory during development

H Thomassin  1 , M Flavin, M L Espinás, T Grange
Affiliations

Glucocorticoid-induced DNA demethylation and gene memory during development

H Thomassin et al. EMBO J. .

Abstract

Glucocorticoid hormones were found to regulate DNA demethylation within a key enhancer of the rat liver-specific tyrosine aminotransferase (Tat) gene. Genomic footprinting analysis shows that the glucocorticoid receptor uses local DNA demethylation as one of several steps to recruit transcription factors in hepatoma cells. Demethylation occurs within 2-3 days following rapid (< 1 h) chromatin remodeling and recruitment of a first transcription factor, HNF-3. Upon demethylation, two additional transcription factors are recruited when chromatin is remodeled. In contrast to chromatin remodeling, the demethylation is stable following hormone withdrawal. As a stronger subsequent glucocorticoid response is observed, demethylation appears to provide memory of the first stimulation. During development, this demethylation occurs before birth, at a stage where the Tat gene is not yet inducible, and it could thus prepare the enhancer for subsequent stimulation by hypoglycemia at birth. In vitro cultures of fetal hepatocytes recapitulate the regulation analyzed in hepatoma cells. There fore, demethylation appears to contribute to the fine-tuning of the enhancer and to the memorization of a regulatory event during development.

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Figures

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Fig. 1. Prolonged glucocorticoid treatment induces a stable DNA demethylation at the –2.5 Tat GRU. Rat hepatoma cells (H4II) were grown with 10–7 M dexamethasone (+Dex) for the indicated time (0, no hormone addition) and the corresponding genomic DNA was treated with hydrazine and piperidine. The upper strand of the –2.5 Tat GRU was analyzed by LM–PCR.
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Fig. 2. Schematic representation of the –2.5 Tat GRU region. The relative location of the transcription factor binding sites, the dinucleotide CpGs with their methylation status, and the extent of the region where chromatin is remodeled following glucocorticoid stimulation are indicated. The data not described in the present study originate from other sources (Grange et al., 1991; Reik et al., 1991; Espinás et al., 1994; Roux et al., 1995; M.Flavin, L.Cappabianca, H.Thomassin and T.Grange, in preparation). DR0-TF, transcription factor(s) interacting with the DR0 site; MeS-TF, methylation-sensitive transcription factor.
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Fig. 3. The first glucocorticoid stimulation enhances the subsequent hormonal response. Glucocorticoid-induced variations in TAT mRNA levels in H4II cells were analyzed by northern blotting using a TAT cDNA probe (Grange et al., 1985). After stripping of the probe, the blots were reprobed for an internal control with a cDNA of a housekeeping gene, that coding for poly(A) binding protein (PABP) (Grange et al., 1987). The graph shows the variation with time of the corrected TAT mRNA levels following quantitative analysis of the blots using a phosphoimager.
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Fig. 4. DNA demethylation permits glucocorticoid-dependent loading of a transcription factor at the demethylated sites. (A) Genomic footprinting of hepatoma cells using DMS. Methylated cells, naive H4II cells; demethylated cells, H4II cells treated for 4 days with 10–7 M Dex and grown for 1 month in the absence of GR ligand. Cells were grown with Dex for the indicated time prior to treatment with DMS. The upper strand of the –2.5 Tat GRU was analyzed by LM–PCR. (B) Mobility shift analysis of factor binding as a function of DNA methylation. Crude nuclear extracts prepared from H4II cells treated or not with Dex for 24 h were incubated with double-stranded oligonucleotide probes (–2399/–2425) containing either only unmethylated cytosines (lanes 1 and 2) or methylated cytosines at all CpGs (lanes 3 and 4).
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Fig. 5. DNA demethylation is associated to glucocorticoid-dependent loading of another transcription factor nearby a demethylated site. Genomic footprinting of hepatoma cells using DNase I. The lower strand of the –2.5 Tat GRU was analyzed by LM–PCR.
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Fig. 6. Glucocorticoid-induced DNA demethylation during development. (A) Liver-specific demethylation of the Tat GRU occurs before birth and parallels the glucocorticoid peak. DNA methylation was analyzed at various time points in the perinatal period as described in Figure 1. The postnatal days (d) are indicated using birth (B) as the reference. The prenatal stages analyzed are e.d.15 and e.d.19. (B) Glucocorticoid-induced demethylation and chromatin remodeling in primary cultures of e.d.15 fetal rat hepatocytes. After isolation, fetal hepatocytes were cultured in vitro for the indicated time without (0) or with 10–7 M dexamethasone. (a) DNA methylation analysis as described in Figure 1. Similar demethylation was observed using bisulfite-PCR analysis (data not shown). (b) Chromatin remodeling analysis as assessed by restriction enzyme accessibility. Nuclei from fetal hepatocytes were incubated with XbaI for 1 h prior to DNA isolation and indirect-end labeling was performed as described in Materials and methods. The band corresponding to cleavage by XbaI at the –2.5 GRU (at position –2558) is indicated. (C) Liver-specific demethylation of the Tat promoter occurs several days after birth. Data were obtained as described in (A), except that the upper strand of the proximal promoter was analyzed (Rigaud et al., 1991).
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