Characterization of the maternal <i>Kcnq1ot1</i> transcript.

<p>A) Schematic showing the regulatory sequences at the <i>Kcnq1ot1</i> locus. Minimal promoter, enhancer and previously reported transcriptional start sites (*, TSS) are from Fitzpatrick <i>et al.</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002956#pgen.1002956-Fitzpatrick2" target="_blank">[21]</a>. The silencing domain depicted was characterized by Mohammad <i>et al.</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002956#pgen.1002956-Mohammad1" target="_blank">[23]</a>. Primers used for 5′ RACE experiments in this report are designated A, B, C and nested primers, An, Bn and Cn. Transcriptional start sites (TSS) in heart and liver are depicted as bent arrows. A star indicates the relative location of a single nucleotide polymorphism (SNP) used to discriminate between parental transcripts. B) Schematic of the scan to determine the length of the <i>Kcnq1ot1</i> maternal transcript, with primers amplifying fragments located at the indicated distances relative to the <i>Kcnq1ot1</i> transcriptional start site. C) RT-PCRs on RNAs extracted from hearts of F1 hybrid progeny from a BxC cross, followed by allele-specific digestion, showing the maternal transcript absent after 33 kb. M, maternal; P, paternal; ND, non-digested; D, digested; B, C57BL/6J; C, B6(CAST7).</p

Model for regulation of <i>Kcnq1</i> in the embryonic heart.

<p>WT, wild-type; <i>K-term</i>, mutant mouse, in which transcription of <i>Kcnq1ot1</i> is terminated prematurely; IF, methylation sensitive inhibitory factor. Maternal (m) events are shown above and paternal (p) events below the chromosome; filled circles, methylated DNA, empty circles, unmethylated DNA. Curved arrows represent interactions, bent arrows depict transcription. Ovals represent enhancers, which are inactive (light gray) at 10.5 dpc and active (black) at 14.5–16.5 dpc.</p

The <em>Kcnq1ot1</em> Long Non-Coding RNA Affects Chromatin Conformation and Expression of <em>Kcnq1</em>, but Does Not Regulate Its Imprinting in the Developing Heart

<div><p>Although many of the questions raised by the discovery of imprinting have been answered, we have not yet accounted for tissue- or stage-specific imprinting. The <em>Kcnq1</em> imprinted domain exhibits complex tissue-specific expression patterns co-existing with a domain-wide <em>cis</em>-acting control element. Transcription of the paternally expressed antisense non-coding RNA <em>Kcnq1ot1</em> silences some neighboring genes in the embryo, while others are unaffected. <em>Kcnq1</em> is imprinted in early cardiac development but becomes biallelic after midgestation. To explore this phenomenon and the role of <em>Kcnq1ot1</em>, we used allele-specific assays and chromosome conformational studies in wild-type mice and mice with a premature termination mutation for <em>Kcnq1ot1</em>. We show that <em>Kcnq1</em> imprinting in early heart is established and maintained independently of <em>Kcnq1ot1</em> expression, thus excluding a role for <em>Kcnq1ot1</em> in repressing <em>Kcnq1</em>, even while silencing other genes in the domain. The exact timing of the mono- to biallelic transition is strain-dependent, with the CAST/EiJ allele becoming activated earlier and acquiring higher levels than the C57BL/6J allele. Unexpectedly, <em>Kcnq1ot1</em> itself also switches to biallelic expression specifically in the heart, suggesting that tissue-specific loss of imprinting may be common during embryogenesis. The maternal <em>Kcnq1ot1</em> transcript is shorter than the paternal ncRNA, and its activation depends on an alternative transcriptional start site that bypasses the maternally methylated promoter. Production of <em>Kcnq1ot1</em> on the maternal chromosome does not silence <em>Cdkn1c</em>. We find that in later developmental stages, however, <em>Kcnq1ot1</em> has a role in modulating <em>Kcnq1</em> levels, since its absence leads to overexpression of <em>Kcnq1</em>, an event accompanied by an aberrant three-dimensional structure of the chromatin. Thus, our studies reveal regulatory mechanisms within the <em>Kcnq1</em> imprinted domain that operate exclusively in the heart on <em>Kcnq1</em>, a gene crucial for heart development and function. We also uncover a novel mechanism by which an antisense non-coding RNA affects transcription through regulating chromatin flexibility and access to enhancers.</p> </div

Chromatin immunoprecipitation (ChIP) of selected regions of the <i>Kcnq1</i> gene.

<p>A) Schematic of the <i>Kcnq1</i> domain. The blue arrowhead indicates the anchor primer at the <i>Kcnq1</i> promoter and vertical lines represents the primer regions investigated. The purple boxes represent <i>Kcnq1</i> exons, the dark blue box represents the <i>Kcnq1ot1</i> gene. B) ChIP analysis in wild-type heart for p300, H3K4Me1 and H3K27Ac at the interaction peaks observed in the 3C assays. The asterisks indicate frequent interactions in the <i>K-term</i> heart as determined by 3C.</p

<i>Kcnq1</i> expression in the heart during development when the truncated <i>Kcnq1ot1</i> (<i>K-term</i> mutation) is inherited paternally.

<p>A) RT-PCR followed by allele-specific digests in E10.5 heads and bodies and throughout the development of the heart in F1 hybrid progeny of B6(CAST7)×<i>K-term</i> mice. M, maternal; P, paternal; N, non-digested; D, digested; B, C57BL/6J; KT, <i>K-term</i>. B) qRT-PCR analysis of <i>Kcnq1</i> expression in wild-type and <i>K-term</i> mice. Transcripts were normalized to <i>β-actin</i>. A significant difference in expression was seen when comparing wild-type and <i>K-term</i> hearts at E16.5 and P2 Hearts. These differences had a p-value less than 0.05. C) Parental origin of <i>Kcnq1</i> expression throughout cardiac development. The RT-PCR and allele specific bands were quantified and the ratio of paternal to maternal transcript was determined.</p

Chromosome conformation capture (3C) in the <i>K-term</i> mouse.

<p>A) Schematic of the <i>Kcnq1</i> domain. The blue arrowhead indicates the anchor primer at the <i>Kcnq1</i> promoter and vertical lines represent the regions investigated. The purple boxes represent <i>Kcnq1</i> exons, the dark blue box represents the <i>Kcnq1ot1</i> gene. Comparison of the chromatin interaction profile in wild-type and <i>K-term</i> hearts (B), wild-type and <i>K-term</i> brains (C) and <i>K-term</i> hearts and brains (D).</p

Barriers to adoption and scale-up of POC technologies.

<p>Barriers to adoption and scale-up of POC technologies.</p

Diversity of target product profiles, users, and settings within the spectrum of POC testing.

<p>HBV, hepatitis B virus; HCV, hepatitis C virus; UTI, urinary tract infection; MRSA, methicillin-resistant staphylococcus aureus; C. diff, clostridium difficile; RDT, rapid diagnostic test; Strep A, group A streptococcus.</p

Barriers for use of point-of-care tests in India.

<p>Barriers for use of point-of-care tests in India.</p

Additional file 6: Table S4. of Sex chromosomes drive gene expression and regulatory dimorphisms in mouse embryonic stem cells

Expression in undifferentiated murine embryonic stem (ES) cells of genes that escape X chromosome inactivation (XCI) after differentiation (BC cell lines)