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Fig. 5 | Epigenetics & Chromatin

Fig. 5

From: Identification of a pituitary ERα-activated enhancer triggering the expression of Nr5a1, the earliest gonadotrope lineage-specific transcription factor

Fig. 5

ERα controls Nr5a1 expression through epigenetic regulation of the α enhancer. a Deletion of the α enhancer ERE-binding site using CRISPR/Cas9 in immature αT3–1 cells. Deletion of ERE genomic sequence in α enhancer was carried out in αT3–1 cells using CRISPR/Cas9 and a couple of specific gRNA flanking the ERE sequence. Untargeting gRNA was used as control. Two independent homozygous clones for both deletion and for control gRNA were sequenced. The genomic sequences of WT and deleted ERE (Δ ERE) αT3–1 clones are shown along with the ERE motif and the δERE–gRNA positions. b Deletion of the α enhancer ERE prevents ERα binding to the enhancer chromatin in immature αT3–1 cells. ERα binding on α enhancer chromatin was investigated using ChIP assays in WT and Δ ERE αT3–1 clones. Quantitative PCR was performed using primers targeting the α enhancer genomic sequence. Raw qPCR data were normalized to input. The final results were expressed as fold over the control region. Results are the mean ± SEM of six independent experiments. Significant difference with the control region was analyzed using Student’s t-test: “c” p < 0.001. c An intact ERα-binding site in the α enhancer is essential for Nr5a1 expression in immature αT3–1 cells. Nr5a1 expression in WT and Δ ERE αT3–1 cells was measured by RT-qPCR. Nr5a1 expression level was normalized to Gapdh. Data are the normalized mean ± SEM of six independent experiments. Significant difference with the WT using Student’s t-test: “c” p < 0.001. d Abolition of the ERα binding site in α enhancer leads to a strong reduction in the α enhancer chromatin accessibility in immature αT3–1 cells. The α enhancer chromatin accessibility was investigated using DNAse I hypersensitivity (DNase I HS) assay in WT and Δ ERE αT3–1 clones. Quantitative PCR was performed using primers targeting the α enhancer genomic sequence. Raw qPCR data were normalized to input. The final results were expressed as fold over the control region. Results are the mean ± SEM of four independent experiments. Significant difference with the WT using Student’s t-test: “c” p < 0.001. e Abolition of the ERα-binding site in the α enhancer prevents active chromatin marks deposition on the α enhancer and 1G promoter in immature αT3–1 cells. Monomethylation of Lys4 (H3K4me1), acetylation of Lys27 on histone H3 (H3K27ac) and trimethylation of Lys4 (H3K4me3) epigenetic modifications as well as binding of P300 and serine 5-phosphorylated RNA polymerase II (S5P Pol II) to the α enhancer and/or 1G promoter sequences were studied using ChIP assays in WT and Δ ERE αT3–1 clones. Quantitative PCR was performed using sets of primers targeting the α enhancer or 1G promoter sequence. Raw qPCR data were normalized to input. The final results are expressed as fold over the control region. Results are the mean ± SEM of six independent experiments. Significant difference between WT and Δ ERE αT3–1 clones for each mark and each cis-regulatory element was studied using Student’s t-test: “b” p < 0.01; “c” p < 0.001. f Abolition of the ERα-binding site in the α enhancer leads to CpG hypermethylation of the α enhancer chromatin in immature αT3–1 cells. Genomic DNA of WT and Δ ERE αT3–1 clones was extracted and bisulfited. The α enhancer-bisulfited sequences were amplified and cloned. A minimum of nine clones per cell line was sequenced. Top: schematic representation of the α enhancer sequence with location of CpG (open-circle lollipops) and of the ERE site. Below is indicated the state of CpG methylation for each cell line, methylated (black circles) or unmethylated (open circles)

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