Summary of the methylation levels in each transgenic mouse line.

<p>The vertical axis of the bar graph represents the percentage of individuals showing distinct degree of DNA methylation at the transgenic H19 ICR' fragment (3' portion), which was summarized from pedigrees of each TgM line. For each 100% stacked column, the number of individuals analyzed in each pedigree (Fig. 2-4 and Fig. S2-S4) was set at 100 (%) and DNA methylation levels were divided into three categories (highly-, partially- and poorly-methylated). P, paternal transmission; M, maternal transmission.</p

Experimental design.

<p>(A) Generation of <i>Insulated ICR</i>' transgenic mice (top). Genomic structure of the mouse <i>Igf2/H19</i> gene locus. The <i>H19</i> ICR fragment used in this study corresponds to nucleotides from 1126 to 3503 in the AF049091 sequences (GenBank). In the enlarged map, restriction enzyme sites (<i>Mlu</i>I, <i>Bsr</i>GI, <i>Bss</i>HII, <i>Xho</i>I), which were artificially introduced to facilitate cloning procedure, are shown adjacent to the CTCF binding sites (solid rectangles) of the <i>H19</i> ICR (termed the ICR'). Nucleotides are numbered relative to the 5' end (set at 1') of the sequence (bottom). Chicken HS4 core fragment (cHS4c, 237 bp) carries five footprints, FI–FV. Tandem cHS4c fragments (I for insulator, gray rectangles) were floxed by a set of either loxP5171 (solid triangles) or loxP2272 (open) sequences and attached to either ends of the ICR' fragment (termed the insulated ICR'), which was then introduced in reversed orientation 3' to the LCR in the human β-globin locus YAC (A201F4.3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073925#B27" target="_blank">27</a>]). The entire β-globin locus is contained within two <i>Sfi</i>I fragments (8 and 100 kbp). Transgene-specific probes used for long-range structural analysis in (C) are shown as solid rectangles. (B) Two possible models for marking epigenetic signature. The mark can be set inside (left) or outside (right) of the <i>H19</i> ICR. (C) Long-range structural analysis of transgenes. DNA from thymus cells was digested with <i>Sfi</i>I in agarose plugs, separated by pulsed-field gel electrophoresis, and blots were hybridized separately to probes in (A). (D) (left) Structure of the transgene after <i>in vivo</i> cre-<i>loxP</i> recombination. Recombination removed the 5' or both (cHS4c)₂ fragments from the parental insulated ICR' locus and generated either the 5'HS4-del or the pseudo-WT loci, respectively. S, <i>Sac</i>I; A, <i>Ava</i>II; X, XbaI (right). Tail DNA of each TgM subline was digested with <i>Xba</i>I<i>/Sac</i>I (top) or <i>Ava</i>II (bottom), separated on agarose gels, and blots were hybridized with the probe.</p

The Chicken HS4 Insulator Element Does Not Protect the <i>H19</i> ICR from Differential DNA Methylation in Yeast Artificial Chromosome Transgenic Mouse

<div><p>Mono-allelic expression at the mouse <i>IGF2/H19</i> locus is controlled by differential allelic DNA methylation of the imprinting control region (ICR). Because a randomly integrated <i>H19</i> ICR fragment, when incorporated into the genome of transgenic mice (TgM), was allele-specifically methylated in somatic, but not in germ cells, it was suggested that allele-discriminating epigenetic signature, set within or somewhere outside of the Tg <i>H19</i> ICR fragment in germ cells, was later translated into a differential DNA methylation pattern. To test if the chicken β-globin HS4 (cHS4) chromatin insulator might interfere with methylation imprinting establishment at the <i>H19</i> ICR, we inserted the <i>H19</i> ICR fragment, flanked by a set of floxed cHS4 core sequences, into a human β-globin locus YAC and generated TgM (<i>insulated ICR</i>' TgM). As controls, the cHS4 sequences were removed from one side (<i>5'HS4-deleted ICR'</i>) or both sides (<i>pseudo-WT ICR'</i>) of the insulated ICR' by <i>in vivo</i> cre-loxP recombination. The data show that while maternally inherited transgenic <i>H19</i> ICR was not methylated in <i>insulated ICR</i>' TgM, it was significantly methylated upon paternal transmission, though the level was lower than in the <i>pseudo-WT ICR</i>' control. Because this reduced level of methylation was also observed in the <i>5'HS4-deleted ICR</i>' TgM, we speculate that the phenotype is due to VEZF1-dependent demethylation activity, rather than the insulator function, borne in cHS4. Collectively, although we cannot rule out the possibility that cHS4 is incapable of blocking an allele-discriminating signal from outside of the transgene, the epigenetic signature appears to be marked intrinsically within the <i>H19</i> ICR.</p> </div

Methylation status of the pseudo-WT ICR' fragment in somatic and germ cells (line 29).

<p>(A) Partial restriction enzyme map of the ICR' (pseudo-WT) transgene. (B–D) Southern blot analysis of the transgene in F1-3 generations. Genomic DNA was prepared from a tail-tip of TgM (<i>pseudo-WT</i> TgM) and the 3' portion of the transgenic <i>H19</i> ICR' was analyzed (<i>Bam</i>HI+<i>Hha</i>I) as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073925#pone-0073925-g002" target="_blank">Figure 2B and C</a>. (E) Genomic DNA was prepare from testis of the TgM and the 3' portion of the transgenic <i>H19</i> ICR' was analyzed (<i>Bam</i>HI+<i>Hha</i>I) as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073925#pone-0073925-g002" target="_blank">Figure 2B and C</a>. (F) Pedigree depicting a paternally-methylated pseudo-WT ICR' transgene is shown as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073925#pone-0073925-g002" target="_blank">Figure 2G</a>.</p

Methylation status of the 5'HS4-del ICR' fragment in somatic and germ cells (line 29).

<p>(A) Partial restriction enzyme map of the ICR' (5'HS4-del) transgene. (B–D) Southern blot analysis of the transgene in F1-3 generations. Genomic DNA was prepared from a tail-tip of TgM (<i>5'HS4-del</i> TgM) and the 3' portion of the transgenic <i>H19</i> ICR' was analyzed (<i>Bam</i>HI+<i>Hha</i>I) as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073925#pone-0073925-g002" target="_blank">Figure 2B and C</a>. (E) Genomic DNA was prepared from testis of the TgM and the 3' portion of the transgenic <i>H19</i> ICR' was analyzed (<i>Bam</i>HI+<i>Hha</i>I) as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073925#pone-0073925-g002" target="_blank">Figure 2B and C</a>. (F) Pedigree depicting a paternally-methylated 5'HS4-del ICR' transgene is shown as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073925#pone-0073925-g002" target="_blank">Figure 2G</a>.</p

DNA methylation status of the insulated ICR' fragment in somatic and germ cells (line 29).

<p>(A) Partial restriction enzyme map of the Insulated ICR' transgene. Fragments from the transgenic (Tg) and endogenous (endo.) <i>H19</i> ICR can be distinguished by their sizes because of a transgene-specific <i>Bam</i>HI (B) or <i>Bsr</i>GI (Bs) sites (circled). Methylation-sensitive <i>Hha</i>I or <i>Hpa</i>II sites in the <i>Sac</i>I (S)-<i>Bam</i>HI, <i>Bam</i>HI, or <i>Bam</i>HI-<i>Bsr</i>GI fragments (horizontal lines) are displayed as vertical lines. Probes are shown as gray rectangles. (B, C) Genomic DNA from a tail-tip of TgM (<i>Insulated ICR</i>', F1 (B) and F2 (C) generations), inheriting the transgene either paternally (Pat.) or maternally (Mat.) was analyzed by Southern blotting (upper panel). DNA was digested with <i>Bam</i>HI in the presence (+) or absence (-) of <i>Hha</i>I and analyzed by the I5 probe shown in (A) to analyze 3' portion of the transgenic <i>H19</i> ICR'. Parental fragments are marked by asterisks. (middle) DNA was digested with <i>Bam</i>HI+<i>Sac</i>I in the presence (+) or absence (-) of <i>Hha</i>I and analyzed by the cHS4 probe. (bottom) DNA was digested with <i>Bam</i>HI+<i>Bsr</i>GI in the presence (+) or absence (-) of <i>Hpa</i>II and analyzed by the middle probe to analyze middle portion of the <i>H19</i> ICR. (D, E) Methylation status of the 3' portion of the <i>H19</i> ICR in F3 (D) and F4 (E) generations was analyzed (by I5 probe). (F) Genomic DNA from testis of the TgM was analyzed by I5 (top panel), cHS4 (middle), or middle (bottom) probes. (G) Pedigree depicting a paternally-methylated insulated ICR' transgene. Male and female (hemi-zygous) transgenic individuals are denoted as squares and circles, respectively. In the Southern blot data of 3' portion of the transgenic <i>H19</i> ICR' sequences, uncut (top), completely-cut (bottom), and other (intermediate) bands were quantified by Phosphorimager and methylation status of each sample was determined as follows. When ratio of uncut/total (top+intermediate+bottom) bands exceeds 50%, the sample was defined as hyper-methylated and represented by solid marks. When ratio of completely-cut/total bands exceeds 50%, the sample was defined as hypo-methylated (open marks). In other cases, the sample was defined as partially-methylated (shaded marks). Testis samples in (F) were obtained from male individuals marked by dots.</p

Expression of endogenous mouse <i>Renin</i> and human <i>RENIN</i> transgenes in THM.

<p><b>(A)</b> Schematic representation of chimeric renin-angiotensin system in Tsukuba hypertensive mice. mRen, mouse Renin; mAgt, mouse Angiotensinogen; hREN, human RENIN; hAGT, human ANGIOTENSINOGEN; ACE, Angiotensin-converting enzyme. <b>(B)</b> Breeding strategy for obtaining normotensive (control; ctrl) and hypertensive mice (Tsukuba hypertensive mice; THM). h<i>REN</i>, human <i>RENIN</i>; h<i>AGT</i>, human <i>ANGIOTENSINOGEN</i>; Tg, Transgene. <b>(C</b> and <b>D)</b> Total RNA was isolated from the kidney of normotensive (ctrl) or hypertensive (THM) TgM (8-week old). Levels of endogenous mouse <i>Ren</i> (endogenous; C) or human transgenic <i>REN</i> (h<i>REN</i> Tg; D) gene expression were analyzed by qRT-PCR. Each value represents the ratio of endogenous m<i>Ren</i> or h<i>REN</i> Tg gene expression to that of <i>Gapdh</i>. The expression value of male control animals in each group was arbitrarily set at 100. qPCR analyses were repeated three times. Number of animals analyzed is shown in parentheses below each panel and mean ± SD are shown. Statistically significant differences between the control animals and THM were determined using an unpaired t-test (##, <i>P</i> < 0.01).</p

Expression of human and mouse <i>renin</i> Tgs in adult and fetal THM.

<p>Expression of human and mouse <i>renin</i> Tgs in adult and fetal THM.</p

Comparison of systolic blood pressure (SBP) between non-TgM and m<i>Ren</i> TgM.

<p>Comparison of systolic blood pressure (SBP) between non-TgM and m<i>Ren</i> TgM.</p

m<i>Ren</i> gene expression in the 5'-large-del mutant allele.

<p>(<b>A</b>) Expression of m<i>Ren</i> gene in the 5'-large-del mutant allele was analyzed in normotensive (ctrl) and hypertensive (THM) mouse environments. (<b>B</b>–<b>D</b>) Total RNA was isolated from the kidney of normotensive or hypertensive animals (8-week old) and subjected to qRT-PCR analyses. Expression levels of endogenous 5'-large-del m<i>REN</i> (B), endogenous pseudo-WT m<i>REN</i> (C), h<i>REN</i> Tg (D), and <i>Gapdh</i> (data not shown) were determined. Each value represents the ratio of renin genes expression to that of <i>Gapdh</i> and mean ± SD is shown. Values of male control animals in each group was arbitrarily set at 100. Number of animals analyzed is shown in parentheses below each panel. Statistically significant differences between ctrl and THM were determined using an unpaired t-test (N.S., not significant; #, <i>P</i> < 0.05; ##, <i>P</i> < 0.01).</p