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Regulatory Roles of Noncoding RNA in Development and Disease
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Long noncoding RNAs (lncRNAs) are being realized as important players in gene regulation and their misregulation has been considered as one of the underlying causes for tumor initiation and progression in many human pathologies. In the current thesis, I have addressed the functional role of lncRNAs in development and disease model systems.

Genomic imprinting is an epigenetic phenomenon by which subset of genes are expressed in a parent of origin-specific manner. The Kcnq1 imprinted locus is epigenetically regulated by Kcnq1ot1 lncRNA. Deletion of an 890bp region at the 5’ end of Kcnq1ot1 in mouse resulted in the loss of silencing of neighboring ubiqui-tously imprinted genes (UIGs). In addition, we observed loss of DNA methylation at the UIG promoters. We have shown that Kcnq1ot1 RNA establishes CpG methylation by interacting with DNMT1. To explore the stability of lncRNA mediated silencing pathways, we have conditionally deleted Kcnq1ot1 in the mouse in a stage and tissue-specific manner. We have shown that Kcnq1ot1 is continuously required for maintaining the silencing of UIGs, whereas the silencing of the placental im-printed genes is maintained in an RNA independent manner.  

To identify chromatin-associated lncRNA (CARs) on a genome-wide scale, we purified RNA from the sucrose gradient fractionated chromatin and subjected it to RNA sequencing. Our study has identified 141 intronic and 74 long intergenic CARs. Characterization of one of the CARs revealed that it regulates the expression of neighboring genes in cis by modulating the chromatin structure.  

We have explored the functional role of lncRNA in tumor progression and initiation by using pediatric neuroblastoma. By transcriptional profiling of low- and high-risk tumors, we have identified several lncRNAs differentially expressed between these subtypes. We report an uncharacterized RNA NBAT-1, expressed at lower levels in high-risk tumors relative to low-risk tumors.  Using neuroblastoma cell culture system, we demonstrated that NBAT-1 has anti-cell proliferative and anti-invasive properties. In addition, it promotes differentiation of neurons from undifferentiated neuroblastoma cell lines.  

In summary, by employing mouse genetics, cell culture based model system and expression profiling in tumors, we have uncovered new roles of lncRNA in gene regulation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 51 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 940
Keyword [en]
Noncoding RNA, Genomic Imprinting, Epigenetics, Neuroblastoma
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-209596ISBN: 978-91-554-8786-7 (print)OAI: oai:DiVA.org:uu-209596DiVA: diva2:658644
Public defence
2013-12-04, Rudbecksalen, Rudbeck laboratory, Uppsala, 09:30 (English)
Opponent
Supervisors
Available from: 2013-11-12 Created: 2013-10-22 Last updated: 2014-01-23
List of papers
1. Kcnq1ot1 noncoding RNA mediates transcriptional gene silencing by interacting with Dnmt1
Open this publication in new window or tab >>Kcnq1ot1 noncoding RNA mediates transcriptional gene silencing by interacting with Dnmt1
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2010 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 137, no 15, 2493-2499 p.Article in journal (Refereed) Published
Abstract [en]

A long noncoding RNA, Kcnq1ot1, regulates the expression of both ubiquitously and tissue-specific imprinted genes within the Kcnq1 domain. However, the functional sequences of the Kcnq1ot1 RNA that mediate lineage-specific imprinting are unknown. Here, we have generated a knockout mouse with a deletion encompassing an 890-bp silencing domain (Delta890) downstream of the Kcnq1ot1 promoter. Maternal transmission of the Delta890 allele has no effect on imprinting, whereas paternal inheritance of the deletion leads to selective relaxation of the imprinting of ubiquitously imprinted genes to a variable extent in a tissue-specific manner. Interestingly, the deletion affects DNA methylation at somatically acquired differentially methylated regions (DMRs), but does not affect the histone modifications of the ubiquitously imprinted genes. Importantly, we found that Kcnq1ot1 recruits Dnmt1 to somatic DMRs by interacting with Dnmt1, and that this interaction was significantly reduced in the Delta890 mice. Thus, the ubiquitous and placental-specific imprinting of genes within the Kcnq1 domain might be mediated by distinct mechanisms, and Kcnq1ot1 RNA might mediate the silencing of ubiquitously imprinted genes by maintaining allele-specific methylation through its interactions with Dnmt1.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-128879 (URN)10.1242/dev.048181 (DOI)000279828300006 ()20573698 (PubMedID)
Available from: 2010-07-29 Created: 2010-07-29 Last updated: 2017-12-12Bibliographically approved
2. Long noncoding RNA-mediated maintenance of DNA methylation and transcriptional gene silencing
Open this publication in new window or tab >>Long noncoding RNA-mediated maintenance of DNA methylation and transcriptional gene silencing
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2012 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 139, no 15, 2792-2803 p.Article in journal (Refereed) Published
Abstract [en]

Establishment of silencing by noncoding RNAs (ncRNAs) via targeting of chromatin remodelers is relatively well investigated; however, their role in the maintenance of silencing is poorly understood. Here, we explored the functional role of the long ncRNA Kcnq1ot1 in the maintenance of transcriptional gene silencing in the one mega-base Kcnq1 imprinted domain in a transgenic mouse model. By conditionally deleting the Kcnq1ot1 ncRNA at different stages of mouse development, we suggest that Kcnq1ot1 ncRNA is required for the maintenance of the silencing of ubiquitously imprinted genes (UIGs) at all developmental stages. In addition, Kcnq1ot1 ncRNA is also involved in guiding and maintaining the CpG methylation at somatic differentially methylated regions flanking the UIGs, which is a hitherto unknown role for a long ncRNA. On the other hand, silencing of some of the placental-specific imprinted genes (PIGs) is maintained independently of Kcnq1ot1 ncRNA. Interestingly, the non-imprinted genes (NIGs) that escape RNA-mediated silencing are enriched with enhancer-specific modifications. Taken together, this study illustrates the gene-specific maintenance mechanisms operational at the Kcnq1 locus for tissue-specific transcriptional gene silencing and activation.

Keyword
Noncoding RNA, Epigenetics, Chromatin, Kcnq1ot1, Heterochromatin, Mouse, Genomic imprinting
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-177990 (URN)10.1242/dev.079566 (DOI)000306284400018 ()22721776 (PubMedID)
Available from: 2012-07-23 Created: 2012-07-23 Last updated: 2017-12-07Bibliographically approved
3. Characterization of the RNA content of chromatin
Open this publication in new window or tab >>Characterization of the RNA content of chromatin
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2010 (English)In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 20, no 7, 899-907 p.Article in journal (Refereed) Published
Abstract [en]

Noncoding RNA (ncRNA) constitutes a significant portion of the mammalian transcriptome. Emerging evidence suggests that it regulates gene expression in cis or trans by modulating the chromatin structure. To uncover the functional role of ncRNA in chromatin organization, we deep sequenced chromatin-associated RNAs (CARs) from human fibroblast (HF) cells. This resulted in the identification of 141 intronic regions and 74 intergenic regions harboring CARs. The intronic and intergenic CARs show significant conservation across 44 species of placental mammals. Functional characterization of one of the intergenic CARs, Intergenic10, revealed that it regulates gene expression of neighboring genes through modulating the chromatin structure in cis. Our data suggest that ncRNA is an integral component of chromatin and that it may regulate various biological functions through fine-tuning of the chromatin architecture.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-136028 (URN)10.1101/gr.103473.109 (DOI)000279404700004 ()
Available from: 2010-12-10 Created: 2010-12-09 Last updated: 2017-12-11Bibliographically approved
4. Transcriptomic profiling of primary neuroblastomas revealsa high-risk tumor associated long noncoding RNA NBAT1, with functional roles in cell proliferation and neuronal differentiation.
Open this publication in new window or tab >>Transcriptomic profiling of primary neuroblastomas revealsa high-risk tumor associated long noncoding RNA NBAT1, with functional roles in cell proliferation and neuronal differentiation.
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-209582 (URN)
Available from: 2013-10-22 Created: 2013-10-22 Last updated: 2014-01-23

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