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Characterization of the RNA content of chromatin
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Cancer pharmacology and computational medicine)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Cancer pharmacology and computational medicine)
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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.

Place, publisher, year, edition, pages
2010. Vol. 20, no 7, 899-907 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-136028DOI: 10.1101/gr.103473.109ISI: 000279404700004OAI: oai:DiVA.org:uu-136028DiVA: diva2:376146
Available from: 2010-12-10 Created: 2010-12-09 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Epigenetic Regulation by Noncoding RNA
Open this publication in new window or tab >>Epigenetic Regulation by Noncoding RNA
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

High throughput transcriptomic analyses have realized us with the fact that eukaryotic genome encodes thousands of noncoding RNAs (ncRNAs) with unknown function. In my thesis, I sought to address epigenetic regulation of transcription by ncRNA using the Kcnq1 imprinted cluster as a model system. Genomic imprinting is an epigenetic phenomenon whereby one of the parental alleles is silenced by epigenetic mechanism in a parent of origin-specific manner. A long ncRNA Kcnq1ot1 regulates imprinting of nearly 8 protein coding genes in the Kcnq1 imprinted cluster. Expression of Kcnq1ot1 is restricted to the paternal chromosome while that of protein-coding genes to the maternal chromosome.

Kcnq1ot1 is a 91kb long, moderately stable, nuclear localized and RNAPII encoded transcript. We demonstrated that Kcnq1ot1 RNA itself mediates lineage specific silencing on the paternal chromosome by interacting with chromatin and recruiting the repressive chromatin modifiers to the imprinted gene promoters. Previously we identified an 890bp silencing domain (SD) at the 5´end of the Kcnq1ot1 RNA which is responsible for gene silencing. Targeted deletion of the 890SD in mouse resulted in specific loss of silencing of ubiquitously imprinted genes. We have further shown that Kcnq1ot1 interacts with Dnmt1 and recruit Dnmt1 at the somatic DMRs flanking some of the ubiquitously imprinted genes. We next addressed the stability of the Kcnq1ot1 mediated epigenetic silencing using transgenic mouse where we have conditionally deleted the Kcnq1ot1 RNA at different developmental stages and we found that Kcnq1ot1 RNA is required to maintain the silencing of the ubiquitously imprinted genes. In addition, DNA methylation, which controls imprinting of the ubiquitous genes require Kcnq1ot1 for its maintenance.

To characterize the ncRNAs that mediate gene regulation through chromatin interaction we have isolated chromatin associated RNAs (CARs) from sucrose gradient fractioned chromatin. High-throughput sequencing of the CARs resulted in the identification of the 141 intronic and 74 intergenic regions harboring CARs. We characterized one of the intergenic CARs which regulate the transcription of the two neighboring genes by modulating the chromatin marks.

In summary current thesis has uncovered unprecedented role of ncRNAs in gene expression via chromatin level regulation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 719
Keyword
Genomic Imprinting, Noncoding RNA, Epigenetics, DNA methylation
National Category
Medical and Health Sciences
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-160326 (URN)978-91-554-8201-5 (ISBN)
Public defence
2011-12-06, Rudbecksalen, Rudbecklaboratoriet, Uppsala Science Park, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-11-15 Created: 2011-10-21 Last updated: 2011-11-23Bibliographically approved
2. Regulatory Roles of Noncoding RNA in Development and Disease
Open this publication in new window or tab >>Regulatory Roles of Noncoding RNA in Development and Disease
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
Noncoding RNA, Genomic Imprinting, Epigenetics, Neuroblastoma
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-209596 (URN)978-91-554-8786-7 (ISBN)
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

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