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

Characterizing the role of long non coding RNAs epigenetic regulators in disease model

Long non-coding RNAs (lncRNAs) are a class of biological molecules which are transcribed from DNA but are not translated into any protein. LncRNAs have been identified as critical players in gene regulation. Misregulation of lncRNAs has been considered as one of the underlying causes for cancer pathogenesis and in other human diseases. In the current thesis, I have addressed the epigenetic roles of lncRNAs in regulating gene expression in cell line based and disease model systems. We investigated the functional role of lncRNAs in the maintenance of active chromatin by sequencing lncRNAs associated with active chromatin enriched with H3K4me2 and WDR5. We identified 209 lncRNAs to be commonly enriched in H3K4me2 and WDR5 pulldown chromatin fractions and we named them as active chromatin associated RNAs (active CARs). Interestingly, 41% of active CARs mapped to divergent transcription units having transcription factor genes as their partner. CARs were found to regulate the expression of partner protein coding genes at the transcriptional level by recruiting WDR5 to maintain the active histone marks H3K4me2/H3K4me3 at these promoters. Depletion of active CARs results in reduced WDR5and H3K4me2/H3K4me3 occupancy at these promoters. However, in absence of WDR5, we found the levels of H3K4me2 to remain unchanged at divergent promoters. Taken together our findings indicates that, conversion of H3K4me2 to H3K4me3 is mediated via active CARs-WDR5 interaction at the active divergent promoter, whereas, the maintenance of H3K4me2 marks appears to be WDR5 independent. Additionally, we used transcriptome profiling approach, to identify lncRNAs that are differentially expressed between low- and high- risk neuroblastoma tumours. We report NBAT-1 lncRNA as an independent prognostic biomarker in predicting clinical outcome of neuroblastoma patients. The expression profile analysis showed NBAT-1 to be lowly expressed in high-risk tumours relative to low-risk tumours. Using cell line and mouse models we characterized NBAT-1 as a tumour suppressor lncRNA which regulates gene expression by interaction with PRC2 repressive chromatin complex. NBAT-1 lncRNA promotes differentiation and acts as a tumour suppressor by epigenetic regulation of genes to inhibit cell proliferation and invasion. Thirdly, we sought to study genomic imprinting in a disease model. Genomic imprinting is an epigenetic regulation of gene expression in a parent of origin-specific manner. Studies in mouse have identified Kncq1 imprinted domain to be epigenetically regulated by a 91kb long lncRNA Kcnq1ot1 which is expressed from the paternal chromosome to silence imprinted genes in cis. Using BW-syndrome human disease model, we identified a maternal 11p15.5 micro duplication which included the 5′ 20 kb of the non-coding KCNQ1OT1 gene. Its maternal transmission was associated with ICR2 hypomethylation and familial BWS phenotype. Normally ICR2 is methylated to repress KCNQ1OT1, thereby allowing maternal copies of the imprinted genes including growth inhibitor CDKN1C to be expressed. We demonstrated that this duplicated maternal KCNQ1OT1 RNA also interacts with chromatin through its most 5′ 20 kb sequence to silence CDKN1C. This provides a mechanism for biallelic silencing of CDKN1C which contributes to the BWS disease phenotype. In summary, by ChRIP-seq, RNA expression profiling in tumours and human patient-derived cell line based model systems, we have uncovered new roles of lncRNA in epigenetic gene regulation

Understanding Long Noncoding RNA and Chromatin Interactions: What We Know So Far

With the evolution of technologies that deal with global detection of RNAs to probing of lncRNA-chromatin interactions and lncRNA-chromatin structure regulation, we have been updated with a comprehensive repertoire of chromatin interacting lncRNAs, their genome-wide chromatin binding regions and mode of action. Evidence from these new technologies emphasize that chromatin targeting of lncRNAs is a prominent mechanism and that these chromatin targeted lncRNAs exert their functionality by fine tuning chromatin architecture resulting in an altered transcriptional readout. Currently, there are no unifying principles that define chromatin association of lncRNAs, however, evidence from a few chromatin-associated lncRNAs show presence of a short common sequence for chromatin targeting. In this article, we review how technological advancements contributed in characterizing chromatin associated lncRNAs, and discuss the potential mechanisms by which chromatin associated lncRNAs execute their functions

The Risk-Associated Long Noncoding RNA NBAT-1 Controls Neuroblastoma Progression by Regulating Cell Proliferation and Neuronal Differentiation

Neuroblastoma is an embryonal tumor of the sympathetic nervous system and the most common extracranial tumor of childhood. By sequencing transcriptonnes of low- and high-risk neuroblastomas, we detected differentially expressed annotated and nonannotated long noncoding RNAs (lncRNAs). We identified a lncRNA neuroblastoma associated transcript-1 (NBAT-1) as a biomarker significantly predicting clinical outcome of neuroblastoma. CpG methylation and a high-risk neuroblastoma associated SNP on chromosome 6p22 functionally contribute to NBAT-1 differential expression. Loss of NBAT-1 increases cellular proliferation and invasion. It controls these processes via epigenetic silencing of target genes. NBAT-1 loss affects neuronal differentiation through activation of the neuronal-specific transcription factor NRSF/REST. Thus, loss of NBAT-1 contributes to aggressive neuroblastoma by increasing proliferation and impairing differentiation of neuronal precursors

Y chromosomal noncoding RNAs regulate autosomal gene expression via piRNAs in mouse testis.

Abstract Background Deciphering the functions of Y chromosome in mammals has been slow owing to the presence of repeats. Some of these repeats transcribe coding RNAs, the roles of which have been studied. Functions of the noncoding transcripts from Y chromosomal repeats however, remain unclear. While a majority of the genes expressed during spermatogenesis are autosomal, mice with different deletions of the long arm of the Y chromosome (Yq) were previously also shown to be characterized by subfertility, sterility and sperm abnormalities, suggesting the presence of effectors of spermatogenesis at this location. Here we report a set of novel noncoding RNAs from mouse Yq and explore their connection to some of the autosomal genes expressed in testis. Results We describe a set of novel mouse male-specific Y long arm (MSYq)-derived long noncoding (lnc) transcripts, named Pirmy and Pirmy-like RNAs. Pirmy shows a large number of splice variants in testis. We also identified Pirmy-like RNAs present in multiple copies at different loci on mouse Y chromosome. Further, we identified eight differentially expressed autosome-encoded sperm proteins in a mutant mouse strain, XYRIIIqdel (2/3 Yq-deleted). Pirmy and Pirmy-like RNAs have homology to 5′/3′UTRs of these deregulated autosomal genes. Several lines of experiments show that these short homologous stretches correspond to piRNAs. Thus, Pirmy and Pirmy-like RNAs act as templates for several piRNAs. In vitro functional assays reveal putative roles for these piRNAs in regulating autosomal genes. Conclusions Our study elucidates a set of autosomal genes that are potentially regulated by MSYq-derived piRNAs in mouse testis. Sperm phenotypes from the Yq-deleted mice seem to be similar to that reported in inter-specific male-sterile hybrids. Taken together, this study provides novel insights into possible role of MSYq-derived ncRNAs in male sterility and speciation