Epigenetic Regulation by Noncoding RNA

University dissertation from Uppsala : Acta Universitatis Upsaliensis

Abstract: 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.

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