The GRB Model.

<p>GRB has developmental and/or transcription factor gene (target gene, orange) spanned by a cluster of highly conserved non-coding elements (red ovals), which regulates the target gene expression by acting as enhancers/insulators and other un-related neighboring genes (bystander genes, green).</p

The dissimilarity matrix of HCNE content among nuclear receptors and its clustering.

<p>Nuclear receptor genes broadly divided in to two clusters on the basis of higher and lower enrichment of HCNEs around 2(shown below) consists of 25 genes having higher enrichment of HCNE, while cluster 2 consists of the remaining 23 genes.</p

Statistical significance test for H3K4me1 around different genomic distributions.

<p>A) H3K4me1 distribution in different clusters across ±10 kb TSS against the random background distribution. B) H3K4me1 distribution in different clusters across ±1 Mb TSS with respect to random background distribution. C) H3k4me1 distribution in different clusters across ±2 Mb TSS with respect to random background distribution. This figure shows that cluster 1 (shown by red bar) has significantly higher distribution of H3K4me1 in comparison to random selected background region (marked by black bars), CpG and non-CpG region (shown by blue and green bar respectively) and cluster 2 genes (shown by pink bar).</p

Classification comparison of nuclear receptors gene family with respect to sequence homology and transcriptional mechanism and function based.

<p>The GRB target genes (cluster 1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088880#pone-0088880-t001" target="_blank">Table 1</a>) are shown in red, while non-targets are in blue. Nuclear hormone receptors are presented in normal bold text while orphan receptors are underlined and in italics. There are in total 23 nuclear receptor GRB target genes and 25 nuclear receptor non-GRB target nuclear receptor genes. It is clear from the figure that both GRB target and non-target nuclear receptors are dispersed among seven families classified on the basis of sequence homology.</p

The bubble plots for bivalent promoter mark for each gene in human embryonic stem cell line.

<p>The x-axis shows read counts for repression (H3K27me3) mark around ±10 KB TSS. The y-axis shows read counts for active promoter (H3K4me3) mark around ±10 KB TSS. The size of the bubble (yellow) shows RPKM value for respective gene. The left section of the plot comprises all of the genes (black) in cluster 2 (except few cases where cluster 1 gene have very high expression). This shows that cluster 2 genes does not have any enrichment of repression mark around their TSS irrespective of their expression. The top and bottom right sections consist of genes from cluster 1 (red). This shows that when genes in cluster 1 are not expressed they have higher read counts for repression mark while still some of the genes retain repression mark even when they are expressed.</p

Epac2 protein isoforms and genomic organization of <i>Epac2</i>.

<p><b>A)</b><b> </b> Schematic illustration of the protein domains of the Epac2 isoforms. The regulatory domain consists of one (Epac2B and Epac2C) or two (Epac2A1/2A2) cAMP binding domains (cAMP-A and cAMP-B) and a dishvelled, Egl–10 and pleckstrin homology domain (DEP; except for Epac2C). The catalytic region consists of an exchange activity domain that catalyses Rap activation (RasGEF), a Ras-exchange motif (REM) and a Ras association (RA) domain. The full-length isoform (Epac2A1) consists of 1011 amino acids (aa), while Epac2A2 is deduced to contain 993 aa. Epac2A2 is identical to Epac2A1, except for an 18 aa deletion corresponding to exon 7. Epac2B consists of 867 aa, and Epac2C of 696 aa. <b>B)</b> Genomic organization and alternative promoters at the <i>Epac2</i> locus. Coding exons are numbered and illustrated by black boxes. White parts of exons indicate untranslated regions (UTRs). TSSs for the Epac2A, Epac2B and Epac2C isoforms are indicated by bent arrows, while the corresponding translational start sites (ATGs) are indicated by open arrowheads. The filled arrowhead in exon 31 indicates a shared stop codon. The figure is based on the NCBI reference sequence: NC_000068.6, Chr2∶71819344–72094433, and is expanded in 5′-region to include the CpG-island (start; nt 71818997) and in the 3′-region to include the UTR of exon 31 (end; nt 72095526). Note the different scales for exons and introns.</p

The CGI of the Epac2A-promoter is demethylated in all tissues examined.

<p>Bisulfite sequencing analyses of the <i>Epac2</i> A-promoter (containing 50 CpG sites across a region of 441 bp, spanning nucleotides 71980937–71981377 of chr2). DNA was prepared from brain (A), pancreas (B), adrenal cortex (C), liver (D) and kidney (E). Bisulfite sequencing was performed on pooled DNA; from 7 mice in A–B and D–E, and from 8 mice in C. For all tissues, the percentage of methylated CpG sites was ≤1%. The total number of analyzed clones were: 32 in (A), 30 in (B), 12 in (C), 35 in (D) and 34 in (E). Each horizontal line represents one analyzed clone. Black circles: methylated CpG sites, open circles; demethylated CpG sites. Representative clones are shown for A–B and D–E.</p

The Epac2C-promoter is hypomethylated in liver.

<p>Bisulfite sequencing analyses of the <i>Epac2</i> C-promoter (containing 13 CpG sites across a region of 469 bp, spanning nucleotides 72179672–72180140 of chr2). Genomic DNA was prepared from liver (A; 7 mice), freshly isolated hepatocytes (B; 3 mice), hepatocytes cultured for 11 days (C; 3 mice), adrenal cortex (D; 8 mice), endocrine pancreas (E; 5 mice), brain (F; 7 mice), pituitary (G; 5 mice), exocrine pancreas (H; 5 mice) and kidney (I; 7 mice). Bisulfite sequencing was performed on pooled DNA from the number of mice indicated above. The overall percentage of demethylated CpG sites and the number of clones analyzed were (number of clones in parentheses): 78% (52) in (A), 95% (48) in (B), 99.7% (64) in (C), 7% (29) in (D), 12% (15) in (E), 10% (40) in (F), 4% (28) in (G) 12% (15) in (H) and 6% (31) in (I). Each horizontal line represents one analyzed clone. Black circles: methylated CpG sites, open circles; demethylated CpG sites. Representative clones are shown for A-D, F, G and I.</p

The predicted promoters regions of <i>Epac2</i> contains CpG dinucleotides and hold transcriptional activity.

<p><b>A)</b> Schematic drawing of the predicted promoter regions of <i>Epac2</i>. The promoter region directing expression of Epac2A (chr2 71980880–71981553; upper panel) contains a potential INR- (initiator) like element, a TATA-box and two putative TSSs. A CGI with 51 CpG sites (indicated by grey lollipops) covers the promoter region. The promoter region directing expression of Epac2B (chr2 72054237–72054766; middle panel) contains a putative INR element and a M3 element. This region contains 6 CpG sites as indicated by lollipops. The promoter region directing expression of Epac2C (72179738–72180207; lower panel) contains three alternative TSSs (TSS1–3), as well as an INR element as also described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone.0067925-Ueno1" target="_blank">[6]</a>. Transcription is initiated from TSS1/2 and not TSS3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone.0067925-Ueno1" target="_blank">[6]</a>. This region contains 13 CpG sites. The translational start sites are indicated by bent arrows. To identify novel potential promoter regions in <i>Epac2</i> the sequences corresponding to potential promoter regions were analyzed by the FlyBase eukaryotic promoter prediction computational tool (<a href="http://flybase.org" target="_blank">http://flybase.org</a>) and rVISTA evolutionary conservation analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone.0067925-Loots1" target="_blank">[17]</a>. The promoter regions shown correspond to the genomic regions that were analyzed by bisulfite sequencing in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone-0067925-g004" target="_blank">figures 4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone-0067925-g006" target="_blank">6</a>. Note that the CpG-site marked with a “Z” (upper panel) is included in primer sequence and therefore not included in the BSP analysis). <b>B)</b> The reporter gene plasmids containing the <i>Epac2</i> promoter regions (pCpG/prom-Epac2A, pCpG/prom-Epac2B and pCpG/prom-Epac2C; 200 ng), the positive control vector pCpG/CMV-EF1alpha (200 ng) and the pCpG-basic vector (containing no promoter/enhancer; 200 ng), were transfected into Cos-1 cells. Before transfection the plasmids were either <i>in vitro</i> methylated by the <i>Sss</i>I CpG methylase (black bars) or left untreated (grey bars). IVM; <i>in vitro</i> methylation. The luciferase activities are presented as average +/− stdev, n = 9.</p

Expression of Epac2 isoforms.

<p><b>A)</b> mRNA was prepared from mouse brain, adrenal glands, liver, kidney and endocrine and exocrine pancreas, converted to cDNA, and RT-PCR was performed with primers as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone-0067925-t001" target="_blank">table 1</a>. +/−; presence and absence of reverse transcriptase (RT). <b>B–C)</b> Immunoblotting with Epac2 antibodies was performed on protein extracts prepared from Cos1-cells overexpressing Flag-tagged Epac2A, Epac2B or Epac2C (20 μg; B, C; Cos1-cells do not express endogenous Epac2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067925#pone.0067925-Islam1" target="_blank">[9]</a>), and extracts prepared from mouse brain, adrenal gland, liver and kidney (250–300 μg; B) as well as from brain and endocrine and exocrine pancreas (250–300 μg; C). The migration of the different isoforms is indicated by arrows. Immunoblotting against actin is shown below each blot. The apparent absence of actin in the Cos-1 cell extracts in B is explained by a very short exposure time of the blot (as only 20 μg of Cos-1 extract was loaded on the gel compared to 200–250 μg of tissue extract).</p