Expression of members of the miR-290s and pluripotency factors gradually decreases during E14.1 ES cells differentiation. (A) Quantitative RT–PCR analysis of EEmiRC miRs in ES (black bar), neural progenitor cell at Day 4 of differentiation (NPCD4) (white), neural progenitor cell at Day 8 of differentiation NPCD8 (gray) and MEFs (white/gray); expression levels were normalized relative to Sno234. (B) Expression levels of Oct-3/4 and Nanog (relative to Histone4, H4) in ES, NPCD4, NPCD8 and MEF’s. Data are represented as means ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01.

En-II.1a and its vicinity are highly enriched in Oct3/4, Sox2 and CTCF. (A) Schematic representation of the EEmiRC genomic regions (P1–P4) subjected to ChIP-qPCR analysis. P3 and P4 are highly enriched in Oct3/4 (B), Sox2 (C) and CTCF (D) when compared to IgG control (E). Doxycyclin-induced depletion of Oct3/4 in ZHBTc4 ES cells strongly reduces the recruitment of Sox2 (F) and CTCF (G) to P3 and P4 regions when compared to IgG controls (H). Data are represented as mean ± SD from three different experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Identification of En-II.1a enhancer within the EEmiR cluster. (A) Schematic representation of genomic regions used in the luciferase reporter assays; nucleotide positions are relative to the EEmiRC transcriptional start site. The different genomic regions of the inter-hC were cloned in to pGL-4.23 and pGL-4.23dMP (without the minimal promoter) vector and tested for the ability to promote luciferase transcription after transfection in HEK293T, mESCs and P19CL6 cells. (B) Delineation of the inter-hC enhancer using deletion constructs. The 332bp fragment (En-II.1a) shows significant enhancer activity in HEK293T, mESCs and P19CL6 cells. (C) The En-II.1.a fragment cloned downstream of Luciferase enhances transcription from the endogenous miR290 promoter in a pGL-4.23dMP vector transfected in mESCs. Data are normalized to pGL-4.23dMP readings and represented as mean ± SD from three different experiments. (D) Deletion of En-II.1a significantly reduces the level of expression of EGFP from a pEEmiRC-EGFP construct transfected into mESCs. Data are normalized to Renilla transcripts and represented as mean ± SD from three different experiments. *P < 0.05; **P < 0.01.

En-II.1a enhancer is subject to epigenetic regulation. (A) Schematic representation of the 222 bp CpG island within the inter-hC region. (B) DNA methylation analysis of the 12 CG dinucleotides of the CpG island in ES, NPCD4, NPCD8 and MEFs using the bisulphite-sequencing approach. Each column represents a CpG dinucleotide, each row represents the number of clones analysed. Open circles represents the unmethylated CpGs, closed circles represents the methylated CpGs. (C) In vitro methylation of pGL-4.23-En-II.1a construct using M.SssI leads to a loss of the enhancer activity as assesed by luciferase assay. The enhancer activities were normalized to M.SssI-treated and -untreated empty vectors and represented as relative luciferase units (RLU). (D) Treatment of MEFs with the DNA methyltransferase inhibitor 5′-azacytidine (5′-azaC), HDAC inhibitor trichostatin A (TSA) or a combination of both reactivates the expression of the members of the EEmiRC. (E and F) ChIP–qPCR analysis of P1–P4 enrichment in H3K4 (E) and H3K27 (F) trimethylation in ESCs (black bar), NPCD4 (white bar), NPCD8 (gray bar) and MEFs (white/gray bar). Data in (C–F) are represented as mean ± SD from three different experiments. *P < 0.05; **P < 0.01; ***P < 0.001.