The long and the short of it: RNA-directed chromatin asymmetry in mammalian X-chromosome inactivation

AbstractMammalian X-chromosome inactivation is controlled by a multilayered silencing pathway involving both short and long non-coding RNAs, which differentially recruit the epigenetic machinery to establish chromatin asymmetries. In response to developmentally regulated small RNAs, dicer, a key effector of RNA interference, locally silences Xist on the active X-chromosome and establishes the heterochromatin conformation along the silent X-chromosome. The 1.6kb RepA RNA initiates silencing by targeting the PRC2 polycomb complex to the inactive X-chromosome. In addition, the nuclear microenvironment is implicated in the initiation and maintenance of X-chromosome asymmetries. Here we review new findings involving these various RNA species in terms of understanding Xist gene regulation and the establishment of X-chromosome inactivation

HnRNPK maintains single strand RNA through controlling double-strand RNA in mammalian cells

Although antisense transcription is a widespread event in the mammalian genome, double-stranded RNA (dsRNA) formation between sense and antisense transcripts is very rare and mechanisms that control dsRNA remain unknown. By characterizing the FGF-2 regulated transcriptome in normal and cancer cells, we identified sense and antisense transcripts IER3 and IER3-AS1 that play a critical role in FGF-2 controlled oncogenic pathways. We show that IER3 and IER3-AS1 regulate each other\u27s transcription through HnRNPK-mediated post-transcriptional regulation. HnRNPK controls the mRNA stability and colocalization of IER3 and IER3-AS1. HnRNPK interaction with IER3 and IER3-AS1 determines their oncogenic functions by maintaining them in a single-stranded form. hnRNPK depletion neutralizes their oncogenic functions through promoting dsRNA formation and cytoplasmic accumulation. Intriguingly, hnRNPK loss-of-function and gain-of-function experiments reveal its role in maintaining global single- and double-stranded RNA. Thus, our data unveil the critical role of HnRNPK in maintaining single-stranded RNAs and their physiological functions by blocking RNA-RNA interactions

The KCNQ1OT1 imprinting control region and non-coding RNA: new properties derived from the study of Beckwith–Wiedemann syndrome and Silver–Russell syndrome cases

A cluster of imprinted genes at chromosome 11p15.5 is associated with the growth disorders, Silver–Russell syndrome (SRS) and Beckwith–Wiedemann syndrome (BWS). The cluster is divided into two domains with independent imprinting control regions (ICRs). We describe two maternal 11p15.5 microduplications with contrasting phenotypes. The first is an inverted and in cis duplication of the entire 11p15.5 cluster associated with the maintenance of genomic imprinting and with the SRS phenotype. The second is a 160 kb duplication also inverted and in cis, but resulting in the imprinting alteration of the centromeric domain. It includes the centromeric ICR (ICR2) and the most 5′ 20 kb of the non-coding KCNQ1OT1 gene. Its maternal transmission is associated with ICR2 hypomethylation and the BWS phenotype. By excluding epigenetic mosaicism, cell clones analysis indicated that the two closely located ICR2 sequences resulting from the 160 kb duplication carried discordant DNA methylation on the maternal chromosome and supported the hypothesis that the ICR2 sequence is not sufficient for establishing imprinted methylation and some other property, possibly orientation-dependent, is needed. Furthermore, the 1.2 Mb duplication demonstrated that all features are present for correct imprinting at ICR2 when this is duplicated and inverted within the entire cluster. In the individuals maternally inheriting the 160 kb duplication, ICR2 hypomethylation led to the expression of a truncated KCNQ1OT1 transcript and to down-regulation of CDKN1C. We demonstrated by chromatin RNA immunopurification that the KCNQ1OT1 RNA interacts with chromatin through its most 5′ 20 kb sequence, providing a mechanism likely mediating the silencing activity of this long non-coding RNA

LncRNAs join hands together to regulate neuroblastoma progression

Trait associated single nucleotide polymorphisms often overlap with noncoding transcripts but their contribution to disease phenotype is poorly investigated. Our study on neuroblastoma risk associated 6p22.3 locus derived long noncoding RNAs (lncRNAs) demonstrates that functional co-operation between sense-antisense CASC15 and NBAT1 lncRNAs control neuroblastoma progression via regulating SOX9-CHD7-USP36 regulatory axis

Long Non-Coding RNAs: Tools for Understanding and Targeting Cancer Pathways

The regulatory nature of long non-coding RNAs (lncRNAs) has been well established in various processes of cellular growth, development, and differentiation. Therefore, it is vital to examine their contribution to cancer development. There are ample examples of lncRNAs whose cellular levels are significantly associated with clinical outcomes. However, whether these non-coding molecules can work as either key drivers or barriers to cancer development remains unknown. The current review aims to discuss some well-characterised lncRNAs in the process of oncogenesis and extrapolate the extent of their decisive contribution to tumour development. We ask if these lncRNAs can independently initiate neoplastic lesions or they always need the modulation of well characterized oncogenes or tumour suppressors to exert their functional properties. Finally, we discuss the emerging genetic approaches and appropriate animal and humanised models that can significantly contribute to the functional dissection of lncRNAs in cancer development and progression

Elevated Expression of <em>H19</em> and <em>Igf2</em> in the Female Mouse Eye

<div><p>The catalogue of genes expressed at different levels in the two sexes is growing, and the mechanisms underlying sex differences in regulation of the mammalian transcriptomes are being explored. Here we report that the expression of the imprinted non-protein-coding maternally expressed gene <i>H19</i> was female-biased specifically in the female mouse eye (1.9-fold, p = 3.0E−6) while not being sex-biased in other somatic tissues. The female-to-male expression fold-change of <i>H19</i> fell in the range expected from an effect of biallelic versus monoallelic expression. Recently, the possibility of sex-specific parent-of-origin allelic expression has been debated. This led us to hypothesize that <i>H19</i> might express biallelically in the female mouse eye, thus escape its silencing imprint on the paternal allele specifically in this tissue. We therefore performed a sex-specific imprinting assay of <i>H19</i> in female and male eye derived from a cross between <i>Mus musculus</i> and <i>Mus spretus</i>. However, this analysis demonstrated that <i>H19</i> was exclusively expressed from the maternal gene copy, disproving the escape hypothesis. Instead, this supports that the female-biased expression of <i>H19</i> is the result of upregulation of the single maternal. Furthermore, if <i>H19</i> would have been expressed from both gene copies in the female eye, an associated downregulation of Insulin-like growth factor 2 (<i>Igf2</i>) was expected, since <i>H19</i> and <i>Igf2</i> compete for a common enhancer element located in the <i>H19/Igf2</i> imprinted domain. On the contrary we found that also <i>Igf2</i> was significantly upregulated in its expression in the female eye (1.2-fold, p = 6.1E−3), in further agreement with the conclusion that <i>H19</i> is monoallelically elevated in females. The female-biased expression of <i>H19</i> and <i>Igf2</i> specifically in the eye may contribute to our understanding of sex differences in normal as well as abnormal eye physiology and processes.</p> </div

Primers.

<p>The primer sequences are given 5′ to 3′.</p

Gene expression analysis. A.

<p>Schematic model of allelic regulation in the <i>H19/Igf2</i> imprinted domain, and premise for the experimental approach. The imprinting control region (ICR, triangle) is unmethylated on the maternal allele (<i>mat</i>), allowing for the expression of <i>H19_mat</i> and the binding of the CCCTC-binding factor (hexagon) which insulates <i>Igf2_mat</i> from interaction with an enhancer element (circle) located downstream of <i>H19</i>. Thus <i>Igf2_mat</i> is normally silenced when <i>H19_mat</i> is expressed. In contrast, on the paternal allele (<i>pat</i>), the ICR is methylated, preventing expression of <i>H19_pat</i> and blocking CCCTC-binding to the ICR which allows <i>Igf2_pat</i> to interact with the enhancer element and to be expressed. <b>B.</b> Volcano plot, separating female-biased (upper right quadrant) and male-biased (upper left quadrant) autosomal genes in the mouse eye in our microarray screen. <i>H19</i> (red square) is identified as a candidate female-biased gene (p = 1.3E−12, female/male fold-change = 1.5). y-axis: –log₁₀(p-value, two-sided t-test), x-axis: log₂(female/male) expression ratio. The dotted line represents the significance threshold p = 0.001, and numbers within parenthesis denote the number of significant probes : unique genes in each sex. n_females = 88, n_males = 88. <b>C.</b> RT-qPCR assays of female (F) and male (M) eye and lung tissues. Expression is normalized to the geometric mean of <i>Gapdh</i> and <i>Actb</i> and shown relative to the mean male expression in each tissue. P-values are given according to a two-sided t-test and error bars denote standard error of the mean. n_{females, eye} = 19, n_{males, eye} = 19, n_{females, lung} = 16, n_{males, lung} = 18.</p

Additional file 2: of GeneSCF: a real-time based functional enrichment tool with support for multiple organisms

The list of organisms supported by GeneSCF for Gene ontology functional database. (XLS 9 kb